At one point in their life, virtually all humans become infected with EBV, most of them without any acute, severe or lasting health problems. However, in those who do, one can often identify an underlying disease-associated, more or less pronounced genetic susceptibility, which begs for the question whether EBV-associated disease processes indeed afflict also "immunocompetent" individuals, or put the other way around, what in the end will define such an "immune (in)competence" (235).

# PREDISPOSITION TO HEMATOLOGIC NEOPLASMS IN CHILDREN

The role of predisposing germline mutations and sequence variants in children and adults with various types of hematologic malignancies was hitherto largely underappreciated, because not all of them concur with nor create any easily recognizable clinical stigmata or suspicious family history. Especially if one screens neoplastic tissues for disease- and/or therapy relevant somatic mutations, it becomes of critical importance to distinguish those from germline ones, because the latter may also have a profound clinical impact as regards choice of therapy, donor selection in case of transplantations, evaluation of comorbidities as well as surveillance strategies (8, 173).

A recent paper by Duan et al. provides an excellent and very comprehensive overview about all the primary immunodeficiencies that in particular predispose to the development of various types of lymphomas and hematologic malignancies (3). The authors compiled more than 60 conditions, which comprised all subgroups of syndromic and non-syndromic cellular and humoral PID as well as defects in phagocytes and innate immunity.

To pack some of the underlying principles and problems into a practical and newsworthy perspective, I will now briefly turn to recent findings in three categories of childhood leukemia.

# Constitutional Trisomy 21

Trisomy 21 is not only the most common chromosome abnormality in liveborns but in many aspects also one of the most outstanding and fascinating examples of an immune system disorder, although for a long time the precise nature of the respective immunological derangements remained elusive (178). Since it is not a monogenic ailment, it is also hardly ever viewed as a primary immunodeficiency, although it clearly concurs with multiple distinct immunological and developmental defects that affect the myeloid but also the early and committed Blymphoid progenitor compartments in second trimester fetal liver (247). I chose this most intriguing and highly instructive example to explain in which way the predisposing risk score of a kind of "polygenic" disposition can easily reach at least the same magnitude as the one that is otherwise only achievable in a monogenic setting (55).The ensuing variable and clinically often inapparent immunological alterations in Down individuals comprise a mild to moderate decrease in T and B cells, impaired mitogen-induced T cell proliferation, reduced specific antibody responses to immunizations as well as defects of neutrophil chemotaxis (248). Affected individuals suffer from various types of autoimmune and autoinflammation diseases, whereas it is still a matter of debate whether they are also more prone to experience more or severer infections than non-Down individuals (176, 178, 248, 249). The first clues that helped to resolve the functional consequences of this immunological conundrum derived from recent transcriptome and proteome analyses. They revealed that the presence of an extra chromosome 21 leads, amongst others, to an overexpression of the four chromosome 21 encoded interferon receptors and, therefore, place this syndrome into the class of interferonopathies. Interferons are normally produced by cells in response to viral or bacterial infections, regulate genes in neighboring cells and shut down the production of proteins, which activate the immune system and thereby prevent the spread of the infection (250, 251). In line with interferonopathies and autoinflammatory conditions, individuals with Down syndrome display higher levels of many proinflammatory cytokines (including IL-6, IL-22, TNF-α, and MCP-1) as well as complement consumption, a state that indicates that the immune system is constantly fighting viral infections that are in fact not there (250). Whether and to which extent such a faulty overreaction may also participate in promoting the development of hematologic neoplasms perhaps in a similar fashion as in genuine virus infection-triggered malignancies, remains to be elucidated.

Individuals with an inborn trisomy 21 have also an extraordinary risk to acquire special forms of hematologic neoplasms in early life, whereas they are otherwise exquisitely protected against the development of any other malignancies (252, 253). Compared to normal age-matched children, the selflimiting transient myeloproliferative disorder (TMD) together with the acute megakaryoblastic leukemia (AML-M7), is ∼150 times and the B cell precursor ALL ∼33 times more common (99, 101, 102, 253, 254). Of particular note are also the absence of infant ALL, the rarity of T-ALL and, compared to normal children, the different distribution pattern of genetic B-ALL subtypes (255).

The occurrence of specific mutations in the receptive precursor cells determine which form of leukemia will eventually develop. In case of TMD, the perturbation of megakaryocyteerythroid precursor cell differentiation fosters the appearance of a highly specific truncating mutation in exon 2 of the hematopoietic transcription factor GATA1. This in turn provides the receptive cellular and molecular environment for the occurrence of further mutations, primarily in the JAK and RAS signaling pathways as well as in epigenetic regulators and multiple cohesion components, which then facilitate the further progression into AML (99, 101, 104, 254). A reduced lymphoid gene expression in fetal liver hematopoietic precursor cells impairs B-lymphoid development in a similar fashion. The ensuing maturation arrest leads to an ∼10-fold reduction in B cells. The concomitant accumulation of pro-B progenitors (247), on the other hand, increases the likelihood for illegitimate V(D)J recombination-mediated chromosomal rearrangements, in particular CRLF2 gene fusions, that can be found in approximately half of all Down syndrome BCP ALL cases (99, 104). To explore the potential contribution of chromosome 21-encoded and overexpressed genes, a set of 31 triplicated orthologous human genes were tested in germline mouse models (256). Their presence induced progenitor B cell self-renewal in vitro, maturation defects in vivo and the development of especially CRLF2-rearranged and JAK2 pathwayactivated BCP ALL. Out of these 31 genes, the nucleosomeremodeling protein high mobility group nucleosome-binding domain- containing protein 1 (HMGN1), whose protein product suppresses H3K27me3, turned out to be the most relevant candidate. Together with secondary alterations in the CRLF2, JAK2, NRAS, or KRAS genes, it promotes both B cell proliferation in vitro and the development of B ALL in mice in vivo (256).

Given the extraordinary susceptibility and the high incidence of leukemias, one would intuitively expect that both myeloid and lymphoid forms should occasionally occur together by pure chance alone. However, such a coincidence has so far never been reported. On the one hand, this lack of co-occurrence might indicate that the development of a specific type of leukemia requires and is subject to very individual-specific predisposing conditions. On the other hand, it is also in keeping with the fact that these patients virtually never suffer from secondary neoplasms and that they are in a unique and matchless way also protected against the development of any other types of neoplasms (252, 253).

This protective effect also has been put down to a copy number-dependent gene dosage but also context-dependent effect of specific chromosome 21-encoded genes. The presence of three ETS2 copies, for instance, act as tumor repressor in the ApcMin intestinal cancer mouse model, whereas in the PyMT breast cancer mouse model it functions as tumor promoter, albeit within the non-cancerous stromal cells, where it regulates the expression of genes that produce the extracellular matrix, an essential component for tumor growth and metastasis (257–260). Two other relevant genes are the Down's syndrome candidate region-1 (DSCR1), which encodes a suppressor of the vascular endothelial growth factor (VEGF)-mediated angiogenic signaling by the calcineurin pathway, and DYRK1A, which encodes a regulator of cell proliferation (261). In mice, the presence of a single extra copy of Dscr1 is sufficient to diminish tumor growth by suppressing the calcineurin pathway and therefore also angiogenesis, an effect that is significantly enhanced by an extra copy of Dyrk1a. For the sake of completeness, one needs to take at least also note of several other trisomic chromosome21 encoded genes whose presence in the stromal compartment helps to reduce tumor angiogenesis, namely the angiogenic inhibitor ADAMTS1, the transcription regulator ERG and, finally, the endothelial cell-specific genes JAMB and PTTG1IP (257).

# Bone Marrow Failure (BMF), Myelodysplastic Syndromes (MDS), and Myeloid Leukemias

**Table 1** provides a comprehensive summary of the various disease entities together with their causative genetic background. For a more in-depth overview I refer the interested reader to recent publications that deal with these individual subjects in detail (8, 60, 158, 264, 265, 268, 269, 272, 275, 283, 299, 300). Herein, I merely select a few instructive examples to highlight some of the intriguing phenomena that are particularly pertinent for the topic discussed herein.

Fanconi Anemia (FA) is not only the most common inherited BMF disorder but, with a 500- to 700-fold higher incidence of head and neck squamous cell carcinomas in older patients, also a highly penetrant cancer susceptibility syndrome (301). Except for the X-linked FANCB gene, it is due to bi-allelic mutations that can affect one of 21 genes, which encode various components of the evolutionarily conserved FA/BRCA repair complex. Five of these (BRCA2, PALB2, RAD51C, SLX4, and BACH1) specifically predispose also to breast cancer. The encoded proteins participate in biochemical pathways that safeguard not only against the effects of alkylating agents and radiation but, probably even more relevant, also against those of endogenous aldehydes, oxidative stress, inflammation, TABLE 1 | Immunodeficiency syndromes that predispose to the development of bone marrow failure, myelodysplasia and myeloid leukemias.

*Apart of being present in the germ line, somatic mutations of most of these genes can be commonly encountered in sporadic forms of analogous malignancies.*

mitophagy, and virophagy (263, 265, 302, 303). All these factors damage DNA in form of distinct inter-strand DNA crosslinks. The inability to repair these damages is the primary driver of the various biological and clinical problems that define this disease category.

Small aldehydes, such as acetaldehyde and formaldehyde, are not only ubiquitously present in the environment, but also potentially toxic byproducts of the normal cellular metabolism and especially de-methylation reactions (304–306). Given that they provide already a rich source for endogenous interstrand DNA and protein cross links, one can envisage that the pathogenic manifestations and consequences of FA mutations may also be strongly influenced and modified by the functional capability of aldehyde detoxifying enzymes, such as aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 5 (ADH5) (306, 307). In line with this notion, Japanese FA children that carried a functionally deficient ALDH2E504K allele were shown to progress more rapidly to aplastic anemia but not to MDS or AML (307–309). Moreover, malformations were only more severe in two of three homozygous carriers, which indicates that a deficient maternal genetic background might contribute to this outcome (310). Maternal-produced aldehydes diffuse indeed across the placenta and can thus damage the developing embryo's DNA, whereas embryo-derived ones can in turn be detoxified by the maternal organism. An inappropriate in utero exposure, such as an excessive maternal ethanol consumption during gestation, would therefore aggravate not only the formation of congenital abnormalities in FA but it provides also an intriguing etiological link to analogous phenotypic changes that define the alcohol embryopathy (308). Whether a disturbed aldehyde detoxifying system might also be causatively involved in the in utero initiation of childhood leukemias remains currently a matter of speculation (307).

In addition to stalling and destabilizing DNA replication forks directly, formaldehyde also selectively depletes BRCA2 via proteasomal degradation, a circumstance that poses a special risk for heterozygous BRCA2 mutation carriers. In these, formaldehyde-induced degradation can decrease the respective protein levels below the otherwise protective one of normal wild-type individuals and thereby potentiate their mutagenic vulnerability (311).

Taken together, these observations have significant implications for risk awareness and avoidance as well as the clinical management of FA patients. On the positive side, they offer new treatment opportunities, for instance in form of ALDH2 agonists and the widely used diabetes drug metformin, which acts as aldehyde scavenger. At least in mouse models, both of them are able to delay the onset of BMF and malignancies as well as improve hematopoiesis (263, 312, 313).

One remarkable feature of many heritable diseases is that somatic mutations can occasionally autocorrect the particular inherited gene defect in the respective cells (314). Such reverting mutations transform a homozygous or combined heterozygous state again back into a heterozygous functionally compensated state, either through somatic recombination, gene conversion or a compensatory mutation (314, 315). Although this phenomenon is well-known in immunodeficiencies, its effects are most probably still underappreciated. In BMF syndromes such an autocorrection can spontaneously improve or even resolve the specific underlying hematopoietic problem. Amongst others, such spontaneous remissions have repeatedly been documented in FA, dyskeratosis congenita, Diamond-Blackfan anemia, Shwachman-Diamond syndrome and, more recently, in the SAMD9- and SAMD9L-associated Mirage and ataxiapancytopenia syndromes, respectively (273, 287–293, 301, 316). Heterozygous SAMD9L gain-of-function mutations decrease cell proliferation. The loss of the mutation-carrying chromosome 7 in bone marrow cells therefore leads to the development of MDS and acute myeloid leukemias, whereas a compensating duplication of the normal allele in form of an uniparental disomy (UPD) 7 or 7q is able to resolve the cytopenias (287–293).

The somatic appearance of a complete or partial UPD always draws attention to regions that are usually highly relevant for specific disease processes (317, 318). Such UPDs may either contain duplicated gain of function mutations or, as alluded to in the example above, eliminate them (317, 318). A similar important consequence is the mere transformation of a heterozygous to a homozygous state. In case of the HLAcontaining region on the short arm of chromosome 6, for instance, it is clearly exerted through a selective pressure, since the loss of one HLA haplotype is an important immuneescape mechanism. It protects neoplastic cells from the immune surveillance machinery and therefore also plays a crucial role for disease recurrence after haploidentical stem cell transplantations (319, 320). Contrariwise, such a haplotype loss is able to shield hematopoietic cells from the destructive effects of autoimmunity, as has been demonstrated in case of aplastic anemia (321). The practical problems that arise from such a hematopoietic revertant mosaicism is that it may lead to an ascertainment bias and cause difficulties in identifying underlying disease-relevant mutations (322).

The formation of such well-adapted clones might suggest that such compensatory mechanisms are rare events. However, there is ample evidence that this is definitely not the case. Rather than being a life-long stable system the genome is a highly dynamic one that is continuously modified and shaped by ongoing mutational processes, which eventually promote the appearance of cell clones and populations with an increased survival fitness. The formation of somatic mosaicism is therefore the rule rather than the exception, as exemplified by Davis et al., who observed a remarkable clonal heterogeneity and diversity of lymphocytes in a patient with a Wiskott-Aldrich syndrome (323). The continuous generation of such cellular variants and the selective pressures they are exposed to is thus not only a characteristic of the exceptional dynamics of neoplastic but also of normal cells populations (324).

# B-Cell Precursor Acute Lymphoblastic Leukemia (BCP ALL)

Germline lesions that predispose to BCP ALL in children comprise not only those which cause various cancer prone and chromosomal syndromes but also other genuine gene disrupting defects as well as high and low risk variants. The majority of these

TABLE 2 | Chromosomal locations of GWAS-verified SNPs or genuine germline gene defects that predispose to the development of particular types of childhood ALL.

*For a more general overview about ALL predisposition syndromes and ALL predisposing RASopathies see Kratz et al. and Cave et al., respectively (62, 243).* \**Noonan syndromes, Rasopathy,* \*\**Rubinstein-Taybi syndrome,* \*\*\**Li-Fraumeni syndrome.*

affect genes that encode B-cell development and transcription factors as well as components of various signal transduction pathways (**Table 2**) (42, 297, 342).

