**HOW TO IMPROVE IMMUNE RECONSTITUTION IN ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION?**

# **Topic Editors Antoine Toubert and Hermann Einsele**

#### *FRONTIERS COPYRIGHT STATEMENT*

© Copyright 2007-2014 Frontiers Media SA. All rights reserved.

All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA ("Frontiers") or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers.

The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. For the conditions for downloading and copying of e-books from Frontiers' website, please see the Terms for Website Use. If purchasing Frontiers e-books from other websites or sources, the conditions of the website concerned apply. **DOI** 10.3389/978-2-88919-491-92015

Images and graphics not forming part of user-contributed materials may not be downloaded or copied without permission.

Individual articles may be downloaded and reproduced in accordance with the principles of the CC-BY licence subject to any copyright or other notices. They may not be re-sold as an e-book.

As author or other contributor you grant a CC-BY licence to others to reproduce your articles, including any graphics and third-party materials supplied by you, in accordance with the Conditions for Website Use and subject to any copyright notices which you include in connection with your articles and materials.

All copyright, and all rights therein, are protected by national and international copyright laws.

The above represents a summary only. For the full conditions see the Conditions for Authors and the Conditions for Website Use.

**ISSN** 1664-8714 **ISBN** 978-2-88919-308-0 **DOI** 10.3389/978-2-88919-308-0 **ISSN** 1664-8714 **ISBN** 978-2-88919-491-9

## *ABOUT FRONTIERS*

Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals.

## *FRONTIERS JOURNAL SERIES*

The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing.

All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too.

## *DEDICATION TO QUALITY*

Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.

Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view.

By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

## *WHAT ARE FRONTIERS RESEARCH TOPICS?*

Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area!

Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office<EMAIL_ADDRESS>
## **HOW TO IMPROVE IMMUNE RECONSTITUTION IN ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION?**

Topic Editors: **Antoine Toubert,** Université Paris Diderot, France **Hermann Einsele,** University Hospital Würzburg, Germany

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is widely used in the treatment of haematological malignancies as a form of immunotherapy acting through a graft-versus-leukemia (GvL) reaction. This curative allogeneic response can be associated with severe drawbacks, such as frequent and severe graft-versus-host disease (GvHD) and a long-lasting immunodeficiency, especially now with the development of innovative strategies such as umbilical cord blood transplantation or transplants from haplo-identical family donors (Haplo-HSCT). In the long-term follow-up of these patients, severe post-transplant infections, relapse or secondary malignancies may be directly related to persistent immune defects.

Reconstitution of the different lymphocyte populations (B, T, NK, NKT) and antigen presenting cells of myeloid origin (monocytes, macrophages and dendritic cells) should be considered not only quantitatively but especially qualitatively, in terms of functional subsets. Immune deficiency leading to an increased susceptibility to infections lasts for more than a year. Although infections that occur in the first month mostly result from a deficiency in both granulocytes and mononuclear cells (MNC), later post-engraftment infections are due to a deficiency in MNC subsets, primarily CD4 T-cells and B-cells. T-cell reconstitution has been extensively studied because of the central role of T-cells in mediating both GvHD, evidenced by the reduced incidence of this complication following T-Cell depletion, and a GvL effect as shown by DLI. In the recent years there has been renewed interest in the role of NK-cells, especially in the context of Haplo-HSCT, and in B-cell reconstitution.

This Frontiers Research Topic will provide state of the art knowledge of the mechanisms of immune reconstitution in an allogeneic environment, in order to improve monitoring and therapeutic intervention in allo-HSCT patients.

# Table of Contents

A. Forcina, M. Noviello, M. R. Carbone, Chiara Bonini and Attilio Bondanza

*12 Thymic function recovery after unrelated donor cord blood or T-cell depleted HLA-haploidentical stem cell transplantation correlates with leukemia relapse* Emmanuel Clave, Daniela Lisini, Corinne Douay, Giovanna Giorgiani, Marc Busson, Marco Zecca, Francesca Moretta, Gloria Acquafredda,

Letizia P. Brescia, Franco Locatelli and Antoine Toubert

*20 Monitoring of pathogen-specific T-cell immune reconstitution after allogeneic hematopoietic stem cell transplantation*

Shigeo Fuji, Markus Kapp and Hermann Einsele

*26 Alloreactivity of virus-specific T cells: possible implication of graft-versus-host disease and graft-versus-leukemia effects*

Shigeo Fuji, Markus Kapp and Hermann Einsele

*31 Immune reconstitution and graft-versus-host reactions in rat models of allogeneic hematopoietic cell transplantation* Severin Zinöcker, Ralf Dressel, Xiao-Nong Wang, Anne M. Dickinson and

Bent Rolstad

*43 Adoptive T-cell immunotherapy from third-party donors: characterization of donors and set up of a T-cell donor registry*

Britta Eiz-Vesper, Britta Maecker-Kolhoff and Rainer Blasczyk

*52 Natural killer cell mediated antibody-dependent cellular cytotoxicity in tumor immunotherapy with therapeutic antibodies*

Ursula J. E. Seidel, Patrick Schlegel and Peter Lang

*60 Cellular and molecular basis of haploidentical hematopoietic stem cell transplantation in the successful treatment of high-risk leukemias: role of alloreactive NK cells*

Franco Locatelli, Daniela Pende, Maria C. Mingari, Alice Bertaina, Michela Falco, Alessandro Moretta and Lorenzo Moretta

*68 Improving engraftment and immune reconstitution in umbilical cord blood transplantation*

Robert Danby and Vanderson Rocha

EDITORIAL published: 06 January 2015 doi: 10.3389/fimmu.2014.00669

## From immunomonitoring to immune intervention

## **Antoine Toubert 1 and Hermann Einsele2\***

1 Immunology-Histocompatibility, Laboratoire d'Immunologie et d'Histocompatibilité, Laboratoire Jean Dausset, INSERM, Assistance Publique Hôpitaux de Paris, Université Paris Diderot, Paris, France

2 Department of Internal Medicine II, University Hospital Würzburg, Würzburg, Germany

\*Correspondence<EMAIL_ADDRESS>
#### **Edited and reviewed by:**

Effie Wang Petersdorf, University of Washington School of Medicine, USA

#### **Keywords: HSCT, immune reconstitution, thymic function, cell therapy, Haplo-SCT**

Host immune status is a key issue in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the long-term follow-up of these patients, severe post-transplant infections, relapse or secondary malignancies may be directly related to prolonged immune defects, especially in the context of innovative stem cell sources such as umbilical cord blood transplantation (CBT) (1) or transplants from HLA haplo-identical family donors (Haplo-HSCT) (2, 3).

This *Frontiers* Research Topic provides insights into mechanisms of immune reconstitution in an allogeneic environment, in order to improve monitoring and therapeutic intervention in allo-HSCT patients.

Forcina et al. (4) critically review T and NK biomarkers, their threshold and clinical relevance. Many factors from the host, transplant conditioning, stem cell source, and genetic disparity may impact immune recovery. Few biomarkers have reached a clinical consensus: a rapid and potent CD4 T-cell recovery is associated with a favorable clinical outcome, CMV-specific CD8 T-cell counts measured by tetramers can predict a lower incidence of CMV disease. Other phenotypic or molecular markers, still based on small-sized studies, will need further validation in large scale-multicentric cohorts. This is the case of molecular markers of lymphocyte generation, such as T-cells evaluated by the quantification of T-cell rearrangement excision circles (TREC) as a surrogate marker of thymic activity. Clave et al. (3) indicate an impact of thymic function recovery on relapse following CBT as well as Haplo-HSCT in children treated for an hematological malignancy. In order to reach the routine medical practice, immunomonitoring tests need also to be simple and fast. This practical issue is well taken into account by Fuji et al. (5) reviewing T-cell monitoring of viral and fungal infections. CMV is still the most intensively studied virus in immunocompromised hosts. CMV shapes T and NK cell responses in many ways and may escape immune response. Among still unanswered questions, is the role of viral-specific (CMV and EBV) T-cell cross-reactivity against allogeneic targets in graft-versus-host and graft-versus-leukemia (6).

Experimental models have proved their importance in allo-HSCT, especially deciphering mechanisms of allogeneicity in graft-versus-host disease (GVHD). Among rodents, rat models are relevant especially in some autoimmune conditions and solid organ transplantation, sometimes closer to the human disease than the "gold standard" murine models. Zinöcker et al. (7) emphasize the interest of rat model in experimental GVHD, especially the rat skin explant assay as a tool for functional

evaluation of GVHD. Experimental models are also required to provide the basis and for preclinical evaluation of immune-based therapies.

This *Frontiers* Research Topic also reviews in some strategies already proposed in the clinics. Adoptive T-cell therapies have been conducted successfully by several groups to control lifethreatening viral (EBV, CMV) reactivations. This leads to "off the shelf" strategies based on third party HLA-typed T-cell donors registries, which may have a major impact in transplant recipients (8). The development of therapeutic antibodies in cancer is one of the most active fields in clinical immunology. Many strategies are underway to improve their action, especially through their antibody-dependent cellular toxicity mediated by natural killer (NK) cells (9). One approach especially relevant in allo-HSCT would be to take advantage of the killer-immunoglobulin-like receptor (KIR) ligand incompatibility in addition to ADCC to boost NK functions.

The impact of these different concepts, from basic knowledge to translational medicine, is integrated in state-of-the art reviews of two main "success stories" in allo-HSCT: CBT (1) and Haplo-HSCT (2).

## **REFERENCES**

9. Seidel UJ, Schlegel P, Lang P. Natural killer (NK) cell mediated antibodydependent cellular cytotoxicity (ADCC) in tumour immunotherapy with therapeutic antibodies. *Front Immunol* (2013) **4**:76. doi:10.3389/fimmu.2013.00076

**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.

*Received: 05 November 2014; accepted: 13 December 2014; published online: 06 January 2015.*

*Citation: Toubert A and Einsele H (2015) From immunomonitoring to immune intervention. Front. Immunol. 5:669. doi: 10.3389/fimmu.2014.00669*

*This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology.*

*Copyright © 2015 Toubert and Einsele. 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) or licensor 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.*

## Predicting the clinical outcome of allogeneic hematopoietic stem cell transplantation: the long and winding road toward validated immune biomarkers

## **A. Forcina, M. Noviello, M. R. Carbone, Chiara Bonini \* and Attilio Bondanza\***

Experimental Hematology Unit, San Raffaele Scientific Institute, Milan, Italy

#### **Edited by:**

Seiamak Bahram, Université de Strasbourg, France

#### **Reviewed by:**

Rupali Das, Children's Hospital of Philadelphia, USA Laurent Garderet, Hopital Saint Antoine, France

#### **\*Correspondence:**

Chiara Bonini and Attilio Bondanza, Experimental Hematology Unit, San Raffaele Scientific Institute, Via Olgettina 58, 20123 Milano, Italy. e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
The clinical outcome of allogeneic hematopoietic stem cell transplantation (HSCT) is strongly influenced from the potential complications arising during the delicate phase of post-transplant immune restoration. The quantitative aspects of immune-cell repopulation after HSCT and the qualitative features their functional restitution have been extensively reported. Nevertheless, measurable immune biomarkers predicting the clinical outcome of HSCT await formal validation. The aim of this review is an appraisal of most studies published so far on the predictive value of different T and NK-cell biomarkers after HSCT with emphasis on defined thresholds endorsed by multivariate analysis.

**Keywords: allogeneic hematopoietic stem cell transplantation, immune reconstitution, biomarkers, opportunistic infections, graft-versus-host disease**

#### **INTRODUCTION**

The restoration of a functional immune system is one of the main factors influencing the clinical outcome of allogeneic hematopoietic stem cell transplantation (HSCT). The post-transplant period is characterized by multiple immune defects that expose the patient to a high risk of opportunistic infections and, eventually, disease relapse. The duration of this period may vary according to several variables, including patient age and immune status before transplant, the degree of donor compatibility, the intensity of the conditioning regimen, the source of stem cells, eventual graft manipulation, and pharmacological immune suppression. The normalization of granulocytes, monocytes, and NK-cell numbers usually occurs within the first weeks and, with the possible exception of NK-cells, coincides with their full competence. Conversely, the normalization of T and B cell numbers may take much longer and does not necessarily associate with their immediate functional restitution (Shiobara et al., 1982). Although often used interchangeably, it is therefore important to distinguish between *immune reconstitution*, which refers to quantitative immunecell repopulation, and *immune recovery*, which pertains to their qualitative restitution.

The quantitative reconstitution of T cells post-transplant occurs through two main mechanisms: (i) the early peripheral expansion of donor-derived memory T cells present in the graft, which happens in weeks (ii) the late emergence of host-tolerant naïve T cells originating from donor stem cells after thymic education, which, depending on donor age, occurs in months (van den Brink et al., 2004). The qualitative recovery of T cells may follow their quantitative reconstitution with a delay of many years and implies *de novo* pathogen encounter, with ensuing differentiation into effector and memory T cells.

Although the quantitative aspects of immune reconstitution post-transplant and the qualitative features of immune recovery have been the subject of several studies, a tight association between measurable immune biomarkers and the clinical outcome of HSCT, is currently missing. So far, the specific issue of validating thresholds of immune measurements that may help predicting the incidence of major post-transplant events, such as opportunistic infections, graft-versus-host disease (GVHD), and disease relapse, has been approached by single-center, necessarily small-sized studies. While sometimes sufficiently powered to obtain statistically significant results, these studies often failed to draw definitive conclusions that may be relevant to daily clinical practice. Conversely, the lack of harmonized methods for immune biomarker measurements and the great heterogeneity of the transplant populations between the different studies have prevented from meaningful meta-analysis.

