IMMUNE PROFILE AFTER AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR AUTOIMMUNE DISEASES: WHERE DO WE STAND?

EDITED BY : Kelen Cristina Ribeiro Malmegrim, Maria Carolina Oliveira, Antoine Toubert and Dominique Farge-Bancel PUBLISHED IN : Frontiers in Immunology

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# IMMUNE PROFILE AFTER AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR AUTOIMMUNE DISEASES: WHERE DO WE STAND?

Topic Editors:

Kelen Cristina Ribeiro Malmegrim, University of São Paulo, Brazil Maria Carolina Oliveira, University of São Paulo, Brazil Antoine Toubert, Paris Diderot University, France Dominique Farge-Bancel, Assistance Publique Hopitaux De Paris (AP-HP), France

Autologous hematopoietic stem cell transplantation (AHSCT) was proposed 20 years ago as an alternative therapeutic approach for patients with severe and refractory autoimmune diseases. Since then, this innovative therapy has been successfully used to treat several autoimmune diseases, such as neurological disorders (Multiple Sclerosis), connective tissue diseases (Systemic Sclerosis and Systemic Lupus Erythematous), gastrointestinal inflammatory diseases (Crohn's Disease) and others (Juvenile Arthritis, Type 1 Diabetes, Vasculitis).

Experience in phase I-II and III clinical trials over the years has led to increased safety and efficacy of this procedure. Recent studies have demonstrated superior therapeutic efficacy of AHSCT versus conventional therapies, showing that transplantation can induce long-term disease remission in the absence of any further immunosuppressive treatment. Immune monitoring studies have showed that AHSCT is able to reduce the inflammatory milieu, to reset the immune balance and to promote the generation of a new auto-tolerant immune repertoire. However, some patients fail to remain in remission, and disease is re-activated after some time post-transplantation. This scenario indicates that further immunological interventions may be still required to improve AHSCT efficacy. Clear understanding of the operating immune mechanisms that contribute to specific clinical outcomes is vital to enable improvement of AHSCT protocols for autoimmune diseases.

Citation: Malmegrim, K. C. R., Oliveira, M. C., Toubert, A., Farge-Bancel, D., eds. (2020). Immune Profile after Autologous Hematopoietic Stem Cell Transplantation for Autoimmune Diseases: Where Do We Stand?. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-472-9

# Table of Contents

Kristofor K. Ellestad, Jiaxin Lin, Louis Boon and Colin C. Anderson

Kristina M. Harris, Tingting Lu, Noha Lim and Laurence A. Turka

*42 Regenerating Immunotolerance in Multiple Sclerosis With Autologous Hematopoietic Stem Cell Transplant*

Jennifer C. Massey, Ian J. Sutton, David D. F. Ma and John J. Moore

*62 Immune Reconstitution After Autologous Hematopoietic Stem Cell Transplantation in Crohn's Disease: Current Status and Future Directions. A Review on Behalf of the EBMT Autoimmune Diseases Working Party and the Autologous Stem Cell Transplantation In Refractory CD—Low Intensity Therapy Evaluation Study Investigators*

Alan Graham Pockley, James O. Lindsay, Gemma A. Foulds, Sergio Rutella, John G. Gribben, Tobias Alexander and John A. Snowden On behalf of the EBMT Autoimmune Diseases Working Party (ADWP) and the ASTIClite Study Investigators

*72 A Future for Autologous Hematopoietic Stem Cell Transplantation in Type 1 Diabetes*

Kayleigh M. van Megen, Ernst-Jan T. van't Wout, Stephen J. Forman and Bart O. Roep

Carlos E. B. Couri, Kelen C. R. Malmegrim and Maria C. Oliveira

*98 The Role of Endothelial Progenitors in the Repair of Vascular Damage in Systemic Sclerosis*

Nicoletta Del Papa and Francesca Pignataro

#### *108 Prostaglandin E2 Stimulates the Expansion of Regulatory Hematopoietic Stem and Progenitor Cells in Type 1 Diabetes*

Moufida Ben Nasr, Francesca D'Addio, Amir Mohammad Malvandi, Silvia Faravelli, Eduardo Castillo-Leon, Vera Usuelli, Francesca Rocchio, Teresa Letizia, Abdel Basset El Essawy, Emma Assi, Chiara Mameli, Elisa Giani, Maddalena Macedoni, Anna Maestroni, Alice Dassano, Cristian Loretelli, Moira Paroni, Giuseppe Cannalire, Giacomo Biasucci, Marco Sala, Alessandra Biffi, Gian Vincenzo Zuccotti and Paolo Fiorina

*120 Autologous Hematopoietic Stem Cell Transplantation for Autoimmune Diseases: From Mechanistic Insights to Biomarkers*

Kelen Cristina Ribeiro Malmegrim, João Rodrigues Lima-Júnior, Lucas Coelho Marlière Arruda, Júlia Teixeira Cottas de Azevedo, Gislane Lelis Vilela de Oliveira and Maria Carolina Oliveira

# Editorial: Immune Profile After Autologous Hematopoietic Stem Cell Transplantation for Autoimmune Diseases: Where Do We Stand?