The development of B lymphocytes, in particular, is coordinated by specific regulatory transcription networks that activate the respective B-lymphoid program and at the same time oppress alternate cell fates (343). Somatic mutations in several of these key regulators, such as IKZF1, TCF3, EBF1, and PAX5, induce leukemic transformation by blocking normal B cell differentiation, which then leads to the accumulation of leukemic B-cell precursors. Although their role in leukemogenesis has been known and explored already for quite some time, the awareness that otherwise apparently inconsequential germline variants may also exert a predisposing effect is quite new. The biological relevance and functional consequences of some of these variants has been confirmed in the meantime with appropriate in vitro and in vivo experiments and test systems (23–26, 28, 332, 333).

Take for instance PAX5, a member of the "paired box" family of transcription factors, which encodes the B cell lineage specific activator protein that is expressed at early but not late stages of B-cell differentiation (195). Out of the three up to now reported families with highly penetrant germline variants, 13 carriers developed ALL (48, 328). In the two families, in which the respective information was provided, all unaffected carriers as well as those with ALL had normal immunoglobulin levels and no evidence of an impaired B cell function at diagnosis (328). Moreover, in line with Greaves two step model, leukemia developed in mice only after exposure to common pathogens and the acquisition of second hits in the IL7R/JAK3/STAT5 signaling axis (344).

Another revealing example is IKZF1, which encodes IKAROS, a member of a hematopoietic zinc-finger transcription factor family (345–347). The mutational spectrum of human IKZF1 associated diseases ranges from somatic to germline and from haploinsufficient to dominant negative forms (171). Somatic mutations occur in overall 15% of BCP-ALL and especially in prognostic adverse genetic subtypes (348, 349). Heterozygous germline mutations cause two different forms of immunodeficiency. The haploinsufficient, autosomal dominant late onset form of common variable immunodeficiency (CVID) has an incomplete penetrance, is clinically mild and concurs with a marked decrease in B-cell numbers and immunoglobulin levels as well as autoimmunity (175, 350, 351). The early-onset dominant negative CVID, on the other hand, is characterized by innate and adaptive immune defects of the B, T and myeloid cell lineage (171). Notably, 2/29 of patients with the late onset form developed B-ALL and 1/7 patients as well as another independently reported one with the early onset form developed T-ALL (171, 346, 351). Based on these observations, Churchman and colleagues screened remission samples from 4,963 childhood ALL cases, identified a total of 28 unique IKZF1 variants in 43 and succeeded to prove a functional consequence in 22 of them (172, 173). Evans et al. even attempted to elucidate the influence of a parental environmental exposure on such leukemia-predisposing risk alleles (352). They found some preliminary albeit hitherto unexplainable evidence that the IKZF1 risk genotype might have a stronger effect if the mother took folic acid or if the father did not smoke prior to pregnancy (352).

Finally, Auer et al reported a first intriguing example of a "double-hit one-pathway" scenario, in which the biparental inherited combination of two rare germline variants, JAK2 (G571S) and STAT3 (K370R), whose products synergistically interact in the same disease-relevant JAK/STAT signal transduction pathway, is obviously sufficient to induce a Ph-like BCP-ALL (49).

#### CONCLUDING REMARKS

During their entire development and ongoing existence, both the immune system as well as malignant diseases need to adapt themselves to highly variable and continuously fluctuating environmental conditions, which requires a high flexibility that is largely driven by a combination of interacting antagonistic as well as synergistic deterministic events and regulatory probabilities. "Immunodeficiency" and "tumor susceptibility" are thus two closely intertwined concepts, whose original understanding was based on easily explicable clinical symptoms as well as certain genetic norms. As long as these were based on such more or less simple phenotypic and genotypic features, one did not require any further explanatory definitions. However, the switch from phenotypic to genetic ascertainment programs include now much less obvious disease categories, healthy carriers as well as only vaguely defined potentially predisposed individuals. Although this approach enables of course unprecedented insights into the fine-scale structural and regulatory organization of biological system, the boundaries of classification standards get increasingly blurred, which goes hand in hand with the awareness that it becomes increasingly difficult and virtually impossible to define either of these terms in an unambiguous manner anymore. The more closely one looks, the harder it gets to find genes that are not in one or the other way part of this game.

#### REFERENCES

1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell (2011) 144:64674. doi: 10.1016/j.cell.2011.02.013

In his highly recommendable and readable book "Tending Adam's garden," Irun Cohen portrays the immune system as a cognitive system (353). Like its prototypic equivalent, the brain, it learns through individual experience and thereby forms a functionally highly efficient, flexible, and to some extent also redundant interactive structure. As Cohen pointed out, a particular gene may only become essential once the system has organized itself around it so that thereafter the system becomes dependent on it. In case this particular gene is already defective at the beginning, the system often can compensate for this loss and organize itself around an alternative gene, which then becomes the essential one.

Thus, organizational entities depend not only on distinct features of particular sub-units but even more so on their functional interactions. In case of the immune system, such multi-component, self-emergent networks comprise a variety of distinct cellular as well as humoral host constituents, but also a manifold of environmental factors, such as the maternal immune system, the microbiome, infectious agents, as well as physical and chemical agents, that co-govern and modulate, but often also interfere and disrupt its normal development during different stages.

Thus, organizational entities depend perhaps less on the appropriate function of particular genetic sub-units alone but much more on the successful interaction of their cellular and humoral products, an observation that is readily evident in case of genetically determined developmental disorders that can cause both immune system deficiencies as well as malignant diseases.

The essential implication of Cohens' model is that we only may become more successful in curing such disease when we begin to understand the decision-making processes of the immune system rather than that of the effects of individual components alone.

# AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and has approved it for publication.

# ACKNOWLEDGMENTS

This paper is dedicated to my late friend and colleague Andreas Heitger, a devoted immunologist, with whom it was always fun to discuss the philosophical part of the activity and behavioral pro and cons of the immune system.

I acknowledge the networking support by the COST Action CA16223 LEukaemia GENe Discovery by data sharing, mining and collaboration (LEGEND) as well as by the IBFM Leukemia & Lymphoma Genetic Predisposition Committee, the Österreichische Kinderkrebsforschung for the continuous funding of my research work and Fikret Rifatbegovic's invaluable help in designing and drawing the Figure.

2. Sonnenschein C, Soto AM. Theories of carcinogenesis: an emerging perspective. Semin Cancer Biol. (2008) 18:372–77. doi: 10.1016/j.semcancer.2008. 03.012

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patients: an easy-to-use selection tool. Eur J Med Genet. (2016) 59:116–25. doi: 10.1016/j.ejmg.2016.01.008

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and are associated with loss of chromosome 7. Nat Genet. (2016) 48:792–7. doi: 10.1038/ng.3569

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**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Haas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# CVID-Associated Tumors: Czech Nationwide Study Focused on Epidemiology, Immunology, and Genetic Background in a Cohort of Patients With CVID

Pavlina Kralickova1†, Tomas Milota<sup>2</sup> \* † , Jiri Litzman<sup>3</sup> , Ivana Malkusova<sup>4</sup> , Dalibor Jilek <sup>5</sup> , Jitka Petanova<sup>6</sup> , Jana Vydlakova<sup>7</sup> , Alena Zimulova<sup>8</sup> , Eva Fronkova<sup>9</sup> , Michael Svaton<sup>9</sup> , Veronika Kanderova<sup>9</sup> , Marketa Bloomfield<sup>2</sup> , Zuzana Parackova<sup>2</sup> , Adam Klocperk <sup>2</sup> , Jiri Haviger <sup>10</sup>, Tomas Kalina<sup>9</sup> and Anna Sediva<sup>2</sup>

#### Edited by:

Fabian Hauck, LMU München, Germany

#### Reviewed by:

Roshini Sarah Abraham, Nationwide Children's Hospital, United States Jolan Eszter Walter, University of South Florida, United States

#### \*Correspondence:

Tomas Milota<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Primary Immunodeficiencies, a section of the journal Frontiers in Immunology

Received: 11 September 2018 Accepted: 19 December 2018 Published: 22 January 2019

#### Citation:

Kralickova P, Milota T, Litzman J, Malkusova I, Jilek D, Petanova J, Vydlakova J, Zimulova A, Fronkova E, Svaton M, Kanderova V, Bloomfield M, Parackova Z, Klocperk A, Haviger J, Kalina T and Sediva A (2019) CVID-Associated Tumors: Czech Nationwide Study Focused on Epidemiology, Immunology, and Genetic Background in a Cohort of Patients With CVID. Front. Immunol. 9:3135. doi: 10.3389/fimmu.2018.03135 <sup>1</sup> Department of Allergology and Clinical Immunology, Faculty of Medicine, Charles University and University Hospital in Hradec Kralove, Hradec Kralove, Czechia, <sup>2</sup> Department of Immunology, Second Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czechia, <sup>3</sup> Department of Allergology nad Clinical Immunology, Faculty of Medicine, Masaryk University and St Anne's University Hospital in Brno, Brno, Czechia, <sup>4</sup> Department of Allergology and Clinical Immunology, Faculty of Medicine in Pilsen, Charles University and University Hospital Pilsen, Pilsen, Czechia, <sup>5</sup> Department of Allergology and Clinical Immunology, Institute of Health, Usti nad Labem, Czechia, <sup>6</sup> Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czechia, <sup>7</sup> Department of Clinical Immunology and Allergology, Institute for Clinical and Experimental Medicine, Prague, Czechia, <sup>8</sup> Department of Pneumology, Regional Thomas Bata Hospital, Zlin, Czechia, <sup>9</sup> Childhood Leukemia Investigation Prague, Second Faculty of Medicine, Charles University, Prague, Czechia, <sup>10</sup> Department of Informatics and Quantitative Methods, Faculty of Informatics and Management, University of Hradec Kralove, Hradec Kralove, Czechia

Background: Common variable immunodeficiency disorder (CVID) is one of the most frequent inborn errors of immunity, increased occurrence of malignancies, particularly lymphomas, and gastric cancers, has long been noted among CVID patients. Multifactorial etiology, including immune dysregulation, infections, chronic inflammation, or genetic background, is suggested to contribute to tumor development. Here, we present the results of the first Czech nationwide study focused on epidemiology, immunology and genetic background in a cohort of CVID patients who also developed tumors

Methods: The cohort consisted of 295 CVID patients followed for 3,070 patient/years. Standardized incidence ratio (SIR) was calculated to determine the risk of cancer, and Risk ratio (RR) was established to evaluate the significance of comorbidities. Moreover, immunophenotyping, including immunoglobulin levels and lymphocyte populations, was assessed. Finally, Whole exome sequencing (WES) was performed in all patients with lymphoma to investigate the genetic background.

Results: Twenty-five malignancies were diagnosed in 22 patients in a cohort of 295 CVID patients. SIR was more than 6 times greater in comparison to the general population. The most common neoplasias were gastric cancers and lymphomas. History of Immune thrombocytopenic purpura (ITP) was established as a potential risk factor, with over 3 times higher risk of cancer development. The B cell count at diagnosis of

**96**

lymphoma was reduced in the lymphoma group; moreover, post-treatment B and T cell lymphopenia, associated with poorer outcome, was found in a majority of the patients. Intriguingly, no NK cell depression was observed after the chemotherapy. WES revealed heterogeneous genetic background among CVID patients with tumors, identifying gene variants associated with primary immunodeficiencies (such as CTLA4, PIK3CD, PMS2) and/or increased cancer susceptibility (including BRCA1, RABEP1, EP300, KDM5A).

Conclusions: The incidence of malignancy in our CVID cohort was found to be more than 6 times greater compared to the general population. Gastric cancers and lymphomas were the most frequently diagnosed tumors. ITP was identified as a risk factor for malignancy in CVID patients. WES analysis confirmed a wide genetic heterogeneity among CVID patients. The identified causative or modifying gene variants pointed to errors in mechanisms contributing to both immunodeficiency and malignancy.

Keywords: common variable immunodeficiency, malignancy, lymphoma, gastric cancer, whole exome sequencing

# INTRODUCTION

Immune control of tumor development and growth requires a functional immune system capable of complex immune responses necessary for recognition and elimination of malignant cells. As such, inborn errors resulting in immunodeficiencies may convey an increased risk of cancer. In general, the spectrum of malignancies in primary immunodeficiency (PID) patients is clearly biased when compared to malignant diseases in the general population. Malignancies in PIDs tend to be restricted to certain oncological entities, and their pathophysiology is often linked to the mechanism underlying the particular immunodeficiency (1). For example, immunodeficiencies associated with gain-of-function mutations in PIK3 are associated with a high risk of lymphoma (2). The role of this signaling pathway in cancer genesis and immunodeficiency is validated by therapeutic success of targeted PI3K/mTOR pathway inhibition used in activating PIK3 syndrome, as well as in malignant diseases (3). Similarly, immunodeficiencies arising from developmental defects of stem cells, myeloid cells or lymphocytes are associated with an increased incidence of leukemia or lymphomas, implying errors in the corresponding pathways (1). Recent advances in understanding of molecular mechanisms underlying primary immunodeficiencies, as well as tumors, has provided evidence for such associations. However, in immunodeficiencies that are not yet precisely defined by their molecular/genetic cause, the situation is more complex. Common variable immunodeficiency disorder (CVID) is one of the most frequent forms of antibody deficiencies; yet, its pathophysiology remains largely unknown. The hallmark of CVID is the impairment of the B cell compartment, typically manifesting as a reduction of mature forms of B cells and expansion of less differentiated stages of B lymphocytes (4). The T cell compartment is also usually skewed in CVID, specifically toward terminally differentiated forms, including senescent cells, typically affecting both CD4and CD8 T cells, which are crucial for anti-tumor immunity (5). The mechanisms of B cell involvement in anti-tumor immunity are largely unknown. B cells may promote both pro-tumourigenic responses (e.g., specific subsets of B cells may produce IL-10 or TGF-beta with immunosuppressive properties, B cells may promote tumor genesis and tumor progression by alteration of the angiogenic and proinflammatory microenvironment), as well as anti-tumourigenic responses (e.g., B cells may enhance cytotoxic T cell activity, indirectly mediate antibody dependent cytotoxic mechanisms or serve as professional antigen-presenting cells, initiating the T cell response) (6). Inborn impairment of the B cell lineage, along with T cell dysregulation, may facilitate the genesis of tumors in CVID patients. Furthermore, the immunologic defect is accentuated by recurrent and chronic infections. Chronic viral infections, particularly EBV, are strongly associated with lymphoproliferative diseases and lymphoma (7). Additionally, chronic inflammatory response, per se, represents a risk factor for tumor development, especially in patients genetically predisposed to malignancy.