The aim of this review is an appraisal of the studies published so far on the predictive value of different T and NK-cell biomarkers after HSCT with emphasis on the thresholds chosen for statistical analysis. A comprehensive **Table 1** detailing the main results from the different studies has also been included. Descriptive studies based on the comparison between different groups as the only statistical approach, studies in the setting of autologous HSCT and immune biomarkers whose predictive value has not been endorsed by multivariate analysis, have been purposely excluded from this review.

#### **T LYMPHOCYTES AND INVARIANT NKT CELLS**

The absolute lymphocyte count (ALC) derived from routine blood-cell testing has been the first immune biomarker explored for predicting the clinical outcome of HSCT. In patients undergoing T cell-depleted HSCT from an HLA-identical sibling (HLAsib), an ALC above the median [>690/µL (Montero et al., 2006) or >450/µL (Savani et al., 2007a)] at day 30 posttransplant was found to be independently associated with a lower

ALC, absolute lymphocyte count; HLA-sib, HLA-identical sibling; TRM, transplant-related mortality; LFS, leukemia-free survival; OS, overall survival; RI, relapse incidence; MUD, matched unrelated donor; Haplo, HLA-haploidentical; UCB, umbilical cord-blood; ROC, receiver operating characteristic curve analysis; aGVHD, acute graft-versus-host-disease; TREC, T cell receptor excision circles; pre-tx, pre-transplant; TNC, total nucleated cells; cGVHD, chronic graft-versus-host-disease; PBL, peripheral blood lymphocytes; Tet+ , tetramer-positive cells; cyt+ , intracellular cytokine-positive; S.I., stimulation index.

transplant-related mortality (TRM) and longer leukemia-free survival (LFS) and overall survival (OS). The association between an higher ALC at early time points after transplant and a favorable clinical outcome was confirmed by taking an arbitrary threshold of 1000/µL in T cell-replete HSCT from matched unrelated donors (MUD) (Le Blanc et al., 2009) and CD34-selected HSCT form HLA-haploidentical donors (Ciurea et al., 2011), or by taking an arbitrary threshold of 200/µL in umbilical cord-blood (UCB) transplantation (Burke et al., 2011). Investigating the same issue in patients that received a reduced-intensity regimen, however, has found conflicting results (Matthews et al., 2010; Burke et al., 2011), suggesting that the type of conditioning may influence the predictive value of the ALC.

The predictive value of T lymphocyte subsets assessed by flow cytometry, rather than the simpler ALC, has been examined in more sophisticated studies. At day 90 after T cell-replete HSCT form a HLA-sib or a MUD, a CD4+ T cell count above 200/µL, a threshold derived from the HIV field, was independently associated with a lower NRM, less opportunistic infections, and a longer OS (Kim et al., 2006). The role for a rapid reconstitution of CD4+ T cells in protecting from transplant morbidity and mortality was confirmed in three subsequent studies using slightly different approaches for statistical analysis. The first study found that at day 30 post-transplant, a CD4+ T cell count above the median (>86/µL) was associated with a lower TRM (Berger et al., 2008). The second study confirmed the association and observed no impact on relapse incidence (Buhlmann et al., 2011). The third study used receiver operator curve (ROC) analysis of CD4+ T cell counts at day 20 post-transplant for determining a threshold of 115/µL, which was retrospectively found to be associated with a lower TRM (Fedele et al., 2012).

Differently from CD4+ T cells, the predictive value of CD8+ T cell biomarkers is less studied. In a combined series of HLA-sib, MUD, or HLA-haploidentical pediatric HSCT, reaching a CD8+ T cell count above the fifth percentile of age-matched controls within the first year post-transplant was found to be independently associated with a longer OS and a trend toward a lower relapse incidence (Koehl et al., 2007).

The pattern of invariant natural killer T cells (iNKT) reconstitution has been explored for predicting the clinical outcome at earlier time points after HSCT and independently from conventional T cells. The reconstitution of iNKT cells after HLA-sib or MUD HSCT was found to precede that of T and NK-cells (Rubio et al., 2012). Accordingly, at day 15 post-transplant an iNKT/T cell ratio above 0.58 × 10−3 , a threshold identified after retrospective ROC analysis, was associated with a zero likelihood of GVHD. Moreover, reaching an iNKT/T cell ratio above 10−3 within the first 3 months after transplantation was independently associated with a lower NRM and a longer OS. In a concomitant study, reaching an NKT/T cell ratio above 10−4 within the first 18 months after CD34-selected HLA-haploidentical pediatric HSCT associated with the maintenance of disease remission in all children (de Lalla et al., 2011; Casorati et al., 2012).

#### **TREC ANALYSIS**

The molecular analysis of TCR excision circles (TRECs) in circulating T cells allows to quantitatively assess host thymic function, a parameter that has been shown to play a fundamental role in the rapidity of T cell immune reconstitution after HSCT (Talvensaari et al., 2002). After categorization of data from a retrospective cohort, the group of Antoine Toubert has prospectively shown that a pre-transplant TREC content above the threshold of 172 per 150,000 CD3+ T cells is an independent factor associated with less infections, including Cytomegalovirus (CMV) reactivation, and a longer OS after T cell-replete HLA-sib HSCT (Clave et al., 2005). The same group has found that at 6 months after CD34 selected HLA-haploidentical pediatric HSCT, a TREC value below detection levels (<0.1 per 150,000 CD3+ T cells for sjTREC and <0.001 per 150,000 CD3+ T cells for βTREC) was associated with a higher relapse incidence (Clave et al., 2012).

## **NATURAL TREGS**

In animal models of HSCT, natural regulatory T cells (Tregs) have a key role in promoting tolerance and, in particular, in protecting from GVHD (Nguyen et al., 2006). In humans, however, there are a number of controversial issues that so far have prevented from confirming the value of Tregs assessment for predicting the risk of GVHD, its grading and response to therapy. These include how to discriminate Tregs from activated T cells and what is the most appropriate way to express Tregs measurements.

In patients with acute GVHD after HLA-sib or MUD HSCT, Tregs frequencies measured at disease onset as the percentage of CD4+CD25brightFoxp3+ T cells over total nucleated cells were reported to inversely correlate with acute GVHD grading (Magenau et al., 2010). Moreover, Tregs frequencies above the median, i.e., >0.5%, were associated with complete response to first-line therapy, resulting in a lower TRM and a longer OS. In another study considering patients with gastrointestinal GVHD, however, peripheral blood as well as mucosal Tregs frequencies, measured as the percentage of CD4+ co-expressing Foxp3, were not found to correlate with disease severity (Lord et al., 2011).

The evaluation of Tregs biomarkers for predictive purposes has also yielded conflicting results in chronic GVHD. Some authors have found a paradoxical increase in Tregs measured both as the percentage and as the absolute count of CD4+CD25bright (Clark et al., 2004). These T cells were later found to be suppressive *ex vivo*, ruling out that they were activated T cells in disguise. On the contrary, other authors have reported that Tregs frequencies measured as the percentage of CD4+CD25bright T cells over peripheral blood lymphocytes below 3%, a threshold derived from linear and logistic regression, were associated with chronic GVHD. This threshold was derived from linear regression models based on data from healthy donors (Zorn et al., 2005). In a phase I/II trial investigating the administration of low dose IL-2 in chronic GVHD, the same group has found that changes in the median count of Tregs somewhat correlated with the probability of responding to the treatment (Koreth et al., 2011).

#### **PATHOGEN-SPECIFIC T CELLS**

The value of pathogen-specific T cell responses as an immune biomarker predictive of the risk and the severity of opportunistic infections after HSCT is still controversial. This is mostly due to the use of different methods for measurement (MHC-peptide tetramers, intracellular cytokine staining, ELISPOT assays) and to the lack of harmonized protocols between the different studies. Other contentious issues are whether it is sufficient assessing either CD8+ or CD4+ responses or it is needed considering both, and whether complex and costly biomarkers are worthy compared with easier, cheaper, and already validated tests, such as serology (Ljungman et al., 2003).

Since CMV disease is a major complication after HSCT, the majority of the studies have focused their attention on CMVspecific responses. In an early study investigating the use of tetramers, it was found that reaching 10 CMV-specific CD8+ T cells/µL within the first 100 days after HSCT from an HLA-id or a MUD with discordant serology associated with a zero likelihood of CMV disease (Cwynarski et al., 2001). The predictive value of the CMV-specific CD8+ T cell count measured with tetramers was confirmed in a multicenter, prospective study including HLA-sib and MUD HSCT where, after categorization, it was found that a value above the threshold of 7 cells/µL associated with a lower incidence of CMV disease (Gratama et al., 2010).

Other studies have examined the predictive value of CMVspecific CD4+ T cell responses showing comparable results. In a pioneering study by the group of Hermann Einsele, it was found that a positive CD4+ T cell proliferative response defined as a stimulation index above the arbitrary value of 2.5 within the first 120 days after HLA-sib or MUD HSCT associated with a reduced incidence of CMV disease (Krause et al., 1997). The predictive value of studying CD4+ T cell responses was confirmed by using intracellular cytokine staining. In a similar setting, reaching 0.4 CMV-specific CD4+ T cells/µL within day 30 and 50, for example, was found to associate with complete prevention from subsequent CMV reactivation (Pourgheysari et al., 2009). In another study, a positive proliferative response to either CMV, HSV, or VZV defined as a stimulation index above 7 was associated not only with a zero probability of opportunistic infections, but also with the maintenance of disease remission, indicating that the recovery of pathogen-specific immunity may serve as a surrogate biomarker of immune restoration (Parkman et al., 2006).

In certain studies, the concomitant exploration of both CD4+ and CD8+ CMV-specific T cells allowed determining the predictive thresholds for the two subsets in a compared manner. In a pediatric population, after ROC analysis of intracellular cytokine staining data from a retrospective cohort (Lilleri et al., 2006), it was found that reaching a CMV-specific T cell count above the threshold of 1/µL for CD4+ T cells and of 3/µL for CD8+ T cells within the first year after HLA-sib,MUD, or HLA-haploidentical pediatric HSCT associated with a zero likelihood of CMV reactivation up to 2 years thereafter (Lilleri et al., 2012). These thresholds were found to be remarkably similar (1.2 and 1/µL for CD4+ and CD8+ CMVspecific T cells, respectively) in HLA-sib and MUD adult HSCT (Tormo et al., 2011) suggesting that measuring pathogen-specific responses in both subsets may be of predictive value, although with slightly different thresholds.

The results of the studies on Epstein-Barr virus (EBV)-specific responses are more controversial. Although measuring EBVspecific T cell responses by intracellular cytokine staining was found to have no value in predicting the likelihood of posttransplant lymphoproliferative disease (Hoegh-Petersen et al., 2011), finding at day 56 an EBV-specific T cell score above 1, a

threshold found after categorization of a complex measure including CD4 and CD8 viral epitopes, was associated with a near-zero likelihood of leukemia relapse (Hoegh-Petersen et al., 2012).

## **NK-CELLS**

The discovery that NK alloreactivity plays a major role in preventing disease relapse after CD34-selected HLA-haploidentical HSCT (Ruggeri et al., 2002), has fostered a number of studies investigating the predictive value of NK-cell biomarkers on clinical outcome. The reconstitution of NK-cells post-transplant is slightly slower compared to other cells of the innate immune system, but definitively faster than conventional T cells. At 30 days after T cell-depleted HLA-sib HSCT, an NK-cell count above the median (>150/µL) was found to associate with less acute GVHD, a lower relapse incidence, and a longer OS (Savani et al., 2007b). The association between higher NK-cell counts and lower relapse incidence was however restricted to patients with myeloid leukemia, a disease that is susceptible to NK lysis. The predictive value of NK-cell counts at early time points post-transplant was confirmed in the setting of unmanipulated HLA-haploidentical HSCT, where, after categorization, an NK-cell count above the threshold of 9.27/µL as early as 15 days post-transplant was associated with a longer LFS (Chang et al., 2008). The picture was shown to differ in the context of T cell-replete HSCT, where higher NK-cell counts (>150/µL, a threshold taken from previous studies (Savani et al., 2007b) were associated with a lower TRM at late time points, but not with a lower relapse incidence (Buhlmann et al., 2011).

## **CONCLUSION**

In the era of predictive and molecular medicine, the practice of HSCT is still characterized by many prognostic uncertainties. Since many complications of HSCT derive from the state of temporary, although often prolonged, state of immunodeficiency post-transplant, it is clear that finding a tight correlation between certain immune system defects and the different complications may help predict the overall clinical outcome. This is important not only for improving the care of patients, who may expect benefits from ready and tailored strategies of intervention, such as intensification or discontinuation of antimicrobial and immune suppressive drugs, but also for establishing accepted surrogate markers of immune restoration that may accelerate the clinical development of novel transplant strategies, including the transfer of pathogen-specific T cells generated after *ex vivo* stimulation (Feuchtinger et al., 2010; Heslop et al., 2010).

The road leading to the validation of immune biomarkers answering to this crucial, unmet need is long and winding, and is possibly better traveled by joining forces in multicenter efforts. The recent launch of different, retrospective, and prospective studies coordinated by the Immunobiology Working Party of the European Bone Marrow Transplantation society goes exactly in this direction and is expected to contribute to filling this gap in the near future.