Kelen Cristina Ribeiro Malmegrim1,2, Antoine Toubert 3,4,5, Dominique Farge6,7,8 and Maria Carolina Oliveira2,9 \*

1 Department of Clinical Analysis, Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil, 2 Center for Cell-Based Therapy, Regional Hemotherapy Center of Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil, 3 Université de Paris, Institut de Recherche Saint Louis, EMiLy, Paris, France, 4 INSERM UMRS 1160, Microenvironment, Lymphocyte Development and Homing, Paris, France, 5 Laboratoire d'Immunologie et d'Histocompatibilité, AP-HP, Hopital Saint-Louis, Paris, France, 6 Unité de Médecine Interne, Maladies Auto-immunes et Pathologie Vasculaire (UF 04), Hôpital St-Louis, AP-HP, Paris, France, 7 Université de Paris, IRSL, Recherche Clinique Appliquée à l'Hématologie, Paris, France, 8 Department of Medicine, McGill University, Montreal, QC, Canada, 9 Division of Rheumatology, Allergy, Immunology and Immunotherapy, Department of Internal Medicine, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil

Keywords: hematopoietic stem cell transplantation, autoimmune diseases, immune reconstitution, immune

#### Edited by:

monitoring, cell therapy

**Editorial on the Research Topic**

Aurore Saudemont, GlaxoSmithKline, United Kingdom

#### Reviewed by:

Philippe Saas, INSERM U1098 Interactions Hôte-Greffon-Tumeur and Ingénierie Cellulaire et Génique, France Luca Castagna, Humanitas Research Hospital, Italy

\*Correspondence:

Maria Carolina Oliveira<EMAIL_ADDRESS>
#### Specialty section:

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

Received: 14 November 2019 Accepted: 12 December 2019 Published: 09 January 2020

#### Citation:

Malmegrim KCR, Toubert A, Farge D and Oliveira MC (2020) Editorial: Immune Profile After Autologous Hematopoietic Stem Cell Transplantation for Autoimmune Diseases: Where Do We Stand? Front. Immunol. 10:3044. doi: 10.3389/fimmu.2019.03044 **Immune Profile After Autologous Hematopoietic Stem Cell Transplantation for Autoimmune Diseases: Where Do We Stand?**

Autologous hematopoietic stem cell transplantation (AHSCT) induces long-term remission in autoimmune diseases (AD) without further use of immunosuppression (1, 2). Recently, randomized trials have proven greater efficacy of AHSCT when compared to conventional therapies for multiple and systemic sclerosis (3–6). However, despite the overall positive outcomes, subgroups of patients reactivate the AD after AHSCT due to reasons not yet completely understood, indicating that additional specific immunological interventions may still be required to improve or sustain therapeutic efficacy of AHSCT.

This Frontiers Research Topic, combines reviews, opinions, and original research from the most active researchers in the field of AHSCT for AD. Here, clinical outcomes and immune mechanisms of AHSCT, as well as insights for future studies are presented in the setting of different AD.

Different concepts, from basic knowledge to translational medicine, are integrated in two Perspective articles. Malmegrim et al. critically review the current knowledge about the operating immune mechanisms of AHSCT for AD, and propose strategies for future immune monitoring studies and biobanking. Harris et al. present data from a trial on AHSCT for poor prognosis multiple sclerosis to illustrate post-transplantation immune reconstitution and discuss experimental challenges and strategies to identify biomarkers of clinical response to AHSCT.

Two review articles address mechanistic effects of AHSCT. Massey et al. describe the pathogenesis of multiple sclerosis (MS) and how AHSCT restores immunological balance and, therefore, tolerance. The authors revise most of the available clinical trials on HSCT for MS and discuss aspects such as the lymphopenia-induced proliferation (LIP) that takes place early after transplantation and how it correlates with the later thymic rebound and T-cell diversification. Pockley et al. also describe the dynamics of immune reconstitution that follows AHSCT in AD

**5**

patients and how it can be extrapolated to Crohn's disease patients. Post-transplantation evidence of immune rejuvenation, with thymic rebound and improvement of regulatory mechanisms are shown in Crohn's disease patients, as well as increase in T-cell receptor (TCR) repertoire diversity in mucosal biopsies. The authors also discuss both established and potential effects of AHSCT on the innate immune system, which plays an important role in inflammatory bowel disease pathogenesis, and how innate cells may contribute to the high rate of Crohn's disease reactivations after transplantation.