Efforts made to establish the genetic etiology of CVID have thus far been successful in 2–10% of CVID patients (known CVID-associated gene variants are shown in **Table 1**) (8). Some of the CVID-associated genes represent a clear predisposition to cancer, as described in previously published CVID cohorts; most prominently, these mutations are in genes causing alterations in the NFkB or PI3k pathways or in genes affecting B cell receptors (3, 9, 10).

Overall, the factors contributing to increased incidence of malignancy in CVID are complex, encompassing genetics, immune response dysregulation, infections, inflammation, and perhaps other not yet elucidated mechanisms.

Here, we present the results of a complex study on a Czech national cohort of CVID patients who also presented with malignancy. National epidemiological data were collected, immune profiles were analyzed and, in a subgroup of CVID patients with lymphoma, Whole exome sequencing (WES) was performed.

TABLE 1 | List of genes associated with Common variable

immunodeficiency (monogenic causes and modifier genes) and their prevalence, adjusted according to Bogaert et al. (8).

TNFRSF13B (TACI), TNFRSF13C (BAFF-R), MSH2, MSH5, CLECG1, MLH1, RAD50, ORC4L, FCGR2A.

#### METHODS

This study was approved by local ethics committee of Motol University Hospital. Written informed consents were obtained from all enrolled patients.

#### Data Collection

Retrospective clinical and laboratory data of 295 enrolled patients were obtained from medical records of national referral centers for the treatment of adult patients with primary immunodeficiency diseases. The collected data covered the period from 1997 to 2016. They included a total amount and a length of surveillance of all CVID patients fulfilling ESID/PAGID criteria, a number of CVID cancer patients and patient-specific data: year of birth, age at first symptoms associated with CVID and their nature, clinical comorbidities, age at cancer diagnosis, type of cancer, therapeutic regimens, survival rates, cause of death (if applicable), and specific details of cancer diagnostics.

A more detailed set of clinical data was obtained from 11 CVID patients with cancer and from 160 randomly selected cancer-free CVID patients, who represented the reference group. This cohort included 95 females and 65 males with a median age of 48 years (range 19–88). Czech general population data on occurrence of malignancy were obtained from the Czech National Cancer Registry and covered the period from 1994 to 2014 (the last available reports).

# Epidemiology

Prevalence and SIR were calculated to express the probability of cancer diagnosis in a CVID cohort compared to the general population. RR was used to assess the significance of comorbidities in CVID patients with cancer. Confidence intervals (95% CIs) were determined for both parameters. All results for which number 1 was beyond the 95% CI were accepted as statistically significant (11).

## Immunophenotyping

Serum levels of IgM, IgG, IgA were evaluated by nephelometric method using Image 800 systems (Beckman-Coulter, Brea, CA, USA). Basic lymphocyte subpopulations, including T cells, T helper cells, T cytotoxic cells, B cells and NK cells, were distinguished by FACS based on the expression of specific cell surface membrane markers using fluorochromeconjugated monoclonal antibodies CD3-FITC, CD4-PE/Cy, CD8-APC/Cy,CD19-APC, CD16-PE, CD56-PE; KOMBITEST, Exbio, Prague, Czech Republic. B cell subpopulations (including CD21low, naive, transitional, marginal zone-like, class-switched cells, and plasmablasts) and T cell subpopulations (recent thymic emigrants-RTE, naïve, central memory-CM, effector memory-EM, effector memory expressing CD45RA-TEMRA, activated T cells) were performed using antibody-fluorochrome conjugates: CD45-APC-H7, CD3-APC, CD4-PerCP-Cy5.5, CD16-PE, CD56-PE, TCRgd-PE-Cy7, CD38-FITC, CD21-APC, IgM-FITC, CD8-Horizon V-500, CD45RA-PE-Cy7 (BD Biosciences, San Jose, CA, USA), CD5-PE,CD8-FITC, CD27-Brilliant Violet 421, IgM-Brilliant Violet 510, IgD-PerCP-Cy5.5, CD4-Brilliant Violet 510, CD62L-Brilliant Violet 421, HLA-DR-PerCP-Cy5.5 (Biolegend, San Diego, CA), CD19-PE-Cy7, CD24-PE, CD24- APC-Alexa Fluor 750, CD8-APC-Alexa Fluor 750 (Beckman Coulter, Miami, FL, USA), CD27-Pacific Blue, CD38-Alexa Fluor 700, CD45RO-FITC, CD31-PE, CD4-Alexa Fluor 700 (Exbio, Vestec, Czech Republic), CCR7-PE (MiltenyiBiotec, BergischGladbach, Germany), CD3-PerCP-Cy5.5 (Affymetrix eBioscience, ThermoFisher Scientific, Waltham, MA, USA), and IgD biotin (SouthernBiotech, Birmingham, AL, USA) followed by Streptavidin-Qdot 605 (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA).

The absolute and relative counts were assessed for all subpopulations. Examinations of the basic subpopulations and immunoglobulin levels were performed at the time of diagnosis of CVID and at the time of diagnosis of lymphoma. The B and T cell subpopulations were measured prior to the genetic testing. All parameters were compared to the control cohort of 20 randomly selected CVID patients without lymphoma. All obtained data were statistically evaluated. A non-parametric Mann-Whitney test was used to compare independent samples, and a non-parametric Wilcoxon matched-pairs signed rank test was used to compare dependent samples; differences of p < 0.05 were regarded as significant. Median and 95% CIs were calculated for all analyzed parameters.

# CTLA-4 Expression

CTLA-4 expression was assessed in patient with novel mutations using FACS. Intracellular CTLA-4 was detected 16 h following anti-CD3/CD28 stimulation (Dynabeads, Thermo Fisher Scientific, MA, USA) using CTLA4-APC antibody together with the FOXP3 Fix/Perm Buffer set (BioLegend, San Diego, CA, USA) in CD4+CD127dimCD25+ T regulatory cells (Tregs). CD45-APC-H7, CD4-Brilliant Violet 510, and CD127-Brilliant Violet 421 (BD Biosciences, San Jose, CA, USA), CD25-PE-Cy7 and CD8-FITC (Exbio, Vestec, Czech Republic) antibodies were used for detection of Tregs.

# Whole Exome Sequencing

Sequencing libraries were prepared using a SureSelectXT Human All Exon V6+UTR kit (Agilent Technologies, Santa Clara, CA) from DNA isolated from patients' peripheral blood with a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Sequencing was performed by our facility on the NextSeq 500 (Illumina, San Diego, CA) instrument according to the manufacturer's protocols. The reads in resulting Fastq files were aligned against the human reference genome hg19 with BWA (12). Genomic variants were called with samtools and VarScan (13). Variant annotation was performed using SnpEff (14). Variant filtering was performed with Ingenuity <sup>R</sup> Variant AnalysisTM (IVA) software (www.qiagen.com/Ingenuity, QIAGEN). Only variants with read depths of at least 10 and allele frequencies of at least 0.3 were evaluated. Common variants with allele population frequencies of more than 0.1% or homozygous counts of 5 or more in the ExAC or gnomAD databases were filtered out unless reported as disease-causing in the HGMD <sup>R</sup> (BIOBASE GmbH) or dbSNP databases (15, 16). Variants predicted to have low impact by at least 2 out of 3 scores calculated by SIFT, PolyPhen2, or CADD and present in population databases were also discarded (17–19). Remaining variants were manually evaluated in Integrative Genomics Viewer (http://www.broadinstitute.org/igv) to exclude variants in reads with low mapping quality (20). The analysis was then focused on variants in genes reported as causative for inborn errors of immunity in the last International Union of Immunological Societies (IUIS) guidelines, cancerpredisposition genes in children and in-house lists of genes possibly leading to immune dysregulation based on recent publications and close interactions with causative genes reported by IUIS (21, 22).

# RESULTS

# Epidemiology and Clinical Manifestation

Our cohort of patients included 295 patients followed for 3,070 patient/years in total. The average ages at the first CVID-related symptoms and at the time of CVID diagnosis in a subgroup of CVID patients with malignancy were 34.2 and 38.3 years, respectively. A total of 25 malignancies were found in 22 patients (7.4% of all included patients) with SIR 6.3 (95% CI: 4.08– 9.31). These cases included 6/25 (24.0%) gastric carcinoma (GC): SIR 5.7, 95% CI: 2.08–12.32, 4/25 (16.0%), B cell Non-Hodgkin lymphoma (B-NHL): SIR 5.5, 95% CI: 1.50–14.09, 5/25 (20.0%), Hodgkin lymphoma (HL):SIR 30.0, 95% CI: 9.73–69.93 and 10/24 (41.7%) other cancers (SIR 5.0, 95% CI: 2.40–9.16). These were two cases of spinocellular carcinoma, basocellular carcinoma, and T-cell lymphoma, and a single case each of tonsillar carcinoma, breast carcinoma, renal carcinoma and urine bladder cancer. Cancer duplicity was observed in 3 patients (gastric and tonsillar carcinoma, breast cancer and urothelial carcinoma, spinocellular, and gastric carcinoma). Malignancies were diagnosed in 16 males and 6 females; the average age at diagnosis was 52.3 years (15 years after the diagnosis of CVID and 19 years after the first symptoms). The average ages of manifestation of GC, B-NHL, and HL in CVID cancer group were 55–59, 35–39, and 40–44 years, respectively.

Autoimmune cytopaenias, including Immune thrombocytopenic purpura (ITP) and Autoimmune hemolytic anemia (AIHA), were the most common complications in CVID both with and without malignancy. They were found in 8/22 (36.4%) and 27/160 (16.9%), respectively. Interestingly, a strikingly increased risk of malignancy was detected in a subgroup of CVID patients with a history of ITP (RR 3.52, 95% CI 1.42–7.26). Cumulative risk (RR 4.53, 95% CI 1.23–11.59) observed for B-NHL and HL together was similar. However, no risk increase was observed for isolated HL, B-NHL, and GC, probably due to the small number of cancer events and because the direct association with other documented CVID-related comorbidities was not significant (data not shown).

As mentioned above, lymphomas and GC represent the most frequent malignancies diagnosed in CVID patients in our cohort. While five out of six patients with GC (4 males, 2 females) underwent surgery, one received palliative chemotherapy with FLOX regimen (fluorouracil, leucovorin, oxaliplatin) due to highly progressed disease at the time of diagnosis. Five out of six patients had deceased before the study initiation, 2/6 (33.3%) due to disease progression, 2/6 (33.3%) passed away after achieving more than 10 years survival, and one patient died because of malnutrition due to severe enteropathy 2 years after the diagnosis of lymphoma. All patients suffered from various gastrointestinal complications related to CVID, and the majority of them also had splenomegaly.

All 5 patients (4 males, 1 female) with HL received chemotherapy. The following chemotherapeutic regimens were used: R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) in 2/5 (40%) of patients, BEACOPP (bleomycine, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, prednisone), eBEACOPP respectively, in 2/5 (40%) and DBVE-PC (adriamycin, bleomycine, vincristine, etoposide, prednisone, cyclophosphamide) in 1 patient (20%). No therapy-associated deaths or unexpected toxicities were noted. One patient died 3 years after treatment because of severe enteropathy, and 4/5 (80%) patients are still surviving today. All patients had previously described splenomegaly, and 3/5 (60%) had lymphadenopathy described prior to cancer diagnosis.

Similarly, all 4 patients (3 males, 1 female) with B-NHL received chemotherapy; no therapy-associated deaths or unexpected toxicities were noted. The following chemotherapeutic regimens were used: R-CHOP/CHOP [rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone in 3/4 (75%) patients−2 with DLBCL (Diffuse large B-cell lymphoma) and in 1 patient with MALT (Mucosaassociated lymphoid tissue] lymphoma. A GMALL (German multicenter ALL) regimen was used in 1 patient with Burkitt lymphoma. Two of these patients had been regularly followed even prior to the diagnosis of B-NHL for lymphadenopathy and previously reported splenomegaly. All patients are still alive. T-NHL was diagnosed in 2 patients. The clinical features of CVID patients in whom lymphomas were diagnosed are summarized in **Tables 2**, **3**. In this cohort, WES and detailed immunophenotyping were performed as part of further investigation (results presented further).

#### Immunophenotype

Parameters of cellular immunity were investigated, including T cell (CD4 T helpers as well as CD8 T cytotoxic cells), B cell and NK cell counts. No significant differences were registered between the absolute counts of T cells, T helper cells and T cytotoxic cells at the time of diagnosis of CVID in a cohort of patients with lymphoma compared to those without lymphoma. The T cellcounts were also well within the normal reference ranges (T cells 0.8−2.10E9/l, T helper cells 0.3−2.8E9/l, T cytotoxic cells 0.2−1.0E9/l). Unsurprisingly, the chemotherapeutic regiments for lymphoma led to skewing of T cell numbers (median 0.65E9/l, 95% CI 0.46–0.75 vs. 1.22E9/l, 95% CI 1.07–1.47, ∗∗∗p = 0.0004), specifically T helper cells (median 0.34E9/l, 95% CI 0.14–0.36 vs. 0.56E9/l, 95% CI 0.53– 0.76, ∗∗∗p = 0.0004) and T cytotoxic cells (0.26E9/l, 95% CI 0.14–0.36 vs. 0.54E9/l, 95% CI 0.41–0.64, ∗∗p = 0.006).

The number of total B cells at the diagnosis of CVID did not differ significantly from the control group of CVID patients without lymphoma and from normal ranges. No significant difference was noted in the serum levels of IgG in the group of CVID patients with lymphoma (median 2.88 g/l, 95% CI 1.83–3.91, normal values 7.65–13.6 g/l) compared to the CVID control group (median 2.02 g/l, 95% CI1.63– 3.24). In contrast, the number of total B cells at the diagnosis of lymphoma was reduced in the lymphoma group (median 0.01E9/l, 95% CI 0–0.13 vs. 0.195E9/l, 95% CI 0.16–0.29, ∗∗p = 0.006). Absolute B cell counts were further depleted by the chemotherapy (median 0.11E9/l, 95% CI 0–0.46 vs. 0.08E9/l, 95% CI 0.03–0.197, <sup>∗</sup>p = 0.02). Post-therapeutic B cell lymphopenia (B cells count ≤ 0.03E9/l) was found in 6 patients. A complete total B cell count reconstitution was achieved in only 3 patients (median 0.33E9/l, range 0.137–0.654); however, mature forms of B cells, including marginal zonelike, class-switched cells and plasmablasts, remained reduced in these subjects (mean interval after chemotherapy 102 months, range 6–204). The remaining 3 patients failed to re-establish their B cell populations and continued to maintain severely reduced B cell compartments (mean 0.02E9/l, range 0.001–0.08; mean interval after chemotherapy 39 months, range 4–145). Concerning NK cells, their absolute counts were similar to CVID patients without lymphoma and the general population (normal range 0.05–1.0 E9/l) and remained unchanged throughout the disease course. Curiously, no NK cell depression was observed after the chemotherapy. The immunophenotype profiles are summarized in **Figure 1**, and the B cell subpopulations are shown in detail in **Table 4** and **Figure 2**.