#### **ACKNOWLEDGMENTS**

This work was supported by Italian Ministry of Health (GR07- 5 BO, RO10/07-B-1, Eranet Transcan HAPLO-IMMUNE), Italian Ministry of Research and University (FIRB-IDEAS, linked to ERC starting grants), Italian Association for Cancer Research and EU-FP7 program (ATTACK, PERSIST).

## **REFERENCES**

High Epstein-Barr virus-specific Tcell counts are associated with nearzero likelihood of acute myeloid leukemia relapse after hematopoietic cell transplantation. *Leukemia* 26, 359–362.

unrelated donor stem cell transplantation: an EBMT megafile analysis. *Blood* 102, 4255–4260.

graft-versus-host disease and overall survival. *Blood* 120, 2144–2154.

with myeloid leukemias but not with acute lymphoblastic leukemia. *Leukemia* 21, 2145–2152.

T-cell responses affording protection from CMV DNAemia following allogeneic hematopoietic SCT. *Bone Marrow Transplant.* 46, 1437–1443.

**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.

*Received: 24 October 2012; accepted: 04 March 2013; published online: 25 March 2013.*

*Citation: Forcina A, Noviello M, Carbone MR, Bonini C and Bondanza A (2013) Predicting the clinical outcome of allogeneic hematopoietic stem cell transplantation: the long and winding road toward validated immune biomarkers. Front. Immunol. 4:71. doi: 10.3389/fimmu.2013.00071*

*This article was submitted to Frontiers in Alloimmunity and Transplantation, a specialty of Frontiers in Immunology.*

*Copyright © 2013 Forcina, Noviello, Carbone, Bonini and Bondanza. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

## Thymic function recovery after unrelated donor cord blood or T-cell depleted HLA-haploidentical stem cell transplantation correlates with leukemia relapse

*Emmanuel Clave1,2\*, Daniela Lisini 3†, Corinne Douay1,2, Giovanna Giorgiani 3, Marc Busson1,2, Marco Zecca3, Francesca Moretta4, Gloria Acquafredda3, Letizia P. Brescia4, Franco Locatelli 4‡ and Antoine Toubert 1,2,5‡*

*2 Université Paris Diderot, Sorbonne Paris Cité, Paris, France*

*3 Pediatric Hematology-Oncology, IRCCS Policlinico San Matteo Foundation, Pavia, Italy*

*4 Department of Pediatric Hematology and Oncology, University of Pavia, IRCCS Ospedale Bambino Gesù, Rome, Italy*

*5 Hôpital St-Louis, AP-HP, Paris, France*

#### *Edited by:*

*Hermann Einsele, University Hospital Würzburg, Germany*

#### *Reviewed by:*

*Laurent Garderet, Hopital Saint Antoine, France Attilio Bondanza, S. Raffaele Scientific Institute, Italy*

#### *\*Correspondence:*

*Emmanuel Clave, Département d'Immunologie, INSERM UMRS-940, Hôpital St-Louis, 1 Avenue Claude Vellefaux, 75010 Paris, France. e-mail: emmanuel.clave@ univ-paris-diderot.fr*

#### *†Present address:*

*Daniela Lisini, Cell Therapy Production Unit, Foundation IRCCS Istituto Neurologico Carlo Besta, Milano, Italy.*

*‡Franco Locatelli and Antoine Toubert are co-senior authors.*

## **INTRODUCTION**

Whenever an HLA-identical related donor is not available, alternative sources of donors/stem cells are to be used for Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) (Beatty et al., 1995). Both unrelated Cord Blood (CB) and HLA-haploidentical (Haplo) family donors provide an effective treatment for many malignant and non-malignant diseases in children (Rocha and Locatelli, 2008). For the latter, the HLA disparity in the donor/recipient pair may lead to a beneficial and selective graft-versus-leukemia (GVL) effect, associated with natural killer (NK)-cell mediated alloreactivity (Ruggeri et al., 2002). Despite improvement in the graft procedure such as use of T-cell depletion (TCD) and infusion of high doses of CD34+ cells, which allows engraftment and reduces graft-versus-host disease (GvHD) in Haplo-HSCT recipients (Aversa et al., 1994; Handgretinger et al., 2001), a significant delay in immune reconstitution still remains a relevant problem, leading to a high incidence of both opportunistic infection and, in the absence of NK alloreactivity, to relapse (Ball et al., 2005; Wils et al., 2011). In this respect, association between poor clinical outcome and low

Use of alternative donors/sources of hematopoietic stem cells (HSC), such as cord blood (CB) or HLA-haploidentical (Haplo)-related donors, is associated with a significant delay in immune reconstitution after transplantation. Long-term T-cell immune reconstitution largely relies on the generation of new T cells in the recipient thymus, which can be evaluated through signal joint (sj) and beta T-cell-Receptor Excision Circles (TREC) quantification. We studied two groups of 33 and 24 children receiving, respectively, HSC Transplantation (HSCT) from an HLA-haploidentical family donor or an unrelated CB donor, for both malignant (46) and non-malignant disorders (11). Relative and absolute sj and beta-TREC values indicated comparable thymic function reconstitution at 3 and 6 months after the allograft in both groups. Compared to children with non-malignant disorders, those with hematological malignancies had significantly lower pre-transplantation TREC counts. Patients who relapsed after HSCT had a significantly less efficient thymic function both before and 6 months after HSCT with especially low beta-TREC values, this finding suggesting an impact of early intra-thymic T-cell differentiation on the occurrence of leukemia relapse.

**Keywords: HSCT, thymic function, T cells, relapse, leukemia**

lymphocyte counts early after CB- or Haplo-HSCT (Ciurea et al., 2011) or at month 6 in case of T-cell depleted HSCT (Novitzky et al., 2002) has been described. Moreover, we showed recently, in 33 Haplo-HSCT patients an association between low thymic function and an increased risk of leukemia relapse (Clave et al., 2012).

Recently, tools have been developed to monitor thymic function. Signal-joint T-cell-Receptor (TCR) Excision Circles (sjTREC) are small episomal DNA that results from the deletion of the TCR δ region during TCR α locus rearrangement. They are not replicated during lymphocyte cell division and reflect, in the periphery, the number of new lymphocytes generated in the thymus (Douek et al., 1998; Dion et al., 2007). BetaTREC are produced during TCR Dβ–Jβ recombination, occurring before TCR α-chain recombination in αβ T-cell differentiation. BetaTREC counts may therefore reflect the early stages of intrathymic differentiation and sj/beta TREC ratios the T-cell proliferation rate between β- and α-chain recombination events. Quantification of sj and beta-TREC molecules in peripheral blood cells has provided useful information in studies addressing the issues of aging,

*1 Departement d'Immunologie, INSERM UMRS-940, AP-HP, Paris, France*

HIV infection, and HSCT (Dion et al., 2004, 2007), but have not yet been used in the context of studies on leukemia relapse after HSCT.

In view of these considerations, we decided to investigate thymic function recovery in patients given unrelated donor CB transplantation (UCBT) and to compare the results obtained in this cohort of patients with those of children given Haplo-HSCT. In detail, we retrospectively evaluated thymic function recovery through sj and beta TREC quantification in 57 pediatric patients, 33 receiving Haplo-HSCT previously reported and 24 given UCBT. Thymic function recovery was found to be similar in both groups. Moreover, correlation of TREC values with clinical parameters revealed a link between recovery of thymic function and specific disease. Indeed, we found that pre-transplantation sj and beta TREC were lower for patients with malignant disease. Moreover, there was a strong correlation between low thymic function persisting at 6 month after HSCT and a higher incidence of relapse in the Haplo-HSCT group.

## **PATIENTS**

Two groups of pediatric patients, 24 receiving UCBT and 33 given Haplo-HSCT (**Table 1**) for either malignant or non-malignant diseases were studied, the last group being the same reported in our previously published study (Clave et al., 2012). In order to be included, patients had to be alive 6 months after transplantation (so, 3 patients that relapsed before 6 months were also included). All patients had been transplanted between October 2004 and February 2007 after having received a fully-myeloablative conditioning regimen. Details on patient and donor characteristics, as well as on transplantation outcome, are reported in **Table 1**.

*Haplo-HSCT indicates HLA-Haploidentical Hematopoietic Stem Cell Transplantation; UCBT, Unrelated Cord Blood Transplantation; CsA, cyclosporin A; GvHD, graft-versus-host disease; PB, peripheral blood; TBI, total body irradiation.*

*aTBI-based conditioning regimen was employed in 33 children and consisted of: fractionated TBI (12 Gy over 6 fractions in 3 days), Thiotepa (10 mg/Kg in 2 doses) and fludarabine (160 mg/m2 over 4 days). Chemotherapy-based conditioning regimen were as follows: 12 patients received Busulfan (16 mg/Kg in 16 doses over 4 days), Cyclophosphamide (120 mg/Kg in 2 days) and melphalan (140 mg/m2 in single dose); 7 children received Busulfan (16 mg/Kg in 16 doses over 4 days), Thiotepa (10 mg/Kg in 2 doses) and fludarabine (160 mg/m2 over 4 days); and 5 children were given Treosulfan (14 gr/m2 for 3 consecutive days), Thiotepa (10 mg/Kg in 2 doses) and fludarabine (160 mg/m2 over 4 days).*

*bPatients receiving Haplo-HSCT were transplanted with CD34*+ *selected cells and were not given any immune-suppressive drug after transplantation. Patients transplanted with cord blood cells received a combination of Cyclosporine-A (3 mg/Kg/day) and steroids [methylprednisolone (2 mg/Kg/day)] as GvHD prophylaxis. cReactivation of viral infections (i.e., cytomegalovirus and Epstein-Barr virus) and proven/probable invasive aspergillosis.*

No patient given Haplo-HSCT received post-transplantation pharmacologic immune suppression, while those transplanted with CB cells received a combination of CsA and steroids (see also **Table 1** for details). For the purpose of the study, patients with acute leukemia transplanted in 1st complete remission (CR) or in 2nd CR after a relapse occurring more than 6 months after treatment discontinuation, as well as patients affected by refractory cytopenia, were assigned to the early disease group. All other patients were included in the advanced disease group. Acute and chronic GvHD (cGvHD) were diagnosed and graded according to the Seattle criteria (Glucksberg et al., 1974; Storb et al., 1983). Patients surviving more than 14 and 100 days post-transplantation were evaluated for acute and cGvHD, respectively. The study was approved by the Ethical committee of Policlinico San Matteo, Pavia, Italy (approval number 446/DG).

## **METHODS**

#### **DESIGN OF THE STUDY**

DNA samples of patients enrolled in the study were collected before transplantation and at 3 and 6 months after the allograft. At the same time-points, we evaluated absolute number and percentage of the different T lymphocyte subsets.

#### **sjTREC AND beta-TREC QUANTIFICATION**

Genomic DNA was extracted from Peripheral Blood Mononuclear Cells (PBMC) using the QIAamp DNA Blood Minikit (Qiagen, Hilden, Germany). Quantification of sjTREC and beta-TREC was performed by real-time quantitative PCR (qPCR) (ABIPRISM7500, Applied Biosystems, Foster City, CA), as previously described (Clave et al., 2009). Briefly, a first PCR reaction was carried out in multiplex with 3 different outer primer mixes, 1–5μg of genomic DNA, 200μM each dNTP, 2.5 mM MgCl2, 1× buffer and 1.25 unit of *Platinum*® Taq polymerase (Invitrogen, Cergy-Pontoise, France) in 50 μL (10 min at 95◦C, then 19 cycles of 95◦C, 30 s; 60◦C, 30 s and 72◦C, 2 min). Final quantification was made on ABI PRISM 7700, in duplicate with a second multiplex reaction that contains 5μL of a 1/100 or 1/1000 dilution of the first PCR product, inner primers, and probe for sjTREC or one of the Dβ–Jβ segments, inner primers and probe for albumin (alb) gene, 1.25 mM each dNTP, 3 mM MgCl2, 1× buffer and 1.25 unit of *Platinum*® Taq polymerase in 25μL (5 min at 95◦C then 40 cycles of 95◦C, 15 s and 60◦C, 1 min). The sum of the 10 Dβ–Jβ segments was finally multiplied by 1.3 to extrapolate for all the 13 existing Dβ–Jβ segments. All primers and probes have been obtained from Eurogentec (Seraing, Belgium) except the alb vic-labeled probe from Applied Biosystems.

TREC data were validated only if at least 50,000 genome equivalents were detected by alb qPCR; therefore, some time-points were missing for 5 patients but other negative TREC values were not due to absence of genomic DNA. Data were first expressed per 150,000 PBMC and the total number of TREC per μL of blood was calculated using the absolute white blood cells count at time of sample collection.

#### **FLOW CYTOMETRY ANALYSIS**

FITC, PE, PerCP, or APC monoclonal antibodies (MoAbs) specific for the following antigens were employed for the evaluation of lymphocyte subsets: CD45, CD3, CD4, CD8 (BD Biosciences, Mountain View, CA). Appropriate isotype-matched controls (BD Bioscience) were included. Three-color or four-color cytometry, through direct immune fluorescence and FACSCalibur or FACSCanto cytometer (BD Biosciences), was performed.

#### **STATISTICAL ANALYSIS**

Non-parametric Mann–Whitney or Kruskal–Wallis tests were used to correlate the effect of different clinical parameters on TREC counts. Since data did not have a normal distribution, a logarithmic transformation was performed. Difference in cumulative incidence of relapse was estimated using univariate Kaplan– Meier analysis. *P*-values *<*0.05 were considered to be statistically significant. All analyses were performed using SPSS Statistics 20 Software.