# Whole Exome Sequencing

WES was performed in 10 out of 11 CVID patients with lymphoma in whom biological material for genetic testing was available. The WES results were divided into 5 groups. Lymphadenopathy;

 SPLE,

Splenomegaly;

 ITP, Idiopathic

thrombocytopenic

 purpura; RTI, Respiratory

 tract

TABLE 2 |

Characteristics

 of the cohort of 11 CVID patients with lymphoma (M, Male; F, Female; LYM,

TABLE 3 | Characteristics of CVID-related complications in a cohort of 11 patients with lymphoma (ITP, Immune thrombocytopenic purpura; AIHA, Autoimmune hemolytic anemia; RA, Rheumatoid arthritis; LIPS, Lymphocytic interstitial pneumonia; BE, Bronchiectasis; ACOS, Asthma-COPD overlap syndrome; EAA, Exogenous

allergic alveolitis; NLH, Nodular lymphoid hyperplasia; IBD, Intestinal bowel disease; CG, Chronic gstritis; Y, Yes; N, No).

Gene variants previously described in association with CVID or in patients with inborn error of immunity (Group 1) were found in 4 patients. A novel heterozygous missense variant in CTLA4 was identified inpatient Nr. 9, who developed B—NHL (MALT) at the age of 36 years. She was also followed and treated for lymphadenopathy and enteropathy (with features of IBD-like disease), which is in concordance with the expected phenotype of CTLA4 deficiency. The deleterious effect of the mutation was verified by determination of decreased basal and stimulated (CD3/CD28) expression of CTLA4 protein in the patient's T regulatory cells (CD4+CD127dimCD25+) (**Supplementary Figure 1**).

Another type of B—NHL (DBLCL) was diagnosed in patient Nr. 4 at the age of 45 years. A genetic variant in PMS2 was found, which has an important role in the mismatch repair system and class switch recombination (23, 24). In addition to the lymphoma, the patient also manifested with a broad spectrum of non-infectious complications, including autoimmune cytopaenias (both AIHA as well as ITP), celiac– like disease and generalized lymphoproliferation, including lymphadenopathy, splenomegaly, and lymphocytic interstitial pneumonia.

The clinical manifestation of patient Nr. 10, who was found to harbor a PIK3CD mutation, corresponded with the previously described APDS (activated PI3K-delta syndrome) phenotype due to an activating mutation (2). He has been followed for generalized lymphadenopathy, splenomegaly, and nodular lymphoid hyperplasia (NLH) of the lungs and gastrointestinal tract since his childhood. This patient developed Hodgkin lymphoma at the age of 11 years.

TABLE 4 | B cell subpopulations in CVID patients with lymphoma post-chemotherapy (absolute counts in E9/L; reference values for general population in brackets; (↓), decreased count; (↑), increased count; N/A, value not available).

Nodular lymphoid hyperplasia and lymphocytic interstitial pneumonia were also noted in patient Nr. 1, who was followed and treated for splenomegaly and autoimmune thyroiditis before the diagnosis of T cell lymphoma, which developed at the age of 57. A gene variant in TNFRSF13B (TACI), known to increase susceptibility to CVID, was found. TACI variants are not regarded as disease-causing but rather as modifying (Group 2) (8).

Furthermore, several heterozygous variants were identified in genes associated with known primary immunodeficiencies that are inherited in an autosomal recessive manner, such as LYST, LRBA, RAG1, EXTL3, and STX11 (Group 4). The clinical phenotype of these patients did not match the respective disease; nevertheless, we report these variants because of their rarity in the healthy population and their potentially damaging character predicted by in silico tools. Functional assays, which would Kralickova et al. CVID-Associated Tumors-Czech Nationwide Study

elucidate the impact of these variants on protein function, were not performed, as they exceeded the scope of this study. The tumor DNA was not available for analysis of somatic "secondhit" mutations, which might explain the pathogenesis of some of the malignancies.

Variants in genes previously described in association with cancer susceptibility or as likely to increase the risk of cancer development, such as BRCA1, RABEP1, EP300, KDM5A, and others, were found in 6 out of 10 patients. They were divided into variants reported as pathogenic (Group 3) and variants of unknown significance and novel variants predicted as damaging in-silico (Group 5).The summary of WES results and a detailed description of the gene variants is presented in **Table 5** and in **Supplementary Table 1**.

#### DISCUSSION

Immune dysregulation associated with primary immunodeficiencies represents an increased risk of cancer development. We aimed to search for the occurrence of malignant diseases in a nationwide cohort of CVID patients, taking into account relevant epidemiology, immunophenotype, and the genetic background of the patients.

Similarly to published studies, we detected a higher incidence of malignancies among our CVID cohort (25–30). Also in alignment with previous reports, we noted a distinct spectrum of tumors in CVID patients, with Hodgkin and Non-Hodgkin lymphomas and gastric cancers being the most prevalent malignancies (**Table 6** and **Supplementary Table 2**). The overall risk of malignancy was more than 6 times greater in comparison to the general population, while the specific risk of HL was as much as 30 times greater. Curiously, an over 3 times greater risk of malignancy was determined in a subgroup of CVID patients with a history of ITP. Moreover, we noted that the diagnosis of GC (average age 55–59 years vs. 70–74 in general population) and B-NHL (35–39 years vs. 65–69) was established at a much younger age compared to the Czech general population, while HL developed later in life compared to the healthy population (40–44 years vs. 30–34).

Patients with CVID present with a characteristic immunophenotypic profile that is reflected in the diagnostic criteria of CVID. In this context, we specifically searched for potential differences between CVID patients with tumors and CVID patients who did not develop a malignant disease. Malignant hematologic diseases may, in general, reduce lymphocyte counts in up to 60% of patients, and lymphoma in particular may affect an entire spectrum of lymphocyte subpopulations, including CD4+, CD8+, CD19+, and CD56+ cells (31, 32). Nevertheless, in our cohort of CVID patients, we did not observe any significant differences between absolute or relative counts of CD3+, CD4+, CD8+, and CD56+ cells measured at the time of diagnosis of immunodeficiency and those measured at the time of diagnosis of lymphoma. Furthermore, the values of all T cell subpopulations and NK cells were similar to the control group of CVID patients without malignancy. In contrast, chemotherapy regimens had significant impacts on the

TABLE 6 | Summary of tumor prevalence and SIR (Standardized Incidence Ratio, median and 95% Confidence Intervals (CIs) shown) in a cohort of 295 CVID patients.

CD4+ and CD8+ cell counts. A similar observation has already been published in patients with lymphomas without underlying primary immunodeficiencies who underwent chemotherapy with an R-CHOP protocol. In the study, a reduction of CD4+ absolute counts to values <0.343 × 10<sup>9</sup> /l was declared an independent negative prognostic factor with a significant impact on 5-year progression-free survival and overall survival (33). Indeed, in our CVID cohort with lymphoma, the median level of post-chemotherapy absolute numbers of CD4+ was very low, 0.343 × 10<sup>9</sup> /l, 95% CI 0.14–0.36 (R-CHOP being the regiment used in 5 out of 11 patient), which implies that a CVID population treated with chemotherapy should be prognostically regarded as a higher risk group. Quite unexpectedly, NK cell counts were not affected by the chemotherapy. However, the B cell compartment was profoundly depleted in all CVID patients who underwent chemotherapy. The total B cell count normalized in only 3/10 patients. However, even in those, selective reductions of mature forms (including class-switched, marginal zone-like B cells and plasmablasts) persisted. This observation was in striking contrast to patients with autoimmune diseases receiving anti-CD20 therapy (rituximab), in whom the reconstitution is achieved within 5–9 months in up to 90% of patients (34).

B cell deficiency seems to be the hallmark in CVID patients with lymphoma, as they presented with reduced B cell count even at the time of diagnosis of lymphoma, which decreased and remained persistently lower after chemotherapy. Despite this, neither B cell nor T cell detailed immunophenotyping provided a strong enough predictive tool for assessment of the cancerogenic predisposition of CVID patients. Therefore, we set out to search for possible genetic causes of malignancies in CVID using massive parallel sequencing. Out of 10 patients who were available for testing, we identified gene variants previously classified as associated with CVID in 4 patients, namely, CTLR4, PIK3CD, PMS2, and TNFRSF13B. It is noteworthy that mutations in CTLA4 and PIK3CD, which account for the majority of currently known molecular causes of CVID (8), were both found among our small cohort of CVID patients.

CTLA4 heterozygous mutation was first described as a cause of CVID-like syndrome that displayed a significant overlap with CVID phenotype, including hypogammaglobulinemia, low B cell counts and immune dysregulation with variable organ involvement (35). The clinical and laboratory spectra of CTLA4 haploinsufficiency were described in detail in a recently published cohort of 133 patients. In this cohort, 8 mutation carriers developed lymphoma, and 3 had gastric cancer. Thus, our finding of a singleCTLA4 mutation among our small cohort corresponds well with this report. Furthermore, this particular patient also presented with IBD-like gastrointestinal disease that was retrospectively reclassified as a CTLA4-related gastrointestinal presentation. Interestingly, the spectrum of tumors found in the above mentioned CTLA4 study was limited to lymphomas and gastric carcinomas, which also correlates with our findings.

PIK3CD is a well-established genetic cause of APDS, activated PI3K-delta syndrome. Similarly toCLTA4, the clinical presentation of APDS overlaps significantly with the CVID phenotype, and a number of patients originally diagnosed with CVID were found to harbor mutations in PIK3CD. A large study including 53 patients with APDS reported lymphoma occurrence in 13% of patients (36). Furthermore, somatic mutations of PIK3 were found in several types of HL and NHL, thus suggesting the role of PI3K signaling in tumorigenesis.

Finally, the PMS2 protein is involved in complex mechanisms of DNA repair. As such, mutations in PMS2 are directly associated with an increased risk of cancer (37). At the same time, mutations in PMS2 were also implicated in class-switch recombination defects and impaired immunoglobulin production (36). Therefore, our finding of PMS2 mutation among CVID patients with lymphoma corresponds well with these reports.

Overall, we suggest that each of these CVID-associated genes may also convey a predisposition to tumor development.

The role of the TNFSF13B molecule, also known as TACI**,** in CVID has long been discussed. TACI variants have been found to be associated with autoimmune complications in CVID (38). Moreover, given the involvement of TACI in B cell activation, its mutations might contribute to immune dysregulation and lymphoma development (39). However, TACI variants are regarded as modifier genes rather than a monogenic cause of CVID.

Apart of the above mentioned genetic findings, we identified several variants in genes involved in lymphogenesis and immune system regulation. Although sufficient data to postulate their role in immune deficiency or tumorigenesis are lacking, we report them along with our results for the sake of completeness, reflecting the previously described roles of heterozygous mutations in PID and possible epistatic roles of various genes in immune dysregulation (40–42).

Finally, we also detected several variants in genes involved in tumor surveillance in our cohort, such as BRCA1 and others listed in **Supplementary Table 1**. These variants were previously reported in patients with a broad spectrum of solid tumors (including breast, ovarian, colorectal cancer, and others) and may therefore represent another contributory mechanism of malignant susceptibility.

# CONCLUSION

Malignancies belong to the most severe non-infectious complications of common variable immunodeficiency disorder. The prevalence of malignancy in our CVID cohort was found to be more than 6 times greater than in the general population. The spectrum of cancers was characteristically narrow, involving mostly gastric cancers and lymphomas. Moreover, ITP was elucidated as a novel risk factor for malignancy in CVID patients. Post-treatment T and B cell lymphopaenias, associated with poorer prognosis, were found in a majority of CVID patients who received chemotherapy. Surprisingly, NK cells remained unaffected. WES analysis illustrated a wide heterogeneity of potential genetic background of the oncogenic predisposition among CVID patients and identified several causative or contributing gene variants, pointing toward immune system dysregulation. In the future, modern genetic analytic approaches applied on larger cohorts of CVID patients, along with the use of oncogenomic tools, will undoubtedly enable the identification of other CVID-associated genes with increased risk of cancer and elucidate their roles in tumorigenesis.

#### REFERENCES

# AUTHOR CONTRIBUTIONS

PK and TM conceived and designed the study, collected data, and drafted manuscript. JL, IM, DJ, JP, JV, AZ, MB, ZP, and AK collected and provided primary patient data. EF and MS performed analysis and interpretation of data from genetic testing (Whole exome sequencing). VK performed analysis and interpretation of immunophenotyping data. JH provided statistical analysis of the obtained data and its interpretation. TK and AS provided critical revisions of the manuscript and final approval of the version to be published.

# ACKNOWLEDGMENTS

This study was supported by grants from the Grant Agency Charles University in Prague (GAUK) nr. 2120217 and the Czech Health Research Council (AZV) nr.NV18-05-00162.

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2018.03135/full#supplementary-material

TRAF recruitment and reveals new insights into BAFF-R signaling. J Exp Med. (2010) 207:2569–79. doi: 10.1084/jem.20100857

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Kralickova, Milota, Litzman, Malkusova, Jilek, Petanova, Vydlakova, Zimulova, Fronkova, Svaton, Kanderova, Bloomfield, Parackova, Klocperk, Haviger, Kalina and Sediva. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Loss of JAK1 Drives Innate Immune Deficiency

Agnieszka Witalisz-Siepracka<sup>1</sup> , Klara Klein<sup>1</sup> , Daniela Prinz <sup>1</sup> , Nicoletta Leidenfrost <sup>1</sup> , Gernot Schabbauer <sup>2</sup> , Alexander Dohnal <sup>3</sup> and Veronika Sexl <sup>1</sup> \*

*1 Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna, Austria, <sup>2</sup> Center for Physiology and Pharmacology, Institute for Physiology, Medical University of Vienna, Vienna, Austria, <sup>3</sup> Tumor Immunology, St. Anna Kinderkrebsforschung, Children's Cancer Research Institute, Vienna, Austria*

The Janus kinase—signal transducers and activators of transcription (JAK-STAT) signaling pathway is critical in tuning immune responses and its dysregulation is tightly associated with cancer and immune disorders. Disruption of interleukin (IL)-15/STAT5 signaling pathway due to the loss of IL-15 receptor chains, JAK3 or STAT5 leads to immune deficiencies with natural killer (NK) cell abnormalities. JAK1, together with JAK3 transmits signals downstream of IL-15, but the exact contribution of JAK1 to NK cell biology remains to be elucidated. To study the consequences of JAK1 deficiency in NK cells, we generated mice with conditional deletion of JAK1 in NKp46<sup>+</sup> cells (*Jak1fl*/*flNcr1Cre*). We show here that deletion of NK cell-intrinsic JAK1 significantly reduced NK cell numbers in the bone marrow and impaired their development. In line, we observed almost a complete loss of NK cells in the spleen, blood, and liver, proving a crucial role of JAK1 in peripheral NK cells. In line, *Jak1fl*/+*Ncr1Cre* mice showed significantly impaired NK cell-mediated tumor surveillance. Our data suggest that JAK2 is not able to compensate for the loss of JAK1 in NK cells. Importantly, conditional deletion of JAK2 in NKp46<sup>+</sup> cells had no effect on peripheral NK cells revealing that NK cell-intrinsic JAK2 is dispensable for NK cell survival. In summary, we identified that loss of JAK1 in NK cells drives innate immune deficiency, whereas JAK2 deficiency leaves NK cell numbers and maturation unaltered. We thus propose that in contrast to currently used JAK1/JAK2 inhibitors, the use of JAK2-specific inhibitors would be advantageous for the patients by leaving NK cells intact.

 Deficiency of NK cells is a rare but increasingly appreciated subtype of primary immunodeficiency (PID). Classical NK cell deficiency is characterized by the absence of NK cells in the peripheral blood and results in enhanced susceptibility to viral infections (2).

The Janus kinase (JAK)—signal transducer and activator of transcription (STAT) signaling pathway acts downstream of multiple cytokines, growth factors, and hormones thereby critically regulating immune responses (3, 4). Upon binding of a specific ligand to its cognate receptor, conformational changes lead to receptor oligomerization and activation of the receptor-associated JAKs. JAKs auto- and trans-phosphorylate one another and phosphorylate receptor

#### Edited by:

*Markus G. Seidel, Medical University of Graz, Austria*

#### Reviewed by:

*Lisa Renee Forbes, Baylor College of Medicine, United States Jane Oliaro, Peter MacCallum Cancer Center, Australia Olli Silvennoinen, University of Helsinki, Finland*

\*Correspondence: *Veronika Sexl<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Primary Immunodeficiencies, a section of the journal Frontiers in Immunology*

Received: *28 September 2018* Accepted: *17 December 2018* Published: *08 January 2019*

#### Citation:

*Witalisz-Siepracka A, Klein K, Prinz D, Leidenfrost N, Schabbauer G, Dohnal A and Sexl V (2019) Loss of JAK1 Drives Innate Immune Deficiency. Front. Immunol. 9:3108. doi: 10.3389/fimmu.2018.03108*

**107**

chains, providing the docking sites for STAT molecules. STATs then undergo JAK-mediated phosphorylation, dimerize, and translocate to the nucleus, where they regulate the transcription of target genes (5). JAK3 and STAT5 are crucial players in transducing the signal downstream of cytokines which utilize γc receptor (6). "Loss-of-function" (LOF) mutations in genes encoding JAK3 (7) or STAT5B (8) lead to PIDs with an NK cell abnormality underlining the importance of the pathway for innate lymphocytes. The immunodeficiency of these patients has been explained by impaired IL-7 and IL-15 responses (6). Importantly, JAK1 has a dominant role over JAK3 in activating STAT5 downstream of γc-containing cytokine receptors (9). It is attractive to speculate that LOF mutations of JAK1 could also result in PID. To date, only one patient harboring JAK1 germline mutations, where JAK1 was reduced but not absent, has been identified and indeed presented with immune suppression (10).

In mice, complete loss of JAK1 leads to perinatal lethality and newborn mice display a strong reduction of thymocytes and B cells (11). These observations were confirmed in adult mice: inducible deletion of JAK1 leads to impairment of hematopoietic stem cells (HSCs) homeostasis and markedly reduces the frequencies of B cells and the B220+CD11c+NK1.1<sup>+</sup> subset of NK cells (12). However, to date, no study has directly analyzed the effect of loss of JAK1 on conventional NK cells.

The first insights into the contribution of JAK1 to NK cell biology derive from studies using JAK inhibitors—approved drugs for treatment of cancers and autoimmune diseases (13). Both, mice and patients treated with the JAK1/JAK2 inhibitor Ruxolitinib showed reduced NK cell numbers, impaired maturation, and function (14, 15). Since JAK2 has also been implicated in driving NK cell differentiation (14, 16), it remains to be elucidated which of the two kinases is responsible for the observed effects of Ruxolitinib treatment.

Using mice with knockout of Jak1 or Jak2 in NKp46<sup>+</sup> cells, we show here that JAK2 is dispensable for NK cell survival. In contrast, deletion of JAK1 in mature NK cells leads to NK cell deficiency and loss of one allele of Jak1 is sufficient to impair tumor growth control. Thus, we identified JAK1 as a key factor for mature NK cells and generated a mouse model of classical NK cell deficiency.

# MATERIALS AND METHODS

#### Mice and Cell Lines

Jak1fl/fl (C57BL/6N-Jak1tm1c(EUCOMM)Hmgu/H; were kindly provided by Dr. Alexander Dohnal (CCRI, Vienna, Austria). The Jak1tm1<sup>c</sup> allele of the mutant was generated from mice with the Jak1tm1<sup>a</sup> knockout first allele (described by International Mouse Phenotyping Consortium https://www.mousephenotype. org) by excision of the lacZ-neo cassette via Flp-recombination. The conditional potential of Jak1fl/fl mice was activated by Cre-recombination and excision of the loxP-flanked exon 3 of Jak1. Tissue-specific recombination was induced by cross breeding of Jak1fl/fl or Jak2fl/fl [Jak2tm1Kuw; (17)] with B6N-Tg(Ncr1Cre); (18) mice. Stat5fl/fl (19) and Stat5fl/flNcr1Cre (18) mice were described before. Jak1fl/fl , Ncr1Cre, Stat5fl/fl , Stat5fl/flNcr1Cre mice were on C57B6/N background and Jak2fl/fl were on mixed background. The experimental animals were age-matched (8–12 weeks) and maintained under specific pathogen-free conditions at the University of Veterinary Medicine, Vienna according to Federation for Laboratory Animal Science Associations (FELASA) guidelines (2014). The animal experiments were approved by the Ethics and Animal Welfare Committee of the University of Veterinary Medicine Vienna and the national authority (Austrian Federal Ministry of Science and Research) according to §§ 26ff. of Animal Experiments Act, Tierversuchsgesetz 2012—TVG 2012, under licenses BMWF-68.205/0218-II/3b/2012 and BMBWF-68.205/0174-V/3b/2018 and were conducted according to the guidelines of FELASA and ARRIVE. Throughout the paper Jak1WT refers to pooled data from Jak1fl/<sup>+</sup> and Jak1fl/fl mice.

The mouse lymphoma cell lines RMA-Rae1 [kindly provided by Prof. A. Cerwenka; (20)] and YAC-1 were cultured in RPMI1640 (Sigma) complete medium containing 10% FCS (Bio & Sell), 100 U/mL penicillin, 100 mg/mL streptomycin (Sigma), and 50µM 2-mercaptoethanol (Sigma).

# In vivo Tumor Model

Jak1fl/<sup>+</sup> and Jak1fl/+Ncr1Cre mice were injected s.c. with 10<sup>6</sup> RMA-Rae1 cells into both flanks and the tumor growth was monitored every other day. Ten days post injection the mice were sacrificed and the tumor weight was determined. For flow cytometric analysis of tumor infiltrating NK cells, tumors were cut into ∼5 mm<sup>2</sup> pieces and the single cell suspension was obtained using gentleMACSTM Octo Dissociator (Miltenyi Biotec) with digestion buffer containing Collagenase D (1 mg/mL; Sigma Aldrich) and DNAse I (20 mg/mL; Roche).

# NK-Cell Isolation, Expansion, and Stimulation

NK cells were isolated from spleen single-cell suspensions using DX5-labeled MACS beads according to the manufacturer's instructions (Miltenyi Biotec). NK cells were expanded in RPMI1640 complete medium supplemented with 5,000 U/mL rhIL-2 (Proleukin, Novartis) for 7 days. The number of CD3−NK1.1<sup>+</sup> cells was assessed by flow cytometry on day 0, 3, 5, and 7. On day 7 cells were lysed for Western blot analysis. For pSTAT5 analysis 10<sup>6</sup> splenocytes were stimulated with 50 ng/ml rmIL-15 (PeproTech) for 15 min and the cells were fixed in 2% PFA followed by methanol permeabilization and rehydration.

# NK-Cell Cytotoxicity Assay

For in vitro cytotoxicity assays, DX5-MACS–sorted NK cells were expanded for 7 days in IL-2 as described above and mixed at

**Abbreviations:** FACS, fluorescence-activated cell sorting; HSCs, hematopoietic stem cells; ILC, innate lymphoid cells; JAK, Janus kinase; LOF, loss of function; NK, natural killer; PID, primary immunodeficiency; SCID, severe combined immune deficiency; STAT, signal transducer and activator of transcription.

indicated effector: target ratios with carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, CellTrace CFSE Cell Proliferation Kit) labeled target cells. After 4 h of incubation at 37◦C, the cells were stained with Sytox Blue Dead Cell Stain (Thermo Fischer) and the specific target cell lysis was assessed by flow cytometry.

#### Flow Cytometry

Single cell suspensions were prepared from spleen, bone marrow, or liver. Liver was perfused via the portal vein with 5–10 mL sterile PBS. Separation of lymphocytes was performed using 37.5% percoll (GE Healthcare). For blood analysis, the erythrocytes were lysed using BD FACS Lysing Solution according to manufacturer's protocol (BD Bioscience). The antibodies (clones) targeting following proteins were purchased from eBioscience: CD3 (17A2), CD3e (145-2C11), CD11b (M1/70), CD16/CD32 (93), CD19 (eBio1D3) CD27 (LG.7F9), CD49b (DX5), CD122 (5H4), CD226 (10E5), Gr-1 (RB6-8C5), KLRG1 (2F1), Ly49A (A1), Ly49G2 (eBio4D11), NKG2A/C/E (20d5), NKG2D (CX5), NKp46 (29A1.4), NK1.1 (PK136), and Ter119 (TER-119). CD49a (Ha31/8) and pSTAT5 [47/Stat5(pY694)] antibodies were purchased from BD Pharminogen and pan-Rae1 (186107) was purchased from R&D Systems. Total cell numbers were assessed by flow cytometry using counting beads Count Bright Beads (Invitrogen). Flow cytometry experiments were performed

of CD3−NKp46<sup>+</sup> NK cells in the (D) spleen and (E) blood were assessed by flow cytometry. (F) Frequency of conventional NK cells (CD3−NK1.1+NKp46+CD49b+, left panel) and ILC1 cells (CD3−NK1.1+NKp46+CD49a+, right panel) was analyzed in the liver of *Jak1WT* , *Jak1fl*/+*Ncr1Cre,* and *Jak1fl*/*flNcr1Cre* mice by flow cytometry. (A,B,D–F) Bar graphs represent mean ± SEM of 1–2 independent experiments; *n* = 3–11. \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001.

on a BD FACSCanto II (BD Bioscience) or Cytoflex (Beckman Coulter) and analyzed using BD FACSDiva V8.0 (BD Bioscience), CytExpert (Beckman Coulter) or FlowJo V10 (FlowJo, LLC) software.

#### Western Blot

Cell lysis, SDS-PAGE, and Western blots were performed as described previously (21). The detection of chemiluminescence was performed using Clarity Western ECL substrate (BioRad) and the ChemiDocT XRS+ Molecular Imager (BioRad) and analyzed by Image Lab software (BioRad). The following antibodies were used: anti–β-actin (C4, sc-47778) from Santa Cruz as loading control, anti-JAK2 (D2E12; #3230), anti-Perforin (#3693) and anti-JAK1 (#3332) from Cell Signaling Technology.

#### Statistical Analysis

Unpaired t-tests or one-way ANOVA with Tukey post tests were performed using GraphPad Prism version 5.00 (GraphPad Software). The level of significance is indicated for each experiment (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

#### RESULTS

# JAK1 Deletion Reduces NK Cell and ILC1 Numbers in a Dose-Dependent Manner

The JAK1/JAK2 inhibitor Ruxolitinib has been shown to reduce NK cell numbers, maturation, and function (14, 15). To compare and address the contribution of JAK1 and JAK2 for NK cell biology we generated mice with conditional deletion of either JAK1 or JAK2 in NKp46<sup>+</sup> cells. We thus crossed Ncr1Cre (18) mice with Jak1fl/fl or Jak2fl/fl [see Materials and Methods section and (17)] mice, respectively. NK cells develop in the bone marrow from NK cell precursors (NKPs), which are defined as Lin−CD122+NK1.1−NKp46−.  As the Cre recombinase expression in Ncr1Cre mice is driven by the NKp46 promoter, Cre-mediated deletion is restricted to mNK cells. We observed a significant decrease of percentage and total numbers of bone marrow NK cells in Jak1fl/flNcr1Cre mice (**Figure 1A**). The Lin−CD122<sup>+</sup> NK cells in Jak1fl/flNcr1Cre mice showed enriched percentages of NK cell precursors (NKPs) and immature NK cells, while mNKs were significantly reduced in the bone marrow in line with a developmental block at the iNK cell stage preventing progression to mNK cell stage (**Figure 1B**). Deletion of one allele of Jak1 led to intermediate numbers of bone marrow NK cells (**Figure 1A**). Consistently, NK cell development showed an intermediate phenotype suggesting a Jak1 gene-dosage effect on NK cell development (**Figure 1B**).

The block in development of bone marrow NK cells translated into drastically reduced numbers of NK cells in the periphery. Loss of JAK1 led to an almost complete deficiency of splenic and blood NK cells (**Figures 1C–E**). In line with a Jak1 gene dosage effect, Jak1fl/+Ncr1Cre mice displayed reduced NK cell percentages and total numbers to 50% compared to wild-type littermates in the spleen and blood (**Figures 1C–E**). Deletion of the JAK1 downstream effector and transcription factor STAT5 in NKp46<sup>+</sup> cells also leads to a reduction of mature NK cells (18). A direct comparison of Jak1fl/flNcr1Cre and Stat5fl/flNcr1Cre mice revealed that deletion of JAK1 provoked an even more pronounced NK cell deficiency in spleen and blood than deletion of STAT5 (**Figures S1A,B**).