## **RESULTS**

### **THYMIC FUNCTION RECOVERY IS SIMILAR AFTER HAPLO-HSCT AND UCBT IN PEDIATRIC PATIENTS**

The distribution of circulating CD3+, CD4+, or CD8+ T-lymphocyte subsets, 3 and 6 months after HSCT, was variable, especially for patients given Haplo-HSCT (**Table 2**). At month 3, CD3+, CD4+, and CD8+ cell counts/μL blood were lower in Haplo-HSCT than in UCBT patients (*p* = 0*.*003, 0.005, and 0.010, respectively). At month 6, only CD4+ cells remained significantly lower (*p* = 0*.*022) in the former group. These results confirm the detrimental role of TCD of the graft on early reconstitution of mature T cells and the role played by homeostatic expansion of T cells transferred with the graft in the case of UCBT (Roux et al., 1996).

Reconstitution of newly generated naïve T cells relies on thymic differentiation of donor-derived lymphoid progenitors. We, therefore, evaluated recovery of thymic function through the quantification of sjTREC and beta-TREC on the samples collected at the same points in which lymphocyte counts were investigated. Before the allograft, the median number of sjTREC per 150,000 PBMC were 1375 (range 0–27,612) and 2035 (range 0–16,626) for Haplo-HSCT and UCBT patients, respectively (*p* = not significant, NS). Three months after the allograft the median number of sjTREC per 150,000 PBMC had dropped to 27 (0–1973) and 45 (0–1284) for Haplo-HSCT and UCBT patients, respectively. At 6 months, although there was a great inter-patient variability, the median number of sjTREC numbers almost returned to pre-graft levels with 281 (0–31,286) and 647 (0–16,395) sjTREC/150,000 PBMC, for Haplo-HSCT and UCBT patients (**Figure 1A**), respectively. The recovery of betaTREC paralleled that of sjTREC (**Figure 1B**) without any significant difference between the two groups of patients. Accordingly, at each time points, the number of sjTREC highly correlated with the number of beta-TREC (*r* = 0*.*66, *p <* 0*.*001, Pearson correlation).

Since proliferation of memory or effector T cells could affect TREC number per PBMC by dilution, we also expressed sjTREC and beta-TREC counts per microliter of patient blood using absolute cell counts. Absolute numbers of sjTREC (**Figure 1C**) and

**Table 2 | Median number (range) of circulating T-lymphocyte subsets three (M3) and six (M6) months after HSCT (cells ×103/µl).**

*M3 stands for Month 3; M6, Month 6, and CR, Complete remission.*

beta-TREC (**Figure 1D**) followed the same kinetics as numbers of TREC/150,000 PBMC, without difference between Haplo-HSCT and UCBT patients, this ruling out an indirect, confounding effect of T-cell proliferation. Only absolute sj and beta-TREC numbers per μL will be used afterwards.

Since sjTREC value as a marker of thymic output is dependent on age (Douek et al., 1998), we also compared the median age of patients in the two groups. Haplo-HSCT patients were indeed significantly older (*p* = 0*.*0008) than UCBT patients (median age being 7.7 and 4.7 years, range 3–17 and 0.75–16 years, respectively). Thus, from the observed TREC values, we can conclude that, despite an older age, thymic reconstitution was equally effective in patients given either Haplo-HSCT or UCBT.

Since GvHD has a major impact on immune reconstitution after allogeneic HSCT, we studied its influence on both groups of patients. There were less cases of acute GvHD (aGvHD) in Haplo-HSCT patients (**Table 1**). We did not see any significant difference for sjTREC/μL of blood between patients without or with grade I aGvHD and patients with grade II and III aGvHD (**Figure 1E**), in both groups of patients. Incidence of cGvHD was low in both groups with 6 (18%) and 2 cases (8%) in the Haplo-HSCT and UCBT cohorts, respectively. TREC values were lower at month 6, in both groups, when cGvHD occurred, but the difference was not statistically significant (**Figure 1F**).

#### **PATIENTS AFFECTED BY HEMATOLOGICAL MALIGNANCIES HAVE LOWER PRE-TRANSPLANT THYMIC FUNCTION**

Diagnosis may also have an impact on recovery of thymic function (Petridou et al., 2002; Clave et al., 2005). Indeed, TREC values before transplantation were significantly lower in the 46 patients with hematological malignancies (median 10 sjTREC/μL blood, range 0–674) than in the 11 with non-malignant disorders (median, 235 sjTREC/μL blood, range 97–1340, *p* = 0*.*0002) (**Figure 2**). This finding was not influenced by the type of allograft that the patients received (data not shown). There was no significant difference in age between the two groups of patients, affected or not by hematological malignancy (median 6.6 vs. 5.0 years, range 1–9 vs. 1–17, *p* = 0*.*18) that could explain a lower thymic function in patients with malignancy. Before transplantation, patients with early disease had higher sj and beta-TREC than those with more advanced disease (**Figure 2**). Moreover, median numbers of betaTREC/μL of blood were also significantly lower in patients treated for malignancies (median 1.7 vs. 39, *p* = 0*.*004) (**Figure 2**), suggesting an effect of the disease itself or of previous treatment on the T-cell progenitor compartment or before β-chain recombination.

#### **A LOW THYMIC FUNCTION IS ASSOCIATED WITH A HIGHER RELAPSE RISK**

We have previously shown in the group of Haplo-HSCT patients that a low thymic function before transplantation or after 6 months, was associated with an increased risk of relapse (Clave et al., 2012). Here, thymic reconstitution appears similar after both UCBT and Haplo-HSCT. Thus, we decided to merge the 19 UCBT to the 27 Haplo-HSCT cases affected by malignancies. Among the 46 patients treated for malignancies, 11 relapsed: 8 after Haplo-HSCT and 3 after UCBT (see **Table 1**). The median time between transplantation and relapse was 10 months (range 1.6–18 months).

When considering both patients given UCBT and those receiving Haplo-HSCT, the median number of sjTREC/μL blood was low at 3 months and returned to the initial level at month 6. With a median follow-up of 43 months, patients who relapsed had lower values than the relapse-free patients both before transplantation (*p <* 0*.*05) and during follow-up (*p* = 0*.*014 and 0.006, at 3 and 6 months, respectively) (**Figure 3A**). Values of beta-TREC before transplantation and, even more significantly, after 6 months (**Figure 3B**) were also lower in patients who subsequently relapsed (*p* = 0*.*037 and 0.009, respectively).

We also carefully evaluated the occurrence of relapse in patients with a poor thymic function. We calculated the cumulative incidence of relapse in patients with a detectable thymic function compared to those without any thymic function (based on the detection sensitivity of the sj and beta TREC assays less than 0.1 and 0.01/150,000 PBMC, respectively). Because values at month 3 were too low to define this cut-off reliably, we studied only the values before transplantation and those at month 6 post transplantation. Absence of detectable sjTREC at each time

point correlated with an increased incidence of relapse, although this correlation was not statistically significant, due to the limited number of events (data not shown). More notably, lack of detectable betaTREC before and at month 6 after the allograft was strongly associated to a higher incidence of relapse (*p* = 0*.*03 and 0.02, respectively) (**Figures 3C**,**D**). Use of median sj or beta-TREC value to split patient groups as low or high TREC gave similar results (data not shown). We also analyzed, in patients treated for malignancies, other parameters that have been shown to influence relapse rate, but neither age (*p* = 0*.*28), high-risk disease (*p* = 0*.*73), conditioning regimen (*p* = 0*.*24) nor number of CD34+ cells infused (for Haplo-HSCT patients, *p* = 0*.*72)

had a significant impact on relapse in univariate analysis in this relatively small cohort of patients.

### **DISCUSSION**

Alternative sources of stem cells/donors are increasingly used in the treatment of children with both malignant and nonmalignant diseases in need of an allograft (Copelan, 2006). However, their use remains associated with delayed immune reconstitution and a high rate of infectious complications (Aversa et al., 1998). Using sj and beta-TREC quantification, we found that thymic function had the same pattern of reconstitution in patients that had been transplanted from either an HLA haploidentical family donor or an unrelated CB donor despite the difference in stem cell source and transplant procedure. It is known that TCD of the graft (*in vivo* or *ex vivo*) has a huge impact on early reconstitution of the lymphocyte compartment, since patients cannot benefit from the homeostatic expansion of lymphocytes transferred with the graft (Roux et al., 1996). Accordingly, we found that reconstitution of T-cell subset numbers (i.e., CD4+ and CD8+ T cell subpopulations) was faster in UCBT patients than in those given Haplo-HSCT. However, median values of beta and sjTREC were similar in both groups, this indicating that, despite TCD of the graft, a larger use of total body irradiation (**Table 1**) and an older age for Haplo-HSCT recipients, recovery of thymic function was equally effective in patients given either Haplo-HSCT or UCBT. These results also illustrate that the TREC assay provides additional and more detailed information on T-cell reconstitution, in particular on that of newly re-generated T lymphocytes which mainly accounts for the medium and longterm patient's immune competence, than crude T cell subsets counts.

We found a very limited impact of GvHD (Hazenberg et al., 2002; Storek et al., 2002; Clave et al., 2009), and especially of aGvHD, on recovery of thymic function in both groups. This could be explained by both the limited incidence of this complication in the 2 groups and by the young age of our patients. Indeed, in a previous study we reported that young age at time of the allograft correlates with a better thymic function recovery (Clave et al., 2009).

In accordance with what we and others have previously shown (Petridou et al., 2002; Chen et al., 2005; Clave et al., 2005), patients with malignant diseases had lower TREC values already before the allograft than those with non-malignant disorders. However, in those previously published studies, the groups were not matched for age (Petridou et al., 2002; Clave et al., 2005) and only the relative number of TREC was assessed (Petridou et al., 2002; Chen et al., 2005), leaving the possibility of TREC dilution due to peripheral proliferation. Here, the median age in groups of patients affected by either a malignant or a non-malignant disease was similar and the number of TREC was given for unit of blood volume. Moreover, we specifically assessed beta-TREC,

a marker of intrathymic proliferation of T-cells undergoing differentiation. Beta-TREC values were also reduced in patients with malignancies, suggesting a mechanistic impact of the disease itself or its treatment on T-cell generation before beta chain TCR recombination, i.e., at the progenitor level and/or after homing to the thymus. In this respect, previous studies indicating the lack of association between TREC and chemotherapy (Petridou et al., 2002; Chen et al., 2005), could argue for a direct impact of hematological malignancy on thymic function rather than for a role of previous treatment.

In conclusion, we have previously shown an association between low levels of sj and beta-TREC and disease recurrence in the group of 27 haplo-HSCT patients with malignancies (Clave et al., 2012). Given the similarity in thymic function recovery of these patients with those treated with UCBT, by adding to the analysis the results obtained for these patients, we could increase to 46 the total number of patient treated for malignancies. With this addition, patients who relapsed still had a lower count of sj and beta TREC (**Figures 3A**,**B**) and patients with a low value of beta-TREC, both before and at month 6 after transplantation, had a significantly higher incidence of relapse (**Figures 3C**,**D**). Since the cohort size did not allow us to perform a multivariate analysis, we cannot exclude that the effect on relapse could be due to other disease variable. However, none of the different clinical parameters we tested such as disease risk or patient age, had a significant effect on relapse in univariate analysis. Therefore, the significant correlation of beta-TREC with relapse 6 months after transplantation suggest that a post-graft thymic differentiation of naïve T cells with anti-leukemia potential could play a crucial role in preventing relapse and mediating GVL.

#### **ACKNOWLEDGMENTS**

The authors would like to thank Rita Maccario for her helpful contribution. This work was supported by research grants from AIRC (Associazione Italiana Ricerca sul Cancro, special grant 5 per 1.000) to Franco Locatelli, Cancéropôle Ile-de-France and AP-HP "contrat de recherche translationelle en biologie 2010" # RTB1002.

## **REFERENCES**

impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation. *Blood* 113, 6477–6484.

from HLA-mismatched parental donors in children. *Bone Marrow Transplant.* 27, 777–783.

siblings. *Ann. Intern. Med.* 98, 461–466.

**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.

*Received: 23 November 2012; accepted: 11 February 2013; published online: 04 March 2013.*

*Citation: Clave E, Lisini D, Douay C, Giorgiani G, Busson M, Zecca M, Moretta F, Acquafredda G, Brescia LP, Locatelli F and Toubert A (2013) Thymic function recovery after unrelated donor cord blood or T-cell depleted HLAhaploidentical stem cell transplantation correlates with leukemia relapse. Front. Immunol. 4:54. doi: 10.3389/fimmu. 2013.00054*

*This article was submitted to Frontiers in Alloimmunity and Transplantation, a specialty of Frontiers in Immunology.*

*Copyright © 2013 Clave, Lisini, Douay, Giorgiani, Busson, Zecca, Moretta, Acquafredda, Brescia, Locatelli and Toubert. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

## Monitoring of pathogen-specificT-cell immune reconstitution after allogeneic hematopoietic stem cell transplantation

## **Shigeo Fuji 1,2, Markus Kapp1 and Hermann Einsele1\***

1 Department of Internal Medicine II, Division of Hematology, University Hospital of Würzburg, Würzburg, Germany 2 Division of Hematopoietic Stem Cell Transplantation, National Cancer Center Hospital, Tokyo, Japan

#### **Edited by:**

Antoine Toubert, Université Paris Diderot, France

#### **Reviewed by:**

Attilio Bondanza, S. Raffaele Scientific Institute, Italy Jean Villard, Geneva University Hospital, Switzerland

#### **\*Correspondence:**

Hermann Einsele, Department of Internal Medicine II, Division of Hematology, University Hospital of Würzburg, Josef-Schneider-Straße 2, 97080 Würzburg, Germany e-mail: Einsele\_H@medizin. uni-wuerzburg.de

The clinical outcome after allogeneic hematopoietic stem cell transplantation (HSCT) has been significantly improved during the last decades with regard to the reduction in organ failure, infection, and severe acute graft-versus-host disease. However, severe complications due to infectious diseases are still one of the major causes of morbidity and mortality after allogeneic HSCT, in particular in patients receiving haploidentical HSCT or cord blood transplant due to a slow and often incomplete immune reconstitution. In order to improve the immune control of pathogens without an increased risk of alloreactivity, adoptive immunotherapy using highly enriched pathogen-specificT cells offers a promising approach. In order to identify patients who are at high risk for infectious diseases, several monitoring assays have been developed with potential for the guidance of immunosuppressive drugs and adoptive immunotherapy in clinical practice. In this article, we aim to give a comprehensive overview regarding current developments of T-cell monitoring techniques focusing on T cells against viruses and fungi. In particular, we will focus on rather simple, fast, non-labor-intensive, cellular assays which could be integrated in routine clinical screening approaches.

**Keywords: virus, fungi,T cell, immune reconstitution, allogeneic stem cell transplantation**

#### **INTRODUCTION**

The clinical outcome after allogeneic hematopoietic stem cell transplantation (HSCT) has been significantly improved during the last decades with regard to the reduction in organ failure, infection, and severe acute graft-versus-host disease (GVHD). These advances have rendered allogeneic HSCT to an integral part of treatment for hematological malignancies (1, 2). However, severe complications due to infectious diseases are still one of the major causes of morbidity and mortality after allogeneic HSCT, in particular in patients receiving haploidentical HSCT or cord blood transplant due to a slow and often incomplete immune reconstitution. The reduction of immunosuppressive drugs could pave the way to strengthen Tcell responses against pathogens after allogeneic HSCT. However, blind rapid tapering or cessation of immunosuppressive drugs is associated with an increased risk of alloreaction with subsequent clinical consequences such as increase of severe acute or chronic GVHD as demonstrated previously (3, 4). In order to improve the immune control of pathogens without an increased risk of alloreactivity, adoptive immunotherapy using highly enriched pathogen-specific T cells offers a promising approach. Adoptive immunotherapy against several pathogens has been already evaluated within clinical trials as reviewed previously (5).

In order to identify patients who are at high risk for infectious diseases, several monitoring assays have been developed with potential for the guidance of immunosuppressive drugs and adoptive immunotherapy in clinical practice. In this article, we aim to give a comprehensive overview regarding current developments of T-cell monitoring techniques focusing on T cells against viruses and fungi. In particular, we will focus on rather simple, fast, non-labor-intensive, cellular assays which could be integrated in routine clinical screening approaches.

#### **THE ROLE OF PATHOGEN-SPECIFIC IMMUNITY IN PREVENTION AND CONTROL OF INFECTIOUS DISEASES VIRUS-SPECIFIC T-CELL IMMUNITY**

It is well-known that virus-specific T cells are important to prevent and/or control viral infection after allogeneic HSCT. Cytomegalovirus (CMV) is one of the most intensively investigated targets of immunotherapy after allogeneic HSCT (6). After allogeneic HSCT, the first emergence of CMV reactive antigenemia triggers the expansion of donor-derived CMV-specific T cells. These expanded cells usually have a phenotype of effectoror effector-memory type. The presence of CMV-specific T cells in patients after allogeneic HSCT was reported to be protective against the recurrence of CMV antigenemia (7–12). Especially, CMV seropositive patients with profound immunosuppression or CMV seropositive patients who received stem cells from a CMV-seronegative donor are at high risk for a significant delay in reconstitution of functional CMV-specific T cell which is associated with persistent CMV viremia and a higher risk of CMV disease (8–12). Furthermore, adoptive T-cell therapy of CMV-specific T cells was demonstrated to be effective for the prophylaxis and treatment of CMV disease after allogeneic HSCT (13, 14).

The importance of virus-specific T cells has been also demonstrated with regard to other viruses such as adenovirus (15–18), EB virus (19–21), BK virus (22–25), and JC virus (26, 27). Recently, banked third party virus-specific T cells were reported to be safe and effective for the treatment of viral disease after allogeneic HSCT, which circumvents a major obstacle to the wider use of virus-specific T cells, in particular in patients after cord blood transplant (28).

The monitoring of T-cell immunity against these viruses can be useful to assess the risk of viral infections. The benefit of adoptive T-cell therapy as prophylaxis or as treatment should be ideally assessed in prospective clinical trials.

#### **FUNGUS-SPECIFIC T-CELL IMMUNITY**

For a long time fungus-specific T cells have not been regarded as important to control fungal diseases. However, there is growing evidence that CD4+ T cells provide defense mechanisms against fungal infection (29–32). The majority of patients diagnosed with invasive aspergillosis after allogeneic HSCT are not neutropenic which for a long time was considered the only or at least the most important immune mechanism to prevent fungal disease (33, 34). Recent studies have shown that fungus-specific T cells are detectable in healthy individuals and patients with hematological malignancies (29, 32, 35). Due to the paucity of clinical studies which assessed the impact of presence of fungus-specific T cells compared to virus-specific T cells, further prospective studies which assess the importance of fungus-specific T cells on preventing/controlling fungal infection are urgently needed.

In addition, the improved outcome of invasive *Aspergillus* following adoptive T-cell therapy for invasive Aspergillosis demonstrates the clinical value of transfer of fungus-specific T cells from the stem cell donor (36). Furthermore, recent reports showed that the GMP-grade-Aspergillus-specific T cells could be produced for clinical trials using commercially available enrichment protocols (37–40).

#### **HOW CAN PATHOGEN-SPECIFIC IMMUNITY BE MONITORED?**

Up to date, various methods are available to assess T-cell immunity against specific antigens. However, some methods like limitingdilution assays are not feasible due to the labor-intensive works which cannot be a part of routine clinical practice. Here we summarize three simple broadly available methods which can be performed using peripheral blood mononuclear cells (PBMC) or whole blood without long-term *ex vivo* culture, and using commercially available reagents. In addition, PBMC can be frozen without the loss of the function when tested in intracellular cytokine staining (ICS) or Enzyme-linked immunosorbent spot (ELISPOT), which is practically very important with regard to reproducibility and standardization with strict quality control (41). Combinations of these assays are needed for the confirmation of results and comprehensive measurement of different T-cell functions. The advantages and disadvantages of each method are summarized in **Table 1**.

#### **ENZYME-LINKED IMMUNOSORBENT SPOT**

Enzyme-linked immunosorbent spot is one of the most established methods to detect functional immunity (42–44). In brief, PBMC are cultured for 18–24 h on an anticytokine capture antibodycoated membrane in the presence of an antigen. Following culture, each antigen-specific T cells will release cytokines that will bind to the capture antibody on the membrane. The cells are then washed and the secreted cytokines can be detected on the membrane by use of an enzymatically labeled antibody and insoluble chromogenic substrate. In this assay, frequencies of cytokine-secreting T cells can be counted after *in vitro* stimulation of PBMC by defined antigens/peptides without previous*ex vivo* expansion. In addition, ELISPOT assays allow the size and intensity of the spots to be calculated, which correlated with the amount of cytokines secreted by each cell. As shown in **Figure 1A**, we are able to detect the induction of IFN-γ after the stimulation with CMV pp65 IE-derived peptides in patients after allogeneic HSCT.

Enzyme-linked immunosorbent spot offers several advantages: (1) many samples can be tested simultaneously using one plate; (2) the secretion of cytokines can be assessed in contrast to the

#### **Table 1 | Comparison of threeT-cell assays.**

artificially retained cytokines in ICS; (3) the cell numbers can be downscaled per well in comparison to flow cytometry-based methods.

Cytotoxic activity can be assessed using granzyme B ELISPOT. Granzyme B ELISPOT has been reported to have excellent correlation with the 51Cr-release assay for measuring cytotoxic activity of T cells (45, 46). Furthermore, multiple-color fluorospot assays make the analysis of single cells secreting several cytokines possible (47, 48). Detecting each cytokine with a different fluorophore, polyfunctionality of T cells can be analyzed, suggested to be important to protect against various infectious diseases.

The disadvantages of ELISPOT are: (1) it is difficult to determine which immune cells secrete IFN-γ. This is critical to assess the immune status after allogeneic HSCT. As Wang and Colleagues reported, the response to 9-mer peptide, which is expected to induce cytokines in an HLA class I-restricted, can also be HLA class II-restricted (49, 50). Therefore, when IFN-γ induction in ELISPOT assay is detected using stimulation with lengths of peptides including peptide-pool, cell of origin of IFNγ secretion has to be determined using CD4/CD8 depletion or HLA blocking assays; (2) sorting of cytokine-secreting cells is impossible.

#### **INTRACELLULAR CYTOKINE STAINING**

Intracellular cytokine staining is also one of the most established methods to detect functional immunity (51, 52). In brief, PBMC are cultured for 6–18 h in the presence of an antigen. To preserve the generated cytokines within the cytoplasm, a Golgiblocking agent (e.g., Brefeldin A or Momensin) is added during the stimulation. After the stimulation, samples are collected, fixated and permeabilized. Consecutively, antibodies against intracellular

cytokines are added. When surface markers whose binding is sensitive to fixation and permeabilization are stained in combination with ICS, they should be stained before fixation and permeabilization. Stained cells were analyzed using a flow cytometer. A representative result is shown in **Figure 1B**.

The advantages of ICS are as follows: (1) the phenotype of each cell which secretes the cytokine can be determined (53); (2) the cytolytic potential of the target cells can be assessed using CD107a degranulation assay in combination with the assessment of multiple cytokine induction.

The disadvantages of ICS are: (1) reagents such as Brefeldin A are required to retain cytokines in the cytoplasm; (2) the cells have to be permeabilized prior to the staining of the cells with antibodies against the cytokines, which makes it impossible to expand the sorted T cells.

#### **MHC-MULTIMER STAINING**

MHC-multimers are synthetic structures made from HLA molecules linked together to form a multimeric complex which are loaded with antigen-specific peptide. Cells stained with multimer and antibodies against surface markers can be analyzed using a flow cytometry. The fluorescence intensity using the tetramer loaded with a high-avidity peptide derived from virus is usually high enough to discriminate the positive population in contrast to the result using the tetramer loaded with a low-avidity peptide derived from autologous antigen (54). A representative result is shown in **Figure 1C**.

The advantages of multimer assays are as follows: (1) combined analysis of phenotyping and specificity can be performed using the antibodies against surface markers. MHC-multimer can detect T cells which do not secrete cytokines, for instance naïve T cells. In combination with the phenotyping and MHC-multimer staining, we can assess the frequency of all antigen-specific T cells including dysfunctional/non-functional; (2) antigen-specific T cells can be sorted with a high purity. For this purpose, the streptamer technology is demonstrated to be useful (54, 55). Sorted cells can be used for adoptive T-cell therapy as a GMP-grade agent without regulatory issues (55).

The disadvantages of multimer staining are: (1) the multimer is not able to assess the functional status of antigenspecific T cells simultaneously. There can be a discrepancy in the frequency of antigen-specific T cells detected by multimer and by ELISPOT/ICS. Several papers reported that T cells detected by ICS were more important than those detected by multimer to control infectious diseases as demonstrated in the study of CMV infection (56–58). Multimer assays can be combined with functional assays, but it is well-known that the stimulation with a respective peptide leads to loss of multimer staining due to the downregulation of T-cell

## **REFERENCES**

**24**:319–37. doi:10.1016/j.idc.2010. 01.008

receptor, in particular when a high-avidity peptide is used (59); (2) MHC-multimer staining is HLA-specific and peptidespecific. Therefore the whole cell repertoire directed against a pathogen cannot yet be determined using MHC-multimer technology.

### **CONCLUSION**

T-cell monitoring against specific targets including viruses and fungi is ready to be integrated in the clinical practice. The monitoring of pathogen-specific T cells may help to define the individual MPE of a patient to develop a certain infections complication and to assess the potential benefit of adoptive T-cell therapy against certain pathogens.

#### **ACKNOWLEDGMENTS**

I appreciate Dr. Julia Fisher and Ms. Elke Baumeister, University of Würzburg, Würzburg, Germany, and Dr. Kohei Tada, National Cancer Center Hospital, Tokyo, Japan for their technical assistance.