Liver NKp46<sup>+</sup> innate lymphocytes comprise two groups of distinct lineages (23). Conventional NK cells (cNK) are characterized by expression of CD49b and circulate freely whereas liver resident type 1 innate lymphocytes (ILC1) are characterized by the expression of CD49a and are restricted to the liver (24). Similarly to spleen and blood, liver cNKs and tissue resident ILC1s were almost completely ablated upon loss of JAK1 (**Figure 1F**). Again the deletion of one allele of Jak1 resulted in an intermediate abundance of liver innate lymphocytes (**Figure 1F**). In summary, these findings led us to conclude that JAK1 expression in NKp46<sup>+</sup> cells is indispensable for NK cell development and maintenance in peripheral organs in a dose-dependent manner.

#### JAK1 Is Crucial for NK Cell Maturation

In the periphery NK cells undergo maturation steps which are characterized by sequential expression of CD27 and CD11b surface markers (22). One allele of Jak1 was sufficient to drive NK cell maturation as we did not detect any differences in percentage of cells in each maturation stage between Jak1WT and Jak1fl/+Ncr1Cre cells (**Figures 2A,B**). The remaining Jak1fl/flNcr1Cre NK cells showed an increase in the immature population (CD27+CD11b−) and a decrease in the mature CD27−CD11b<sup>+</sup> population (**Figures 2A,B** and **Figure S1C**). This result suggests that the remaining cells might just have lost

JAK1 and have not received sufficient IL-15 signaling to fully mature. Indeed, the remaining Jak1fl/flNcr1Cre NK cells showed reduced phosphorylation of STAT5 ex vivo upon short-term stimulation with IL-15 (**Figure 2C**). NK cell activity is controlled by a balance between activating and inhibitory receptors. Deletion of neither one nor both alleles of Jak1 had an effect on the percentage of NK cells expressing following activating and inhibitory receptors: KLRG1, NKG2D, NKG2A/C/E, and Ly49A (**Figure 2D**). The most prominent difference was a slight decrease of Ly49G2<sup>+</sup> NK cells and an increase in DNAM-1<sup>+</sup> NK cells in the Jak1fl/flNcr1Cre mice (**Figure 2D**). Similarly, no gross differences were detected in the expression level (MFI) of each receptor, besides an increase in the MFI of DNAM1 and Ly49G2

(**Figure S1D**). Besides its role as an activating receptor, DNAM-1 expression marks a developmental step; DNAM-1<sup>+</sup> cells give rise to DNAM-1<sup>−</sup> cells (25). We reasoned that the changes in DNAM-1<sup>+</sup> reflect the maturation block of Jak1fl/flNcr1Cre NK cells.

## JAK2 Is Dispensable for NK Cell Survival and Maturation

So far we showed that NK cell-intrinsic JAK1 deletion leads to NK cell deficiency. To elucidate if JAK2 impacts on NK cell survival and maturation, we analyzed splenic NK cells in Jak2fl/flNcr1Cre mice and their wild-type littermates. We failed to detect any impact of JAK2 deletion on the frequency or total numbers of CD3−NKp46<sup>+</sup> cells in the spleen (**Figure 3A**). Furthermore, Jak2fl/flNcr1Cre mice showed normal NK cell maturation, as similar percentages of CD27−CD11b<sup>+</sup> cells were detected in both genotypes (**Figure 3B**). As the deletion of JAK2 protein in NK cells was very efficient (**Figure 3C**), these data unequivocally define that unlike JAK1, NK cell-intrinsic JAK2 is dispensable for NK cell survival and maturation.

# Loss of One Allele of Jak1 Impairs NK Cell Functionality

To get further insights into how JAK1 regulates NK cell functionality, we analyzed the growth of MACS-purified splenic NK cells from Jak1WT , Jak1fl/+Ncr1Cre, and Jak1fl/flNcr1Cre. JAK1-deficient NK cells did not expand, which shows that even under a very high dose of IL-2 other JAKs cannot compensate for the loss of JAK1 (**Figure 4A**). The loss of one allele of Jak1 resulted in a minor growth deficiency (**Figure 4A**). Western blot analysis confirmed the reduced JAK1 protein expression in expanded Jak1fl/+Ncr1Cre NK cells which was paralleled by reduced levels of perforin (**Figure 4B**). In line, Jak1fl/+Ncr1Cre NK cells displayed an impaired cytotoxic activity against target cell lines YAC-1 and RMA-Rae1 (**Figure 4C**). These results prove that JAK1 is not only indispensable for maintaining NK cells in periphery, but also contributes to their cytotoxic activity.

# NK Cell Depletion Induced by Loss of One Allele of Jak1 Impairs Tumor Surveillance

NK cells are crucial for the early recognition and elimination of transformed cells. To investigate whether NK cell reduction

and their impaired functionality in Jak1fl/+Ncr1Cre mice results in increased susceptibility to tumor growth and is not compensated by other means, we made use of RMA-Rae1 lymphoma cells. This cell line is a tool to study NK celldependent tumor surveillance in a robust and efficient way (20). We subcutaneously transplanted RMA-Rae1 lymphoma cells into both flanks of Jak1fl/<sup>+</sup> and Jak1fl/+Ncr1Cre mice. Lacking one allele of Jak1 impaired the ability of NK cells to control the tumor growth as illustrated by increased tumor size (**Figure 5A**) and tumor weight in Jak1fl/+Ncr1Cre (**Figure 5B**). In line, these tumors showed significantly reduced NK cell infiltration (**Figures 5C,D**). We failed to detect any difference in the frequency of NKG2D<sup>+</sup> tumor infiltrating NK cells (**Figure 5E**), which are crucial for the recognition of RMA-Rae1 tumors. In summary, our results show the reduced NK cell numbers combined with an impaired NK cell functionality in Jak1fl/+Ncr1Cre mice are sufficient to significantly impair tumor growth control in vivo.

#### DISCUSSION

Dysregulation of the JAK-STAT signaling pathway is tightly associated with cancer development as well as immune disorders (7). The first JAK-linked disease discovered was the severe combined immune deficiency (SCID) which was characterized by NK cell abnormalities caused by LOF JAK3 mutations (26, 27). We here show that deletion of JAK1 in NKp46<sup>+</sup> cells leads to innate immune deficiency with loss of NK and ILC1 cells in peripheral organs, whereas JAK2 is redundant for NK cell survival and maturation.

Our previous work uncovered that loss of STAT5 in NK cells leads to severe reduction of NK cell numbers in peripheral organs (18). The loss of NK cells in Stat5fl/flNcr1Cre mice was rescued by enforced expression of the pro-survival molecule BCL2 (28). This study defined STAT5 as a crucial survival factor for NK cells. STAT5B-deficient mice largely lack NK cells (29), in line with the fact that STAT5 signals downstream of cytokines that are vital for NK cell biology, such as IL-2 or IL-15 (30). IL-15 is crucial for NK cell development and survival as Il15−/<sup>−</sup> mice are largely devoid of peripheral NK cells (31). IL-15 signals via a receptor complex of γc receptor chain, IL-2Rβ, and IL-15rα (32). Knockout mice of each receptor chain prove an absolutely critical role for signals sent downstream of IL-15 for NK cell development (33, 34). To date, the contribution of γc-associated JAK3 to NK cell development has been well-established. Mice with JAK3 deficiency show a similar SCID phenotype as observed in human patients and NK cell development is blocked at the pre-NK progenitor stage (35, 36). Now we place previously underappreciated JAK1 as a crucial part of the IL-15/STAT5 axis in NK cells. Jak1fl/flNcr1Cre NK cells show a developmental block at the iNK cell stage and an almost complete loss of NK cells in peripheral organs.

Interestingly, the consequences of JAK1 deletion for NK cells exceed the effects of STAT5-deficiency. Impairment of combined STAT3 and STAT5 activation may underlie the more pronounced loss of NK cells, as STAT3 has been shown to induce expression of the crucial NK cell survival gene Mcl1 (37, 38). Alternatively, JAK1 and STAT5 may have different half-lives which account for different frequencies of "just deleters"—NK cells which have just lost the gene but still carry the protein, that may explain differences.

The gene-dosage effect of JAK1-deficiency is reflected in NK cell numbers, while loss of one allele of JAK1 is dispensable for NK cell maturation. This suggests that activated STAT5 is rate limiting for NK cell survival but not maturation. Deletion of one allele of Jak1 is also sufficient to significantly impair tumor surveillance based on decreased numbers of NK cells combined with a diminished functionality of the remaining NK cells. In accordance with our data, NK cell-specific deletion of the STAT5 target gene Mcl1 leads to severe NK cell deficiency which causes a significant increase in metastatic burden (38).

JAKs may exhibit redundant functions and compensate for each other. Downstream of interferon γ receptor JAK1 can partially compensate for loss of JAK2 kinase activity (39). JAK2 has also been shown to phosphorylate STAT5 downstream of IL-15 during in vitro differentiation of NK cells (16). On the other hand, constitutively active JAK2 can only modestly compensate for the loss of JAK1 in stem cells suggesting a non-redundant role of JAK1 and 2 in HSCs. In line, we observe that in the absence of JAK1, JAK2 fails to compensate in activating STAT5, even under high dose of IL-2 in vitro, to allow NK cell survival. It remains to be elucidated whether compensatory effects are achievable by expressing a constitutively active form of JAK2. More importantly, we show that loss of JAK2 in NKp46<sup>+</sup> cells is dispensable for NK cell survival. JAK2-deficient NK cells are fully mature, proving that NK cell-intrinsic JAK2 is not driving NK cell maturation. This also indicates that the impaired NK cell maturation in Jak2fl/flMx1Cre mice (14) is most likely caused by NK cell-extrinsic functions of JAK2. One might speculate that the constitutive deletion of JAK2 alters the cytokine milieu.

#### REFERENCES

Our data provide novel insights into results obtained upon JAK1/JAK2 inhibitor treatment. Inhibition of JAK1/JAK2 reduces NK cell numbers and maturation (14, 15), which according to our data is clearly the effect of inhibiting JAK1 rather than JAK2. We thus propose that the development of JAK2 specific inhibitors may be advantageous as they would leave NK cell-mediated tumor surveillance intact. This might be of particular relevance in the case of JAK2-driven leukemia such as JAK2V617F-induced myeloproliferative neoplasms.

#### AUTHOR CONTRIBUTIONS

AW-S, KK, DP, and NL performed experiments. AW-S analyzed the data. VS, GS, and AD provided the resources. AW-S and VS wrote the manuscript.

#### FUNDING

The work was supported by the Austrian Science Fund FWF grant SFB-F61 to VS.

#### ACKNOWLEDGMENTS

We thank Philipp Jodl for great technical support and Manuela Kindl for maintaining and monitoring the mice. We are grateful to Andrea Hölbl-Kovacic, Dagmar Gotthardt, and Eva Maria Putz for critical feedback on the manuscript.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2018.03108/full#supplementary-material

γc-containing cytokine receptors. Chem Biol. (2011) 18:314–23. doi: 10.1016/j.chembiol.2011

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Witalisz-Siepracka, Klein, Prinz, Leidenfrost, Schabbauer, Dohnal and Sexl. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Extreme Phenotypes With Identical Mutations: Two Patients With Same Non-sense NHEJ1 Homozygous Mutation

Maria J. Recio1,2, Nerea Dominguez-Pinilla2,3, Melina Soledad Perrig1,2 , Carmen Rodriguez Vigil-Iturrate<sup>4</sup> , Nerea Salmón-Rodriguez 2,5,6, Cristina Martinez Faci <sup>4</sup> , María J. Castro-Panete<sup>7</sup> , Javier Blas-Espada2,7, Marta López-Nevado2,7 , Raquel Ruiz-Garcia2,7, Rebeca Chaparro-García<sup>1</sup> , Luis M. Allende2,7† and Luis Ignacio Gonzalez-Granado2,5,6 \* †

#### Edited by:

Andrew R. Gennery, Newcastle University, United Kingdom

#### Reviewed by:

Silvia Clara Giliani, Università degli Studi di Brescia, Italy John Bernard Ziegler, Sydney Children's Hospital, Australia

#### \*Correspondence:

Luis Ignacio Gonzalez-Granado<EMAIL_ADDRESS>
†These authors have contributed equally to this work and share senior authorship

#### Specialty section:

This article was submitted to Primary Immunodeficiencies, a section of the journal Frontiers in Immunology

Received: 05 October 2018 Accepted: 30 November 2018 Published: 07 January 2019

#### Citation:

Recio MJ, Dominguez-Pinilla N, Perrig MS, Rodriguez Vigil-Iturrate C, Salmón-Rodriguez N, Martinez Faci C, Castro-Panete MJ, Blas-Espada J, López-Nevado M, Ruiz-Garcia R, Chaparro-García R, Allende LM and Gonzalez-Granado LI (2019) Extreme Phenotypes With Identical Mutations: Two Patients With Same Non-sense NHEJ1 Homozygous Mutation. Front. Immunol. 9:2959. doi: 10.3389/fimmu.2018.02959 <sup>1</sup> Department of Immunology, Ophthalmology and ENT, School of Medicine, Complutense University, 12 de Octubre Health Research Institute (imas12), Madrid, Spain, <sup>2</sup> Hospital 12 de Octubre Health Research Institute (imas12), Madrid, Spain, <sup>3</sup> Pediatric Hematology and Oncology Unit, University Hospital Virgen de la Salud, Toledo, Spain, <sup>4</sup> Pediatric Hematology and Oncology Unit, University Hospital Miguel Servet, Zaragoza, Spain, <sup>5</sup> Immunodeficiencies Unit, Pediatrics, University Hospital 12 octubre, Madrid, Spain, <sup>6</sup> Complutense University School of Medicine, Madrid, Spain, <sup>7</sup> Department of Immunology, University Hospital 12 Octubre, Madrid, Spain

Cernunnos/XLF deficiency is a rare primary immunodeficiency classified within the DNA repair defects. Patients present with severe growth retardation, microcephaly, lymphopenia and increased cellular sensitivity to ionizing radiation. Here, we describe two unrelated cases with the same non-sense mutation in the NHEJ1 gene showing significant differences in clinical presentation and immunological profile but a similar DNA repair defect.