**116**:5476–85. doi:10.1182/blood-2010-04-259291

study demonstrates an association between JC virus-specific cytotoxic T lymphocytes and the early control of progressive multifocal leukoencephalopathy. *Brain* (2004) **127**:1970–8. doi:10.1093/ brain/awh215

diagnostics. *Methods Mol Biol* (2012) **792**:3–23. doi:10.1007/978- 1-61779-325-7\_1

Scheinberg P, Barrett AJ, et al. Techniques to improve the direct ex vivo detection of low frequency antigen-specific CD8+ T cells with peptide-major histocompatibility complex class I tetramers. *Cytometry A* (2008) **73**:1001–9. doi:10.1002/cyto.a.20642

Cellular responses to cytomegalovirus in immunosuppressed patients: circulating CD8+ T cells recognizing CMVpp65 are present but display functional impairment. *Clin Exp Immunol* (2003) **132**:96–104. doi:10.1046/j. 1365-2249.2003.02098.x

kidney transplant recipients with pp65 antigenemia. *Transplant Proc* (2008) **40**:3500–4. doi:10.1016/j. transproceed.2008.03.176

59. Appay V, Nixon DF, Donahoe SM, Gillespie GM, Dong T, King A, et al. HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function. *J Exp Med* (2000) **192**:63–75. doi:10. 1084/jem.192.1.63

**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.

*Received: 07 August 2013; accepted: 29 August 2013; published online: 17 September 2013.*

*Citation: Fuji S, Kapp M and Einsele H (2013) Monitoring of pathogen-specific T-cell immune reconstitution after allogeneic hematopoietic stem cell transplantation. Front. Immunol. 4:276. doi: 10.3389/fimmu.2013.00276*

*This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology.*

*Copyright © 2013 Fuji, Kapp and Einsele. 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) or licensor 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.*

## Alloreactivity of virus-specific T cells: possible implication of graft-versus-host disease and graft-versus-leukemia effects

## **Shigeo Fuji 1,2\*, Markus Kapp1 and Hermann Einsele1**

1 Department of Internal Medicine II, Division of Hematology, University Hospital of Würzburg, Würzburg, Germany 2 Division of Hematopoietic Stem Cell Transplantation, National Cancer Center Hospital, Tokyo, Japan

#### **Edited by:**

Antoine Toubert, Université Paris Diderot, France

#### **Reviewed by:**

Nicolas Dulphy, Universite Paris Diderot, France Eric Spierings, University Medical Center Utrecht, Netherlands

#### **\*Correspondence:**

Shigeo Fuji, Division of Hematopoietic Stem Cell Transplantation, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-Ku, Tokyo 104-0045, Japan e-mail<EMAIL_ADDRESS>
## **INTRODUCTION**

The reconstitution of functional virus-specific T cells and the importance of these T cells in the control of viral diseases following allogeneic hematopoietic stem cell transplantation (HSCT) has been extensively investigated (1). This led to a successful transfer into the clinical setting within adoptive immunotherapeutic approaches (2).

Among various viruses, immune reconstitution against cytomegalovirus (CMV) has been most intensively studied. Regarding the recovery of CMV-specific T cells after allogeneic HSCT, several reports showed that subclinical CMV antigenemia drives the reconstitution of functional CMV-specific T cells (3–5). The rapid homeostatic expansion of CMV-specific T cells suggests that such T cells might be largely memory T cells, considering the insufficient regeneration of T cells due to the poor thymic function early after allogeneic HSCT. Similar to the immune response after primary CMV infection (6, 7), the proportion of virus-specific T cells including CMV-specific T cells can be high and in some cases CMV-specific T cells make up more than 10% of the circulating T cells after allogeneic HSCT (8, 9). If the number and/or functionality of CMV-specific T cells is insufficient in recipients of an allogeneic stem cell graft, they are at high risk of persistent viremia and CMV disease (10, 11).

Although immune reconstitution of virus-specific T cells has been intensively studied, the fact of possible alloreactivity of virusspecific T cells in the allograft recipient has only been evaluated in few trials (12, 13). However, as suggested in other fields like organ transplantation, virus-specific T cells are assumed to play a role in alloreactivity similarly in the field of allogeneic HSCT (14, 15).

In this review, we focus on crossreactivity of virus-specific T cells against allogeneic targets, and discuss the possible implication of such reactions on the allogeneic immune responses after allogeneic HSCT.

Immune reconstitution of functional virus-specific T cells after allogeneic hematopoietic stem cell transplantation (HSCT) has been intensively investigated. However, the possible role of crossreactivity of these virus-specific T cells against allogeneic targets is still unclear. Theoretically, as in the field of organ transplantation, virus-specific T cells possess crossreactivity potential after allogeneic HSCT. Such crossreactivity is assumed to play a role in graft-versus-host disease and graft-versus-leukemia effects. In this article, we aim to give a comprehensive overview of current understanding about crossreactivity of virus-specific T cells.

**Keywords: allogeneic stem cell transplantation, virus-specificT-cell, GVHD, HLA antigens, GVL, mismatch**

#### **CROSSREACTIVITY OF VIRUS-SPECIFIC T CELLS**

Virus-specific T cells, which dominate the memory pool in humans, have been reported to have the potential of crossreactivity (14). Crossreactivity of T cells is the ability to recognize several different peptide/human leukocyte antigen (HLA) complexes. CD4+/CD8+ and naïve/memory T cells were shown to mediate crossreactivity against allogeneic targets (16, 17). Upon activation, memory T cells proliferate more quickly and produce more cytokines than naïve T cells (18, 19). Considering these rapid and vigorous T-cell responses mediated by memory T cells when compared to naïve T cells, one could assume that such alloreactivity of virus-specific memory T cells could play a role in the pathogenesis of early-onset acute GVHD, in particular hyperacute GVHD, following HLA mismatched HSCT. The difference of these T cells in the ability to expand and express cytotoxic molecules might also contribute to the difference in the outcome in patients with acute GVHD. Virus-specific T cells might have much higher avidity against allogeneic targets after HLA mismatched HSCT when compared to that after HLA-matched HSCT, considering the mechanism of negative selection in thymus (20, 21). Virusspecific T cells possessing high avidity against autologous HLA molecules with a self-peptide should originally be deleted in thymus. However, if virus-specific T cells recognize the complex of a peptide and non-autologous HLA molecule via their T-cell receptor, the avidity of T cells against this complex can be high because this HLA molecule is not expressed in the thymus and thus cannot induce the negative selection in the thymus (22). One hypothesis could be that, if such strong peptide-specific crossreaction exists against tumor-associated antigen (TAA)-derived peptides expressed in leukemia cells accidentally, it should lead to a strong graft-versus-leukemia (GVL) effect. However, there is no data available so far which could support this idea. Furthermore, crossreactivity against non-self HLA presenting a non-polymorphic hematopoietic cell-specific peptide or TAA-derived peptide might provide therapeutic tools for immunotherapy, similar to the concept for minor histocompatibility antigens like HA-1.

In addition, virus-specific T cells which have weak to moderate avidity against autologous HLA molecules with a self-peptide might theoretically remain *in vivo*, considering the mechanism of positive selection in thymus. Such virus-specific T cells might subsequently exert alloreaction in the setting of HLA-matched HSCT, only when the strong activating signals by various cytokines stimulate them (20, 21). However, such crossreactivity by virusspecific T cells against autologous HLA molecules has not yet been demonstrated so far.

Regarding crossreactivity of virus-specific T cells, Epstein–Barr virus (EBV)-specific T cells have been studied in detail (14). Burrows et al. demonstrated crossreactivity of EBV EBNA3A-specific T-cell clones for the immunodominant peptide FLRGRAYGL presented on HLA-B\*08:01 against the alloantigen HLA-B\*44:02 (23). This finding was reconfirmed by other researchers (24, 25). These reports did not demonstrate the requirement of a specific peptide presented on HLA molecule for crossreactivity. Later on, it has been demonstrated that crossreactivity of this EBV EBNA3Aspecific T-cell clones is dependent on the presentation of the self-peptide derived from the ABCD3 gene (EEYLQAFTY) (26). Here, we have to point out the significant difference between the two peptides' sequences, suggesting that crossreactivity does not necessarily require a homology in sequences indicating promiscuity of the T-cell receptor. Another EBV EBNA3A-specific T-cell clone, recognizing the complex of HLA-B\*08:01 and an EBNA3Aderived peptide FLRGRAYGL, has been shown to react with the complex of HLA-B\*35:01 and the self-peptide derived from Cytochrome P450 (KPIVVLHGY) (22). This study demonstrated a similar avidity of the EBNA3A-specific T-cell clone against the complex of HLA-B\*08:01 with EBNA3A-derived peptide and the complex of HLA-B\*35:01 with Cytochrome P450-derived peptide.

Regarding other viruses, Amir et al. reported that crossreactivity of virus-specific T cells against mismatched foreign allogeneic HLA was common (25). They used expanded T cells which were isolated using various combinations of tetramers loaded with a virus-derived immunogenic peptide. The target cells were a panel of lymphoblastoid cell lines (LCL) expressing various combinations of HLA molecules. A major finding was that a large number of virus-specific T-cell clones have crossreactivity potential against various HLA molecules. It is also important that some CD8 T-cell clones showed crossreactivity against HLA class II, even though most CD8 and CD4 T cells were crossreactive against HLA class I and class II molecules, respectively (25). A similar phenomenon showing the recognition of HLA class II by CD8 T cells was also reported by Rist et al. (27).

However, in a clinical trial using expanded virus-specific cytotoxic T-cell lines for the treatment of viral diseases, GVHD was rarely (6.5%) observed even when crossreactivity of expanded virus-specific T cells was observed *in vitro* (13). The fact of rarely observing GVHD clinically in expanded virus-specific T cells in this study might be caused by the absence of the correct crossreactive HLA molecule in the mismatch combinations, the difference in homing capacities and the lack of respective target molecules on the GVHD-target organs. Therefore, the clinical relevance of crossreactivity which was detectable *in vitro* should be further clarified in clinical trials.

Previously, it has been assumed that crossreactivity against allogeneic HLA is independent of the peptides in the HLA groove but that the allogeneic mismatched HLA molecules are the target of this cross-reactivity. In contrast, recent reports support the idea that crossreactivity against allogeneic HLA is peptidedependent as reviewed previously (28). Actually, it is still difficult to demonstrate non-peptide-dependency experimentally, because even transporter-associated with antigen processing (TAP) deficient cell lines, which were believed to be completely deficient in antigen processing- and antigen presenting-capability, are able to load endogenous peptides on HLA molecule (28). Weinzierl et al. have demonstrated the presentation of many peptides by HLA molecules on the TAP-deficient cell line (29).

Peptide-specificity of alloreactive T cells is also supported by tissue/cell type-specific alloreactivity of clinical samples. Various reports showed the presence of tissue-specific alloreactive T cells in patients with graft failure after organ transplant (30– 33). Deckers et al. have reported that the cytotoxic potential of graft-infiltrating CD8+ T cells against proximal tubular epithelial cells (PTEC), gonadal vein endothelial cells (GOVEC), and splenocytes depends on the clone of graft-infiltrating CD8+ T cells in renal allografts (30, 31). In this report (30), 46 graftinfiltrating CD8+ T cells were cloned. Out of 46 clones, 7 lines recognized PTEC but not splenocytes derived from the same donor. Thirty lines recognized PTEC and splenocytes equally. One line preferentially recognized splenocytes over PTEC. Eight lines were not cytotoxic either to PTEC or to splenocytes. Therefore, each clone recognized different targets of the recipient. Jutte et al. also showed the specific cytotoxicity against heart endothelial cells by expanded graft-infiltrating T cells in heart allografts (32, 33). Other reports using virus-specific T cells also support the idea of cell type-specific crossreactivity of virus-specific T cells. For example a VZV-IE62-specific HLA-A2 restricted T-cell clone recognizes allogeneic HLA-B\*57:01-expressing LCLs, phytohemagglutinin (PHA) blasts,and monocyte-derived dendritic cells (DCs), but does not recognize HLA-B\*57:01-expressing B-cells, T cells, monocytes nor fibroblasts in a standard 51Cr release assay. Such tissue/cell type-specific crossreactivity has been also reported by D'Orgogna et al. showing that allogeneic HLA-B\*44:02-positive PTECs and human umbilical vein endothelial cells (HUVECs) are poor targets for EBV EBNA3A-specific T cells due to the lack of EEYLQAFTY peptide presentation (34). Amir et al. also reported that certain cell types with the correct HLA mismatch were recognized by virus-specific T cells while other cell types were not (25).

Regarding T cells in a patient with acute GVHD after HLA mismatched HSCT, single-peptide specificity was documented using a single small hairpin RNA (shRNA) system (12). There is no data available for chronic GVHD. The concept of tissue-specific alloreactivity also might be applied to GVHD following allogeneic HSCT which is also restricted to a few organs (especially skin, gut, and liver), even though other factors such as proinflammatory environment caused by the conditioning regimen affect the specificity of target organs. Tissue damage could change the expression of genes as well as the expression of HLA molecules. Furthermore, the effects of cytokine/chemokine are expected to differ among different organs.

#### **CLINICAL DATA SUGGESTING ALLOREACTIVITY AND VIRAL INFECTION**

Over the last decades, various retrospective studies have shown the possible association between viral infection and graft rejection/GVHD (15, 35). Although there is no published data assessing the impact of CMV prophylaxis on the incidence of GVHD, prospective studies assessing the impact of CMV prophylaxis using ganciclovir in organ transplantation have demonstrated a reduced risk of graft rejection in the group receiving prophylactic CMV therapy, which supports the idea that CMV infection can be associated with an increased risk of graft rejection (35).

One recent report evoked the attention of researchers on the importance of CMV infection regarding the effects on the GVL effect in allogeneic HSCT (36). In this study, Elmaagacli et al. demonstrated a significant association between early CMV reactivation and a reduced risk of relapse in acute myeloid leukemia (AML) patients after allogeneic HSCT. Patients who developed early CMV replication detected by pp65 antigenemia assay had a significantly lower risk of relapse compared with those without early CMV replication. Another group reported a similar finding in patients with chronic myeloid leukemia (CML) (37). A very recent report from Fred Hutchinson Cancer Research Center also supported the hypothesis that early CMV reactivation in AML but not in other diseases including CML may be associated with a reduced risk of relapse, even though the impact was much less in this study compared to the previous report (38). Regarding the difference among the diseases, it is potentially due to the difference in epitopes expressed on HLA molecules. The identification of target molecules recognized by virus-specific T cells might give us a clue to this issue. In contrast to these trials (36–38), persistent CMV antigenemia was associated with a poor clinical outcome possibly due to the fact that the impaired immune status is also associated with an insufficient GVL effects by functional T cells (39).

Previously, Parkman and colleagues reported that the presence of immune response to herpes viruses was associated with a reduced risk of relapse in patients with acute leukemia after cord blood transplantation, which led to a better progression-free survival (40). In this study, there was no association between the absolute count of lymphocytes and the presence of an antigenspecific immune response. Interestingly, neither acute nor chronic GVHD had any significant impact on the likelihood of leukemic relapse, suggesting that virus-specific T cells specifically induced GVL effects and graft-versus-host reaction. Hoegh-Petersen et al. also showed the significant impact of herpes virus-specific T cells at 56 days after HSCT on the incidence of subsequent relapse (41). In this study, in patients without relapse, functional T cells against various viral antigens including BZLF1 and EBNA3 were detected. There is a possibility that anti-viral immunity may be just a surrogate factor for the immunocompetence of the recipient after allogeneic HSCT and thus not have a direct causal relation with GVHD/GVL but being an epiphenomenon of other factors such as inflammation, cytokine storm, and so on. Thus, more detail about the crossreactivity of virus-specific T cells should be clarified in the

setting of allogeneic HSCT. A better understanding of their role in alloreactivity will help to reduce acute and chronic GVHD but also to mediate the important GVL reactivity by more sophisticated immunosuppressive strategies, which makes allogeneic HSCT still the most effective form of immunotherapy – allowing to cure patients with hematological malignancies which are incurable by any other form of treatment.

#### **CONCLUSION**

Virus-specific T cells can recognize and target allogeneic HLA in a peptide-dependent manner. In an HLA mismatched HSCT, the avidity of such crossreactivity can be theoretically high enough to exert clinically meaningful alloreaction. Furthermore, in an HLAmatched HSCT, there is a possibility that virus-specific T cells develop alloreaction, even if virus-specific T cells have only low to intermediate avidity against autologous targets, considering the high frequency of virus-specific T cells and the unique milieu of cytokine storm after a conditioning regimen. Furthermore, intensive prophylaxis of virus infection after allogeneic HSCT might be beneficial to reduce the incidence of GVHD similar to that after organ transplantation because such intervention could reduce the amount of antigen exposure, which is expected to decrease the expansion of donor-derived virus-specific T cells (**Figure 1**).

In conclusion, elaborate basic and clinical research to clarify the detail of crossreactivity of virus-specific T cells after allogeneic HSCT is warranted.

## **REFERENCES**

transfer of adaptive immunity to CMV during hematopoietic stem cell transplantation is dependent on the specificity and phenotype of CMV-specific T cells in the donor. *Blood* (2009) **114**: 5071–80. doi:10.1182/blood-2009- 04-214684

glycoprotein-specific CD4 cytotoxic T lymphocytes: implications for graft-versus-host disease. *Blood* (2005) **105**:1362–4. doi:10.1182/blood-2004-07-2602

human cytomegalovirus replication after transplantation is associated with a decreased relapse risk: evidence for a putative virusversus-leukemia effect in acute myeloid leukemia patients. *Blood* (2011) **118**:1402–12. doi:10.1182/ blood-2010-08-304121

*Blood* (2013) **122**(7):1316–24. doi:10.1182/blood-2013-02- 487074

(2006) **12**:919–27. doi:10.1016/j. bbmt.2006.05.008

41. Hoegh-Petersen M, Sy S, Ugarte-Torres A, Williamson TS, Eliasziw M, Mansoor A, et al. High Epstein-Barr virus-specific T-cell counts are associated with near-zero likelihood of acute myeloid leukemia relapse after hematopoietic cell transplantation. *Leukemia* (2012) **26**:359–62. doi:10.1038/leu.2011.195

**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.

*Received: 03 September 2013; accepted: 29 September 2013; published online: 14 October 2013.*

*Citation: Fuji S, Kapp M and Einsele H (2013) Alloreactivity of virus-specific T cells: possible implication of graft-versushost disease and graft-versus-leukemia effects. Front. Immunol. 4:330. doi: 10.3389/fimmu.2013.00330*

*This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology.*

*Copyright © 2013 Fuji, Kapp and Einsele. 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) or licensor 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.*

## Immune reconstitution and graft-versus-host reactions in rat models of allogeneic hematopoietic cell transplantation

#### **Severin Zinöcker 1,2\*, Ralf Dressel 3 , Xiao-NongWang4 , Anne M. Dickinson4 and Bent Rolstad1**

1 Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway

#### **Edited by:**

Antoine Toubert, Université Paris Diderot and Assistance Publique-Hôpitaux de Paris, France

#### **Reviewed by:**

Philippe Saas, Etablissement Français du Sang, France Pascale Loiseau, Assistance Publique-Hôpitaux de Paris, France

#### **\*Correspondence:**

Severin Zinöcker, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, MSC 8180, Rockville, MD 20852, USA. e-mail<EMAIL_ADDRESS>
Allogeneic hematopoietic cell transplantation (alloHCT) extends the lives of thousands of patients who would otherwise succumb to hematopoietic malignancies such as leukemias and lymphomas, aplastic anemia, and disorders of the immune system. In alloHCT, different immune cell types mediate beneficial graft-versus-tumor (GvT) effects, regulate detrimental graft-versus-host disease (GvHD), and are required for protection against infections. Today, the "good" (GvT effector cells and memory cells conferring protection) cannot be easily separated from the "bad" (GvHD-causing cells), and alloHCT remains a hazardous medical modality. The transplantation of hematopoietic stem cells into an immunosuppressed patient creates a delicate environment for the reconstitution of donor blood and immune cells in co-existence with host cells. Immunological reconstitution determines to a large extent the immune status of the allo-transplanted host against infections and the recurrence of cancer, and is critical for long-term protection and survival after clinical alloHCT. Animal models continue to be extremely valuable experimental tools that widen our understanding of, for example, the dynamics of post-transplant hematopoiesis and the complexity of immune reconstitution with multiple ways of interaction between host and donor cells. In this review, we discuss the rat as an experimental model of HCT between allogeneic individuals. We summarize our findings on lymphocyte reconstitution in transplanted rats and illustrate the disease pathology of this particular model. We also introduce the rat skin explant assay, a feasible alternative to in vivo transplantation studies. The skin explant assay can be used to elucidate the biology of graft-versus-host reactions, which are known to have a major impact on immune reconstitution, and to perform genome-wide gene expression studies using controlled combinations of minor and major histocompatibility between the donor and the recipient.

**Keywords: hematopoietic stem cell transplantation, graft-versus-host disease, rodentia, animal models, skin explant assay**

#### **HEMATOPOIETIC CELL TRANSPLANTATION IN HUMANS AND ANIMALS**

At present, approximately 25,000 allogeneic hematopoietic cell transplantations (alloHCT) are performed each year worldwide, and the total number of such clinical procedures is predicted to rise in the future (Savani et al., 2011). AlloHCT can give rise to considerable toxicity and negative side-effects resulting in relatively high morbidity and mortality with poor quality of life and 5-year survival rates of 40–60% overall. It is however the only curative therapy in patients with relatively severe and advanced diseases for whom other treatment options are limited.

To date, alloHCT is complicated by opportunistic infections, disease relapse and graft-versus-host disease (GvHD). GvHD is an immunological condition that is caused by the reactivity of allogeneic T cells transferred in the stem cell graft from a genetically related or unrelated donor. After activation through recognition of disparate host antigens, alloreactive cells exert pathological damage via cytotoxic mechanisms and cytokine release

on various organs and tissues, primarily the liver, gut, and skin, of the immunocompromised transplant recipient (Ferrara et al., 2008). In the clinical setting, relatives are favored over unrelated volunteer stem cell donors because of genetic similarity with the patient, reducing the overall risk of transplant failure and adverse graft-versus-host reactions (GvHR).

Immunity is deficient after alloHCT due to underlying disease, pre-transplant conditioning, or both, and it takes weeks to several months or years for the patient's immune system to recover (Storek, 2008; Seggewiss and Einsele, 2010; Bosch et al., 2012). Early after transplantation, the reconstitution of immune cells in lymphoid organs is inhibited: the primary sites of white and red blood cell generation, bone marrow and thymus, are damaged by chemotherapy and radiation conditioning, and they are targeted by GvHD (Krenger and Holländer, 2008; Shono et al., 2010). Furthermore, immune function is impaired in elderly patients (Krenger et al., 2011) as both the regeneration of hematopoietic cells in the bone marrow and the thymic

output of T lymphocytes decrease with age (Dorshkind et al., 2009).

Deficient and delayed restoration of immunity post-transplant contributes to the patient's susceptibility to opportunistic infections, recurrence of the original disease and occurrence of secondary malignancies (Toubert et al., 2012). The individual contributions of natural killer (NK) cells, CD8+ cytotoxic T cells, CD4+ helper T cells, B cells, and innate immune cells, and their combined effects on these complications are still not fully understood (Geddes and Storek, 2007; Bosch et al., 2012).

Moreover, T cell depletion of the graft, when applied to reduce the risk of GvHD and essential in haploidentical alloHCT between family members (Kolb, 2008), compromises immune reconstitution, illustrating that grafted "passenger" T cells are needed for successful engraftment. Depletion of T cells also abrogates the graft-versus-tumor (GvT) effect, as GvH and GvT effects are strongly correlated and transplanted donor T cells can either mediate beneficial GvT effects or cause GvHD (Appelbaum, 2001). The crucial question of how to avoid detrimental GvHR without compromising the curative potential of alloHCT through a strong GvT effect has yet to be solved by clinical hematologists and HCT researchers.

#### **THE USEFULNESS OF ANIMAL MODELS IN STUDYING HEMATOPOIETIC CELL TRANSPLANTATION AND GRAFT-VERSUS-HOST DISEASE**

The wish to better understand the dynamic biological processes between graft and host following transplantation, with the overall aim to improve the clinical management of alloHCT, has driven research in the development of adequate laboratory methods and animal models. The use of such models in transplantation research has contributed substantially to our current understanding of the nature of GvHR and its underlying principles in GvHD (Welniak et al., 2007; Socié and Blazar, 2009). Experimental alloHCT using mice and dogs have been particularly instrumental for the discovery of a variety of new therapies and for testing their feasibility, toxicity, and efficacy (Ferrara et al., 2008; Socié and Blazar, 2009).

Inbred rodent strains can be systematically established and maintained, yielding genetically homogeneous and biologically well-characterized strains for experimentation. Experiments in live animals facilitate and accelerate, for example, the study of genetic effects and mapping of gene associations with observable pathological phenotypes. An array of murine experimental models that employ donor and host strain combinations designed for mismatches only in the major histocompatibility complex (MHC) gene region (*H-2* in the mouse), only in minor histocompatibility antigens, or both, are available for the study of immune reconstitution and GvHD (Schroeder and DiPersio, 2011). Fully incompatible strain combinations, such as the popular [C57BL/6 (*H-2b* )→Balb/C (*H-2d* )] mouse model, are most often applied. Combinations of mice strains that are MHC matched but differ in minor histocompatibility antigens,for example [B10.D2 (*H-2d* )→Balb/C (*H-2d* )], [DBA/2(*H-2d* )→B10.D2 (*H-2d* )], or [C3H.SW(*H-2b* )→C57BL/6 (*H-2b* )] transplantation models (Korngold and Sprent, 1987), can mimic fully human leukocyte antigen (HLA) matched HCT between unrelated individuals, but not semi-HLA compatible HCT between sibling pairs. The latter scenario of haploidentical HCT between first-degree relatives can be modeled by inter-crossing two inbred strains and using the resulting hybrid progeny of the F1 generation as recipients of a transfusion of parental leukocytes in so-called parent-into-F1 models.

The mouse is historically and presently the most widely used model in biomedical research. However, other animal models have the potential to complement, validate, and extend the findings from experiments on mice. Studies of alloHCT in dogs make a case in point as they have added important knowledge in this field of research (Storb and Thomas, 1985), e.g., the use of delayed donor lymphocyte infusions to induce a GvT reaction and non-myeloablative ("reduced intensity") conditioning regimens to reduce toxicity and morbidity in patients. Murine models have been employed to test non-myeloablative conditioning regimens with reduced irradiation intensity and/or combination chemotherapy (fludarabine, busulfan, cyclophosphamide, etc.; Santos and Owens, 1969; Ruggeri et al., 2002; Sadeghi et al., 2008). Similarly, rat alloHCT models based on chemotherapeutic conditioning without the need for irradiation have been developed (Santos and Owens, 1966; Tutschka and Santos, 1975; Okayama et al., 2004).

Rats offer all the advantages of a rodent experimental model, such as short generation time and cost effectiveness. Rats are bigger than mice (about 10-fold in body weight) and thus yield more grams of biological material per animal and per cage unit that can be sampled in the course of experimentation. Their spleens, lymph nodes, efferent lymph, and peripheral blood contain lymphocytes that can be readily harvested and serve as organ sources of donor grafts for transplantation. Hematopoietic stem cells are commonly obtained from the marrow of large bones (e.g., femurs and tibiae). Cell grafts enriched in Sca-1+ and CD34+ hematopoietic stem cells that are being used in patients and experimentally in mice have not been available for rat models due to the lack of specific antibody markers for targeted cell purification.