Keywords: XLF/Cernunnos, NHEJ1 mutation, DNA repair, severe combined immunodeficiency, lymphomagenesis, radiosensitive SCID (RS-SCID)

# INTRODUCTION

# Clinical Presentation and Laboratory Test Results

We report two patients harboring the same homozygous mutation in NHEJ1 gene. Strikingly, their clinical phenotypes differed markedly. One presented with severe combined immunodeficiency whereas the other only had isolated thrombocytopenia and macrocytosis. Clinical characteristics are summarized in **Table 1**. Both patients presented severe lymphopenia: immunophenotyping of Patient 1 (P1) showed severe T and B-cell lymphopenia and normal to elevated NK cells (T−B <sup>−</sup>NK<sup>+</sup> phenotype) in contrast to Patient 2 (P2) who presented a milder immunophenotype. T-cell compartment also showed differences between the two patients: P1 had a more senescent T-cell phenotype with increase in CD4<sup>+</sup> and CD8<sup>+</sup> effector memory (CCR7−CD45RA−) and decrease naïve and recent thymic emigrants (RTE) (CD4+CD45RA+CD31+) T cells and a higher proportion of activated T-cells (CD3+HLA-DR+) (**Table 2**). Of note, P1 had a severe decrease in CD8+ population mimicking the one reported in ZAP70 or HLA class I deficiencies among patients with combined immunodeficiency/severe combined immunodeficiency.

B cells were severely reduced in both patients. The B-cell profile in P2 showed a normal proportion of naive, unswitched memory, switched memory, transitional, and plasmoblast B-cells. Serum immunoglobulins were reduced in P1. Due to age limitation, specific antibody response was

TABLE 1 | Clinical features of the patients with Cernunnos/XLF deficiency.

only tested in P2, showing a specific antibody deficiency: IgG and IgG2 levels against pneumococcus antigen were reduced while IgG levels against tetanus toxoid antigen showed normal values after vaccination. Regarding to the immunophenotype in T and B-cells, P1 had very low TRECs and KRECs copies, whereas P2 had preserved levels (**Table 2**).

The clinical presentation and immunologic features of both patients lead to suspicion of a primary immunodeficiency (PID). Accordingly, an in house targeted NGS sequencing panel for 192 PID related genes (**Table 3**) was performed and revealed a homozygous nucleotide substitution in exon 2 of NHEJ1 gene (NM\_024782, c.169C>T) that affects codon 57 and is predicted to result in a severely truncated protein (p.R57X), this mutational change has been previously described in two Cernunnos /XLF defective patients similar to P2 patient displaying microcephaly and slight lymphopenia (1–6). Sanger sequencing confirmed the variants in the patients which were inherited in autosomal recessive fashion from their healthy parents (**Figure 1**).

Increased sensitivity and decreased double strand breaks (DSB) rejoining to ionizing irradiation (IR) is a feature of Cernunnos /XLF deficient cells. Accordingly, a cell-survival assay revealed an increased IR sensitivity in fibroblasts from P2 similar to that observed in LIG4-deficient fibroblasts (**Figure 2A**). We also analyze DSB rejoining in primary fibroblasts from both patients (P1 and P2) by enumerating the rate of loss of γ-H2AX foci following exposure to γ-IR. The results showed impaired DSB rejoining after treatment with γ-irradiation and etoposide in both patients (**Figure 2B**). These findings are in agreement with previously published results in Cernunnos and LIG4-defective patients (7).

Comparative testing of DNA repair was also performed on T cells from the second patient (P2). Upon exposure of PBMCs to 10 Gy, induction and resolution of DNA damage was measured at various time points and a delayed kinetics of DNA repair were observed in P2 compared to a healthy control (**Figure 2B**).

# METHODS

# Cell Culture

Primary fibroblasts were grown in minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS), penicillin and streptomycin.

#### Immunofluorescence and Antibodies

To characterize the repair capacity of the patient's cells we scored the in situ modification of the histone variant H2AX, which is phosphorylated proximal to sites of DNA double-strand breaks. The number of phosphorylated H2AX (γH2AX) foci in a nucleus is reported to be directly proportional to the number of DSBs, and de-phosphorylation coincides with DSB repair. Primary skin fibroblasts were irradiated with ionizing irradiation (137Cs) or treated with 20 mM Etoposide for 1 h. After the indicated treatments, the slides were washed with PBS, fixed using 4% formaldehyde for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 5 min. Cells were incubated with primary antibodies for 1 h, the slides were washed with PBS and the bound antibodies were revealed by IgG Alexa fluor antibodies (Invitrogen). Nuclei were counterstained with DAPI, and slides were mounted for immunofluorescence. Images were taken with a fluorescent microscopy (Zeiss AxioImages.A1, Carl Zeiss). In a single experiment at least 30 cells per sample were counted.

#### Survival Assay

Primary skin fibroblasts were irradiated with ionizing radiation (137Cs). After irradiation, the cells were seeded at a density of 1 × 10<sup>4</sup> cells/mL in T75 flasks in triplicate. To evaluate cell sensitivity to γ-IR (1 and 3 Gy), adherent cells were trypsinized and counted 11 days later.

TABLE 2 | Immunologic features of patients with Cernunnos/XLF deficiency.

ND, not determined.

#### Flow Cytometry

Proportions and lymphocyte count of T-, B-, and NK-cells were determined in blood samples using conjugated mouse antihuman monoclonal antibodies and data were collected by flow cytometry using a Navios Cytometer (Beckman Coulter, Madrid, Spain) and analyzed with Kaluza 1.5a software (Beckman Coulter, Indianapolis IN, US).

PBMCs from patient and healthy controls were irradiated with 10Gy, fixed and stained for CD3, CD19, and phosphohistone H2AX. Mean fluorescence intensities (MFI) of γH2AX were evaluated on gated CD3+ lymphocytes.

#### Immunoglobulins

Total serum immunoglobulins (IgG, IgA, IgM, and IgE) were measured by nephelometry (Beckman Coulter, Madrid, Spain).

#### NGS and Sanger Sequencing

Genomic DNA was extracted from EDTA whole blood using a MagNa Pure Compact Nucleic Acid Isolation Kit (Roche, Madrid, Spain). Missense NHEJ1 mutation was identified by targeted next-generation sequencing with an in-house designed panel of 192 genes involved in primary immunodeficiencies (PID) (Ampliseq, Thermo Fisher, Madrid, Spain) and confirmed

#### TABLE 3 | Gene-panel related to PID.

#### TRECs and KRECs

TRECs, KRECs, and beta-actin (ACTB) copy numbers were determined from dried blood spots (DBS, punches of 3.2 mm) using triplex real-time quantitative polymerase chain reaction (RT-PCR) (TIB MOLBIOL) and run in a Light Cycler 480 II from Roche Diagnostics.

#### Ethics Statement

The protocols of this study were approved by the Institutional Review Board of Hospital Universitario 12 de Octubre (Madrid, Spain) and written informed consent was obtained from all subjects/caregivers in accordance with the Declaration of Helsinki.

# BACKGROUND

DNA non-homologous end-joining (NHEJ) is the major DNA double strand break (DSB) repair pathway in mammalian cells. NHEJ also functions during immune development rejoining the programmed DSBs introduced during V(D)J recombination (7). Most of the patients deficient in NHEJ components display radiosensitivity and severe combined immunodeficiency (SCID), a phenotype which has been called radiosensitive SCID (RS-SCID). To date, mutations in five genes encoding components of the NHEJ pathway, LIG4

(encoding DNA ligase IV), NHEJ1 (encoding Cernunnos), PRKDC (encoding DNA-PKcs), DCLRE1C (encoding Artemis), and XRCC4 (encoding XRCC4) have been identified in patients. Although RS-SCID represents the extreme phenotype, radiosensitivity coupled with variable immunodeficiency ranging from Omenn's Syndrome to CID has been observed. Additional features are also observed in some patients, including microcephaly and severe growth delay. However, all of these

H2AX signal at 24 h post-treatment in P2 is indicative of a general DNA repair defect. \*p < 0.05, \*\*p < 0.01.

patients are at risk of malignancies, particularly lymphoma (6).

# History

Cernunnos, was identified through both cDNA complementation of cells derived from a IR-sensitive immunodeficient patient (1) and through a yeast two-hybrid screen for XRCC4 interacting partners (8). Cernunnos is a homolog of Nej1, one of the NHEJ factors identified in yeast (9, 10). Data from several studies suggest that although Cernunnos may not be strictly required for NHEJ, the loss of Cernunnos does affect NHEJ and interestingly DNA repair defect in these patients is quite similar to that found in LIG4 syndrome patients (11).

#### Review of Similar Cases

Cernunnos/XLF deficiency in human results in extreme sensitivity to IR, microcephaly, and growth retardation, but the effect on the immune system is variable (1–4, 8). Mutations in NHEJ1 have been described previously in 27 patients with clinical features comparable to LIG4 deficiencies. Of contrast, XRCC4 deficiency shares the radiosensitivity and neurological impairment but not overt immunodeficiency (5, 12, 13).

Most of the Cernunnos/XLF deficient patients are hypersensitive to IR and have a significant NHEJ defect (1, 14) similar to LIG4 deficiency, which belong to NHEJ ligation complex; immunodeficiency, however, is milder compared with defects in NHEJ factors involved in hairpin opening as Artemis and DNA-PKcs.

In this study, we reported two patients who present the same c.169C>T mutation in NHEJ1 gene but different immunologic features (**Table 2**). P2 presented with mild T lymphopenia, hypersensitivity and NHEJ repair defect, typical for patients with Cernunnos/XLF defects (1, 5). On the other hand, P1 presented a more severe phenotype (T-B-); hypersensitivity and NHEJ repair defect, however, was similar to P2. These findings indicate that patients with same NHEJ1 mutation and DNA repair defect may show great variability in the clinical phenotype.

Patients with homozygous mutations (p.R178X) in NHEJ1 gene have been previously reported. Two patients died at 1.5 and 4 years (1, 2), while another of the patients remains alive at the age of 8 years (without HSCT) (5). However, none of these patients presented the severe T lymphopenia observed in our first patient.

One explanation for the different degree of lymphopenia found in Cernunnos-deficient patients (P1 and P2) might be the existence of alternative DNA repair proteins that only work to repair DSBs generated during lymphocyte development (3). It has been described that RAG complex and Cernunnos functionally overlap in the repair of DNA breaks during antigen receptor assembly ensuring stabilization of DNA ends after DNA cleavage by RAG (15). ATM and/or ATM-dependent DRR factors, as 53BP1, would contribute to the RAG-DSB stabilization and the recruitment of NHEJ pathway proteins. Severely impaired joining of RAG-generated DSBs in cells that are deficient for Cernunnos and either ATM, 53PBP1or H2AX has been observed (16). Thus, genetic factors such as mutations or polymorphisms in some of these proteins could affect the VDJ recombination process and explain the severe T lymphopenia observed in P1. As Cernunnos is not usually found in the context of a patient with a SCID phenotype we cannot rule out a digenic cause (particularly 53BP1, as other VDJ recombination genes were wild-type in our panel).

# DISCUSSION

# Diagnosis and Treatment

The assignment of a timely and accurate diagnosis is of paramount importance in the management of patients with defects in DNA repair, as HSCT is the only curative therapy available. Usually the repair defect in these disorders is assessed by immunofluorescence assays of irradiation-induced γ-H2AX foci using skin fibroblasts. Flow cytometry (FC) can be applied as a rapid diagnostic tool for DNA repair disorders (17, 18). Therefore, we have used flow cytometry to analyze PBMCs from P2 and the results showed a DNA repair defect similar to that obtained in skin fibroblasts (data not shown) (19).

Hence, a high throughput, sensitive and reliable assay to quantify γ-H2AX foci in PBMCs isolated from blood samples would be a valuable tool to diagnose these patients and thus allow HSCT without delay.

In addition, it would also be helpful in cancer patients to individualize and to guide the dosing of ionizing radiation (IR) and/ or genotoxic agents to avoid accumulation of cells with genomic instability that could accelerate cancer development.

In the era of newborn screening an abnormal TREC assay should be followed by NGS approach as Cernunnos/XLF deficiency may present early in life as SCID, as other RS-SCID defects (20). Since genetic diagnosis takes time, functional radiosensitivity assays in peripheral blood may lead to the correct diagnosis and avoid exposure to alkylating agents during the conditioning regimen even in the absence of a genetic diagnosis (21).

The patients presented in this work are alive and well and both patients after undergoing HSCT. Of note, P2 has survived to age 7 years. It has been reported that the Cernunnos/XLF deficient patients may survive the first years of life, or even up to 18 years, without HSCT. However, due to the comorbilities that these patients face, it is highly recommended HSCT pre-emptively, rather than expect the appearance of a malignant refractory disease disregarding the age at diagnosis (21).

Given the extreme rarity of the disorder, the appearance of two unrelated homozygous cases in non-consanguineous family with the same mutation seems unlikely. However, we can offer three reasons to discard consanguinity in both kindreds: First. Geographical: Both families are more than 450 km apart each other. Both kindreds deny any consanguinity (even far) ties. Second, Molecular: HLA typing were done in both families. It is shown HLA haplotypes generated by segregation analysis in both families: Family 1, Father: A ∗ 30, B<sup>∗</sup> 18, DRB1<sup>∗</sup> 03 and A<sup>∗</sup> 02, B<sup>∗</sup> 40, DRB1<sup>∗</sup> 08; Mother: A ∗ 01, B<sup>∗</sup> 37, DRB1<sup>∗</sup> 13 and A<sup>∗</sup> -, B<sup>∗</sup> 57, DRB1<sup>∗</sup> 07. Family 2, Father: A<sup>∗</sup> 02, B<sup>∗</sup> 39, DRB1<sup>∗</sup> 11 and A<sup>∗</sup> 29, B<sup>∗</sup> 51, DRB1<sup>∗</sup> 04; Mother: A<sup>∗</sup> 11, B<sup>∗</sup> 40, DRB1<sup>∗</sup> 04 and A<sup>∗</sup> 11, B<sup>∗</sup> 07, DRB1<sup>∗</sup> 15. In conclusion, both families did not share any HLA haplotype (**Table 4**). Third, No other mutations in PID genes that associate radiosensibility (LIG4, PRKDC, DCLRE1C, ATM, RNF168) were found.

TABLE 4 | HLA typing in P1 and P2 families.