Many genetically defined and well-characterized inbred rat strains are available for research in transplantation immunology, and a range of rat alloHCT models have been developed from these strains during the past decades. Especially the strains *Brown-Norway* (BN) and *Lewis* (LEW) are widely used for fully MHC mismatched alloHCT (Santos and Owens,1966;Clancy et al.,1976; Pakkala et al., 2001; Okayama et al., 2004; Zhu et al., 2011; Lin et al., 2012). Also HCT between haploidentical parental and filial generations, e.g., transplantation of LEW or BN bone marrow into F1 (BN × LEW) recipients, has been modeled in the rat (Clancy et al., 1983; Kimura et al., 1995; Ohajekwe et al., 1995; Peszkowski et al., 1996; Vaidya et al., 1996; Goral et al., 1998; Kobayashi et al., 1998; Sasatomi et al., 2005; Wolff et al., 2006; Kitazawa et al., 2012).

The MHC is the dominant genomic region that governs mutual tolerance, rejection, and GvHR between the donor and the host in alloHCT. The mouse and rat MHC regions are closely related and share overall similarity with the human MHC (*HLA*; Kelley et al., 2005a), however, some important structural differences exist. In particular, the rat MHC (*RT1*) differs from the mouse MHC region in that it encompasses a large number of polymorphic nonclassical MHC class I genes which serve as potential ligands for NK cell receptors (Vance et al., 1998; Naper et al., 2002; Naper et al., 2005; Kveberg et al., 2010; Andrews et al., 2012). In addition, the rat genome encodes a broader spectrum of inhibitory and activating Ly49 receptors expressed by NK cells compared to the mouse (Kelley et al., 2005b). Therefore, alloreactive rat NK cells and NK cell subsets may behave differently than mouse NK cells in the context of transplantation. Transplanted donor-versus-recipient NK cells mediate both GvT and GvH effects in patients (Ruggeri et al., 2002). They are thus a key element in the risk of leukemia relapse after haploidentical alloHCT (Velardi, 2012) and could influence T cell alloreactivity and the development of GvHR. The considerable differences especially in the NK cell system even between closely related species like rat and mouse illustrate the importance of relying on more than one experimental animal model in HCT research.

The complete genome sequence of *Rattus norvegicus* has been resolved in 2004 (Rat Genome Sequencing Project Consortium, 2004). With the advent of commercial cloning technology for rats (Huang et al., 2011) this species will likely be applied more frequently as a study object in the future. In the following sections, we will discuss some contributions by which rat models have helped to advance our understanding of immune reconstitution and GvHR following alloHCT.

#### **THE RAT AS AN IN VIVO MODEL OF IMMUNE RECONSTITUTION AND GRAFT-VERSUS-HOST REACTIONS AFTER HEMATOPOIETIC CELL TRANSPLANTATION**

To study the role of GvHR on immune reconstitution *in vivo*, we employed experimental transplantation protocols using combinations of rat strains with defined genetic incompatibilities (Engh et al., 2001; Nestvold et al., 2008; Zinöcker et al., 2012). The genetic composition regarding the MHC region of relevant rat strains are listed in **Table 1**. For experiments on fully incompatible (mismatched at the MHC region as well as non-MHC genes) alloHCT, we employed a model using *Piebald Virol Glaxo* (PVG) rats as the donor strain and BN rats as recipients of transplants. Conveniently, the genetic make-up of the donor PVG.7B strain encodes an allele (the RT7.2 allotype) of the CD45 gene (Kampinga et al., 1990), which facilitates later detection of donor-derived cells distinguishable from host-derived cells by flow cytometry using

**Table 1 | MHC haplotypes of inbred congenic and recombinant rat strains.**

allotype-specific antibody. The genetic background of the PVG.7B strain is identical with the original PVG strain; both carry the *c* haplotype of the rat MHC, i.e., *RT1c* (**Table 1**). The recipient BN strain has a different genetic background and carries the *n* haplotype of *RT1*, *RT1n* .

The reconstitution of blood and immune cells, especially of T lymphocytes, has been extensively studied in patients (Holland et al., 2008; Toubert et al., 2012), however, the mechanisms behind reconstitution and immunodeficiency and the complex interactions between donor and host cell types in this context remain incompletely understood. Many useful insights into the underlying cellular and molecular processes have been gained from experiments done mainly in mice. Immune reconstitution and post-transplant immune responses have been characterized also in the laboratory rat to some detail, facilitating the design of new transplantation protocols (Nestvold et al., 2008). The main rat blood cell types and cell subsets with relevance to HCT and their standard phenotypic characterization are summarized in **Table 2**.

One important aim of our studies was to describe in detail the reconstitution of the lymphocytes that mediate GvT and GvH effects, i.e., T cells and NK cells, in bone marrow recipients and in experimental GvHD. The experimental protocol used in these studies (Zinöcker et al., 2011a; adapted from Nestvold et al., 2008) is schematically outlined in **Figure 1**. Recipient rats received a lethal dose of 900 cGy total body irradiation from a Cesium-137 source emitting β and γ irradiation at an approximate rate of 4 Gy min–1. This conditioning regimen made recipient rats susceptible to donor engraftment. Donor bone marrow containing hematopoietic stem cells was obtained from leg bones (femurs and tibiae). The graft consisted of mononuclear bone marrow cells that were depleted of T cells by negative selection using magnetic beads with anti-αβTCR and anti-CD5 antibodies (cf.**Table 2**) prior to transplant (Nestvold et al., 2008). Thus, the primary graft served to establish stable donor engraftment. In this model, the transfer of donor-versus-recipient reactive NK cells (Løvik et al., 1995) contained in the graft should facilitate engraftment and donor reconstitution. GvT effects can be studied after injecting BN acute myelocytic leukemia cells, a model of human acute myeloid leukemia (Martens et al., 1990), into BN recipients (Nestvold et al., 2008). Transplanted rats were convalescent for 14 days before

**Table 2 | Immunophenotype of rat leukocyte populations.**

Phenotypic markers of rat leukocyte populations and subpopulations are summarized together with available antibodies commonly used for their flow cytometric detection. Comprehensive lists of rat antigens and specific antibodies have been published by Puklavec and Barclay (2001) and van den Berg et al. (2001). A list of OX antibodies can be found online at http://users.path.ox.ac.uk/∼ciu/mrcmabs.html

aNo CD designation is assigned for the cognate antigen.

**in the MHC mismatched rat model. (A)** BN rats served as recipients and PVG.7B rats as bone marrow and lymph node donors for bone marrow transplantation (BMT) and donor lymphocyte infusion (DLI), respectively, in fully MHC-incompatible alloHCT performed as shown in **(B)**. Recipient rats were lethally irradiated (900 cGy), rescued by an intravenous injection of donor bone marrow cells and 14 days later received a graded dose of donor lymph node cells to invoke fatal GvHD. Controls received sham treatment (injection without cells) instead of DLI. Blood samples were collected in weekly intervals from the day of DLI for subsequent analysis of reconstitution and chimerism.

GvHD was induced in a cell-dose dependent manner by a single infusion of donor lymphocytes (DLI; cf. **Figure 1**) from lymph nodes.Alternatively,GvHD can be invoked by transfusion of donor cells derived from the spleen or peripheral blood, and the latter source is highly relevant with regard to clinical GvHD, which is commonly caused by the transfer of T cells contained in a donor blood graft or donor lymphocyte transfusion.

#### **T cells, NK cells, and NKT cells**

With respect to lymphocyte reconstitution in recipients of clinical HCT, NK cells recover relatively swiftly (weeks) and T cell reconstitution happens more slowly (months to years; Storek et al., 2008). In our hands, both NKT and NK cells (as defined by CD3 and NKR-P1 surface expression; cf. **Table 2**) of donor origin were rapidly generated in animals transplanted with T cell-depleted bone marrow. NKT and NK cells of host origin were not detected in the circulation 2 weeks after HCT (Zinöcker et al., 2011a). Donor T cells showed a significantly slower pace of reconstitution in these animals, and reached approximately 90% donor chimerism in the blood only several months after transplant (Zinöcker et al., 2011a). Thus, the overall patterns of NK, NKT, and T lymphocyte reconstitution were similar to those observed in patients, albeit with accelerated kinetics as might be expected from a relatively short-lived rodent species.

Donor CD4+ T cells showed a tendency to reconstitute slightly more slowly than did donor CD8+ T cells (Zinöcker et al., 2011a), which is also comparable with known clinical data (Seggewiss and Einsele, 2010). Rats that received an infusion of donor lymph node cells and subsequently developed GvHD displayed full donor chimerism of CD4+ and CD8+ T cells shortly after DLI, coinciding with the onset of disease symptoms. This increase very likely reflected the activation by allo-antigens and rapid expansion of donor CD4+ and CD8+ T cell clones in the host. Any remaining host T cells were probably cleared by alloreactive donor cells.

#### **Memory T cells**

We also noted that donor-derived CD62L-expressing naive T cells were less frequent in the peripheral blood of rats that developed GvHD compared with healthy bone marrow-transplanted controls (Zinöcker et al., 2011a). This finding may likely be due to the loss of CD62L surface expression in the course of T cell activation as measured by the concomitant increase of CD25 and CD134 activation markers on CD4+ and CD8+ T cell populations (cf. **Table 2**). Although we did not specifically investigate a memory T cell phenotype in our study, we speculate that donor memory T cells may expand in the presence of host allo-antigens and allo-activation as CD62L− T cells were more frequent in the circulation in rats with GvHD. CD44+ CD62L− memory T cells that are generated in response to pathogens during infection as well as other antigens have been reported not to cause GvHD (Anderson et al., 2003; Chen et al., 2004). In line with these studies, Xystrakis et al. (2004a) have employed a parent-into-F1 HCT model and showed that CD4+ CD45RC*lo* T cells containing mainly memory T cells, in contrast to CD4+ CD45RC*hi* T cells containing mainly naive T cells, failed to induce acute or chronic GvHD in recipient rats. Conversely, transplanted memory T cells not specifically primed to allo-antigens can have a considerable potency to induce GvHD (Zheng et al., 2009). Therefore, the capacity of differentiated memory T cells to elicit a GvHR may depend on the nature of their primary antigen encounter.

#### **Regulatory T cells**

Furthermore,we found that host-derived FoxP3-expressing CD4+ CD25+ T cells, comprising a regulatory T cell subtype (Beyersdorf et al., 2005), reconstituted relatively quickly and were present at normal levels in peripheral blood at 4 weeks post-transplant (Zinöcker et al., 2011a). It has been shown previously in mice that recipient CD4+ CD25+ T cells can resist irradiation and regulate chronic GvHD (Anderson et al., 2004). Donor-type FoxP3+ regulatory T cells, on the other hand, did not amount to significant numbers in the blood of transplanted rats at any time point post-transplant. Strikingly, and in contrast to transplanted controls which did not receive DLI, rats that suffered from GvHD failed to reconstitute FoxP3+ regulatory T cells completely. We speculate that the absence of regulatory T cells is a major contributing factor in the lack of ability of these animals to avert alloreactivity as they uniformly developed lethal GvHD. Another regulatory T cell subtype that was defined as CD8+ CD45RC*lo* FoxP3+ in the rat (cf. **Table 2**) suppressed alloreactive donor CD4+ T cells*in vitro* and prevented experimental GvHD *in vivo* (Xystrakis et al.,2004b). Decreased FoxP3 expression (Miura et al., 2004) and frequency of regulatory T cells (Zorn et al., 2005; Rieger et al., 2006; Magenau et al., 2010) has been associated with the incidence of GvHD in patients. Furthermore, CD4+ CD25+ regulatory T cells have been shown to suppress host-reactive T cells and reduce acute GvHD in murine models (Cohen et al., 2002; Hoffmann et al., 2002; Taylor et al., 2002; Edinger et al., 2003). Alleviation of GvHD after infusion of donor-derived CD4+ CD25+ regulatory T cells has recently been reported in some patients (Brunstein et al., 2011; Di Ianni et al., 2011), although these phase I clinical trials were not powered to assess the efficacy of this treatment modality and did not include control subjects. The therapeutic potential of regulatory T cell induction or adoptive transfer in GvHD prevention is currently under clinical exploration (Leventhal et al., 2012).

#### **B cells and antibodies**

In transplanted rats, B cells recover gradually in host lymphoid organs and peripheral blood with kinetics similar to that of T cells (Renkonen, 1986). This is consistent with what is known of B cell reconstitution in patients who receive an allogeneic donor transplant (Bosch et al., 2012), as is the finding that B cell reconstitution is delayed during acute GvHD (Renkonen, 1986; Bosch et al., 2012). B cells did not recover in the skin, lungs, or the gut of host rats that developed acute GvHD, but accumulation of immunoglobulin-producing cells was observed in the inflamed liver (Renkonen et al., 1986). B cells are thought to participate in the pathogenesis of GvHD (Shimabukuro-Vornhagen et al., 2009), possibly by presenting allo-antigens and by secreting auto- and alloreactive antibodies.

Disparate MHC molecules and minor histocompatibility (non-MHC) antigens of both donor and host type co-exist in radiation chimeras, by which B cells can be activated to produce alloantibodies either against donor antigens (on hematopoietic cells) or against host antigens (mainly non-hematopoietic tissue). Putative donor-derived allo-antibodies will therefore bind mainly to tissue components of the host and may be difficult to detect in the serum unless circulating donor cells are screened for B cell receptor specificities. We know that the transfusion of PVG T cells into semi-allogeneic F1 (PVG × DA) hybrid recipients induces autoantibodies against basal membranes of the skin (Rolstad, 1985). Increased serum levels of antibodies against heat-shock proteins are associated with acute and chronic GvHD (Goral et al., 1995, 2002).