#### CONCLUDING REMARKS

We report two unrelated cases diagnosed with Cernunnos/XLF deficiency. Both patients showed a similar DNA repair defect and increased cellular sensitivity to ionizing radiation as per in vitro assays. However, clinical presentation and immunological profile were extremely different. The first patient showed a senescent phenotype with decreased TRECs, RTE and naive Tcells counts suggesting that the sustained self-renewal of T-cell pool was impaired, while P2 presented only a slight reduction in T-cell counts as it has been reported in Cernunnos/XLF deficient patients reported up to date with normal TRECs/KRECs and IgG levels (22).

#### AUTHOR CONTRIBUTIONS

JB-E and RR-G performed the laboratory work for this study and computational predictions. MR, RR-G, ML-N, RC-G, MC-P, MP, and JB-E performed the laboratory work for this study. CR, CM, ND-P, NS-R, CR, and LG-G were responsible for the clinical

#### REFERENCES

management of the patients. LA, MR, JB-E, and LG-G designed the research and drafted the manuscript. All authors approved the final version of this manuscript.

# FUNDING

This research was supported by grants from the Spanish Health Research Fund FIS PI16/2053 to LA and LG-G, PI16/0044 to LG-G, MINECO grants SAF2014-54708-R, SAF2016-81876-REDT, and RTI2018-095673-B-I00. CAM grant B2017/BMD3673. The project has been co-financed with FEDER funds.

## ACKNOWLEDGMENTS

We thank both families and Pablo Morales, Isabel Castro, María José Díaz-Madroñero, Marta Martín Cabezudo, and Maria Teresa Fernandez González for their personal and technical assistance and Jaime de Inocencio for critical review of the article.

break repair disorders. J Allergy Clin Immunol. (2018) 141:322–8.e10. doi: 10.1016/j.jaci.2017.02.036

22. Carrillo J, Calvete O, Pintado-Berninches L, Manguan-García C, Sevilla Navarro J, Arias-Salgado EG, et al. Mutations in XLF/NHEJ1/Cernunnos gene results in downregulation of telomerase genes expression and telomere shortening. Hum Mol Genet. (2017) 26:1900–14. doi: 10.1093/hmg/ddx098

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Recio, Dominguez-Pinilla, Perrig, Rodriguez Vigil-Iturrate, Salmón-Rodriguez, Martinez Faci, Castro-Panete, Blas-Espada, López-Nevado, Ruiz-Garcia, Chaparro-García, Allende and Gonzalez-Granado. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Epstein-Barr Virus (EBV)-Related Lymphoproliferative Disorders in Ataxia Telangiectasia: Does ATM Regulate EBV Life Cycle?

Moussab Tatfi, Olivier Hermine and Felipe Suarez\*

INSERM U1163/CNRS ERL8254 - Laboratory of cellular and molecular mechanisms of hematological disorders and therapeutic implications, IMAGINE Institute, Paris, France

#### Edited by:

Fabian Hauck, LMU München, Germany

#### Reviewed by:

Henri-Jacques Delecluse, German Cancer Research Center (DKFZ), Germany Claire Booth, Great Ormond Street Institute of Child Health, University College London, United Kingdom Christoph Walz, Ludwig Maximilian University of Munich, Germany

> \*Correspondence: Felipe Suarez<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Primary Immunodeficiencies, a section of the journal Frontiers in Immunology

Received: 21 September 2018 Accepted: 10 December 2018 Published: 04 January 2019

#### Citation:

Tatfi M, Hermine O and Suarez F (2019) Epstein-Barr Virus (EBV)-Related Lymphoproliferative Disorders in Ataxia Telangiectasia: Does ATM Regulate EBV Life Cycle? Front. Immunol. 9:3060. doi: 10.3389/fimmu.2018.03060 Epstein-Barr virus (EBV) is an ubiquitous herpesvirus with a tropism for epithelial cells (where lytic replication occurs) and B-cells (where latency is maintained). EBV persists throughout life and chronic infection is asymptomatic in most individuals. However, immunocompromised patients may be unable to control EBV infection and are at increased risk of EBV-related malignancies, such as diffuse large B-cell lymphomas or Hodgkin's lymphomas. Ataxia telangiectasia (AT) is a primary immunodeficiency caused by mutations in the ATM gene and associated with an increased incidence of cancers, particularly EBV-associated lymphomas. However, the immune deficiency present in AT patients is often too modest to explain the increased incidence of EBV-related malignancies. The ATM defect in these patients could therefore impair the normal regulation of EBV latency in B-cells, thus promoting lymphomagenesis. This suggests that ATM plays a role in the normal regulation of EBV latency. ATM is a serine/threonine kinase involved in multiple cell functions such as DNA damage repair, cell cycle regulation, oxidative stress, and gene expression. ATM is implicated in the lytic cycle of EBV, where EBV uses the activation of DNA damage repair pathway to promote its own replication. ATM regulates the latent cycle of the EBV-related herpesvirus KSHV and MHV68. However, the contribution of ATM in the control of the latent cycle of EBV is not yet known. A better understanding of the regulation of EBV latency could be harnessed in the conception of novel therapeutic strategies in AT and more generally in all ATM deficient EBV-related malignancies.

Keywords: ataxia telangiectasia, Epstein Barr virus, latency, B-cell, primary immune deficiency, lymphomagenesis, hodgkin lymphoma, non-hodgkin lymphoma

# INTRODUCTION

Epstein-Barr virus (EBV) is a γ-herpesvirus that infects 95% of adults worldwide. EBV targets epithelial cells where lytic replication occurs, and B-cells where latent infection is established. The distinct phases of EBV infection are carefully controlled throughout the infected host's life, and chronic infection in immunocompetent individuals is mostly asymptomatic (1). Control of chronic EBV infection in immunocompromised patients may fail, leading to lymphoproliferative disorders as well as bona fide lymphomas (hereafter referred to as EBV-LPD) (2).

Several primary immune deficiencies (PID) are associated with poor responses to EBV and are also associated with a high risk of EBV-LPD (3). Inherited genetic abnormalities causing PID are often associated with poor or absent EBV-specific cytotoxic Tcell response and studies on PID and their underlying molecular mechanisms have led to a better understanding of immunological and cellular processes that control herpesvirus infection.

EBV infects B-cells both in vivo and in vitro and can lead to their immortalization. EBV latent genes drive the activation and differentiation of B-cells (4). Deregulation of this complex and dynamic interaction of viral gene expression and cellular activation may lead to cell transformation.

Ataxia telangiectasia (AT) is a rare PID caused by mutations in the ATM gene. AT patients are at increased risk of cancer, including EBV-LPD (5). However, the extent of immune compromise in AT is variable, and many patients have only minor immunological alterations (6). ATM is involved in many functions ranging from DNA repair to gene expression. Based on the paradoxical observation that EBV-LPD frequency is increased in ATM patients while they do not exhibit major T-cell deficiency, we raise the hypothesis that the defect of ATM in EBV-infected cells could play a role per se in the control of EBV latency, favoring a latent program more prone to lymphomagenesis. We review here the characteristics of AT and discuss the immunological and cellular abnormalities that may confer susceptibility to EBV-related malignancies.

# CLINICAL AND IMMUNOLOGICAL FEATURES OF AT

AT is an autosomal recessive disorder caused by biallelic mutations in the Ataxia-telangiectasia mutated (ATM) gene. Its estimated incidence is about 1/300.000 live births (7). AT was first reported by Syllaba and Henner in 1926 (8), further characterized by Denise Louis-Bar (9), and finally named by Boder and Sedgwick (10).

AT is characterized by progressive neurodegeneration leading to ataxia, oculo-cutaneous telangiectasia, variable degrees of immune deficiency, and susceptibility to cancer. AT is clinically heterogeneous, the classic form starts typically around 4 years of age, most patients becoming wheelchair-bound by the age of 10. Milder forms of AT may appear later and develop slowly. AT patients have a reduced life expectancy with a median survival of 19 to 25 years (11, 12). Mortality is mostly due to respiratory tract infections and cancers (6).

AT patients have a variable immunodeficiency, rarely progressive, with some patients not affected at all. Complete loss of ATM kinase activity leads to a more severe immunologic phenotype. B-cell and T-cell lymphopenia may be present in ∼70% of AT patients (6, 13). Over 60% of patients also have abnormal serum immunoglobulin levels, most notably a deficiency of IgG4 (65%), IgA (63%), and IgG2 (48%) (6).

# GENETIC AND MOLECULAR BASIS OF AT

The ATM gene (∼160kB) was cloned in 1995 (14). Over 400 mutations of ATM have been reported, spanning all 66 exons of the gene (Leiden Open Variation database). Most of these mutations lead to complete loss of ATM protein expression, but missense, and splice mutations can lead to the expression of a protein with residual kinase activity (15).

The ATM gene encodes a 350 kDa serine/threonine kinase belonging to the phosphatidylinositol 3-kinase-related kinase (PIKK) family (16). ATM is mostly located in the nucleus, but ∼20% are found in the cytoplasm, mainly in peroxisomes, endosomes, and as soluble proteins (17). ATM is involved in many cellular functions, including cell cycle checkpoint, apoptosis, oxidative stress, mitochondrial metabolism, gene regulation, and telomeres maintenance, but one of its major roles is its involvement in double strand break (DSB) repair (18). DSB can occur by endogenous processes, during replication fork collapse, V-(D)-J recombination or class switching, and can be induced by exogenous factors such as chemotherapy. In the canonical pathway, ATM is partially activated few seconds after DSB, probably after the relaxation of chromatin adjacent to the break. The MRN complex (Mre11/Rad50/Nbs1) recognizes the site of DNA break and in turn recruits ATM (**Figure 1**). Autophosphorylation of the ATM dimer occurs after its association with the MRN complex and precedes the formation of the fully active monomer forms (17).

ATM then phosphorylates H2AX, a variant of the histone H2A family, forming γH2AX foci that serve as a scaffold for the recruitment of DNA repair proteins such as MDC1, 53BP1, and BRCA1. Several other important partners are phosphorylated by ATM, such as ChK2 and p53, which initiate the downstream events of DNA repair and induce cell cycle arrest or apoptosis if DNA repair fails (18). The repair mechanism involves non-homologous end joining, an error-prone process occurring in G1/S phase (19), or homologous recombination, a faithful process in G2/M when sister chromatid is available (20).

As a result, defective DSB repair in AT patients elicit genomic instability that leads to B-cell and T-cell lymphopenia, premature senescence, and cancer. ATM plays a key role in the development of lymphocytes, allowing DSB repair occurring during B-cell, and T-cell differentiation. However, DSB repair may be possible via the alternative end-joining, an error prone, and poorly understood ATM independent pathway, which could explain the modest degree of lymphopenia in AT patients (21). Some lymphoid malignancies, such as mantle cell lymphoma, diffuse large B-cell lymphomas, and Hodgkin lymphomas, are also associated with acquired ATM mutations (22).

Patients with AT are at increased risk of cancer, especially lymphoid malignancies (23). To estimate precisely the risk of cancers in patients with AT, we conducted a retrospective study of cancers of 279 AT patients from the registry of the French National Reference Center for Primary Immune Deficiencies (CEREDIH) (5) and found that 25% of AT patients develop malignancies, the most common of which were aggressive non-Hodgkin's lymphomas (55% of all cancers), followed by Hodgkin's lymphoma (17%), leukemia (16%), and various solid tumors (12%). EBV was associated with 100% of Hodgkin's lymphomas and 50% of B-cell non-Hodgkin's lymphomas.

# EPSTEIN-BARR VIRUS

EBV belongs to the herpesviridae family of large enveloped double-stranded DNA viruses, and was first identified in Burkitt's lymphoma in the 1960s (24). All herpeviruses have 2 distinct phases of infection, lytic, and latent. The lytic EBV infection occurs in oropharyngeal epithelial cells and leads to viral replication and production of multiple virions that spread into the underlying lymphoid tissues and infect naive B-cells (25). In the latter, EBV enters latency, a phase during which the viral genome is maintained as a nuclear episome, and only a few viral genes are transcribed (26).

involved apoptosis induction. CHK2 also phosphorylates P53 to promote cell-cycle arrest or apoptosis.

Expression of the latency genes is tightly controlled by cellular and viral factors. Cellular immunity is also strongly induced during EBV infection and contributes to the elimination of infected cells expressing immunogenic lytic and latent antigens (27). Analysis of viral gene expression in EBV-associated cancers has led to a model in which EBV transitions through several latency gene expression programs (latency III, II, I) (28). Depending on the stage of latency, cells can express nuclear proteins (EBNA-1,−2,−3a,−3b,−3c, and -LP), latent membrane proteins (LMP-1,−2A, and−2B), non-coding RNAs (EBER-1 and−2), and several microRNAs (4). According to this model, EBV uses different latency programs to exploit B cell maturations pathways in the germinal center, leading the infected B-cell to the long-lived memory B-cell pool (28). The viral proteins LMP1 and LMP2 replicate the signals induced in B cells during the germinal center reaction and cause their proliferation. LMP1 mimics the signal of the activated CD40 (29) and induces EBV-LPD in transgenic mice (30), and LMP2 mimics the signal of the antigen activated B-cell receptor (31) and allow B-cell development even in the absence of normal B-cell receptor signaling (32).

EBV latency genes and non-coding RNAs may have oncogenic properties or interfere with cellular activation pathways leading to proliferation of B-cells as immunoblasts (33). Indeed, in vitro EBV infection of naïve B-cells leads to immortalized and fully transformed lymphoblastoïd cell lines in latency III that can induce tumors in nude mice (34). During latency III, EBNA-2 interacts with the target of the Notch pathway RBP-Jκ (35) and recruits co-activators, allowing the transcription of many genes involved in proliferation including MYC (36). EBNA-2 also promotes the transcription of all other latency genes, including the EBNA-3 family which gradually replaces EBNA-2 in its association with RBP-Jκ (37). The EBNA-3 family recruits co-repressors and inhibits the transcription of EBNA-2 induced genes. Indeed, prolonged expression of MYC could induce early senescence and be harmful to the virus (38). Infected B-cells migrate to the germinal center and progressively lose the expression of EBNA-2 and EBNA-3, escaping the immune control (latency II). During their transit through the germinal center to finally reach the memory B-cell pool, infected B-cells further restrict the expression of latency genes to express only EBNA1, a viral protein involved in the tethering of the viral episome to the nuclear chromatin (latency I) (39). EBV persists then indefinitely in memory B-cells (40). When these infected cells differentiate into plasma cell following an encounter with their cognate antigen, the transcription factor XBP-1 induced during plasma cell differentiation activates the expression of BZLF1, a viral protein sufficient to induce the lytic cycle (41). Virions are then released into the bloodstream and can re-infect the oropharynx epithelium to perpetuate the infectious cycle.