Del Papa and Pignataro provide a very detailed and updated review on the mechanisms associated with vascular damage and repair in systemic sclerosis patients. The roles of mature endothelial cells and of endothelial progenitor cells (EPCs) are thoroughly described in the context of a disease characterized by diffuse microvasculopathy and endothelial damage. The authors discuss future EPC-based approaches, using either direct cell transplantation or pharmacological stimuli, aiming to promote endothelial repair. This important overview helps us understand possible pathogenic targets for future strategies involving cell therapy in systemic sclerosis.

Couri et al. and van Megen et al. contribute with their opinions on AHSCT for insulin-dependent diabetes mellitus (T1D). Although very consistent and evidence-supported, the opinions of these two groups differ slightly. The former discuss how AHSCT is able to promote temporary, but meaningful pancreatic beta cell preservation, and thus improve glycemic control. However, since the beneficial effects of AHSCT are transient, more intense immunosuppressive strategies may be warranted in future studies, possibly combined with cellreplacement approaches. On the less full side of the glass, van Megen et al. describe AHSCT as a still controversial issue. Ethical concerns such as transplant-associated risk, long-term toxicity, and enrolment of children are discussed, as well as the lack of substantial evidence for irrefutably beneficial results, despite the appeal of reducing long-term complications of T1D. The authors discuss the available information of immunological analyses before and after AHSCT and suggest a more personalized approach to enroll patients for transplantation.

Autologous HSCT for multiple sclerosis leads to abrogation of new clinical relapses and brain lesions. In parallel, there is selective reduction of Th17, but not Th1, cell population and activity. Darlington et al. demonstrate an increase in the kinetics of natural killer (NK) cell reconstitution, when compared to CD4+ T cells, in MS patients post-AHSCT. The resulting increased NK cell:CD4+ T cell ratio correlated with a decrease in Th17 responses. The authors suggest that rapid reconstitution of NK cells following AHSCT contribute to the suppression of Th17 re-emergence, highlighting the importance of NK cells in the post-transplantation setting.

Ben Nasr et al. report that ex vivo modulation of hematopoietic stem and progenitor cells with prostaglandins (PGs) increases their immunoregulatory properties by upregulating expression of the immune checkpoint-signaling molecule PD-L1. When tested in murine and human in vitro autoimmune assays, PG-modulated progenitor cells were shown to diminish the autoreactive T cell response. The use of PG-modulated progenitors may thus become an attractive and novel treatment for T1D, thus circumventing immunosuppression-related toxicity.

Two other contributions demonstrate that the programmed death-1 (PD-1) signaling pathway may control autoimmunity. T cells in a lymphopenic environment undergo LIP to fill the available "niche" as defined by (self) peptide:MHC (pMHC) complexes with which the TCRs interact and receive at least a weak "tonic" signal to promote T cell survival. The numbers of cells and diversity of the peripheral T-cell pool are controlled by intra and interclonal competition for resources, which together define T-cell "space." Ellestad et al. found that PD-1 controls pMHC-dependent tonic signals to T cells, independently of IL-7 signaling, at least when available pMHC is abundant. These data suggest that therapies aimed at reducing TCR signaling during the early phases of T cell reconstitution may be more effective than approaches that aim to limit homeostatic cytokine-mediated signals to T cells.

Finally, Ellestad et al. determine that PD-1 is upregulated on CD4+ T-cells undergoing the natural LIP characteristic of the neonatal period. Newly generated T cells lacking PD-1 maintained an enhanced autoimmune potential even after residence in a lymphoreplete periphery, emphasizing the importance of PD-1 in the establishment of peripheral tolerance. Neither Fas nor perforin-dependent killing mechanisms were required for autoimmunity, while host MHC-II expression was critical, suggesting that LIP-driven autoimmunity in the absence of PD-1 may primarily result from a CD4+ T-cellmediated systemic cytokinemia. Their data suggest that even in a lymphoreplete adult host, peripheral newly generated T cells retain a potential for LIP-driven autoimmunity in the absence of PD-1.

Collectively, the articles from this Research Topic contribute to increase the knowledge of the field. Important aspects about the modulation of the immune system in autoimmune diseases are discussed, from cellular to more molecular approaches, and from bedside to bench, which in the future should reverse back to the bedside.

#### AUTHOR CONTRIBUTIONS

KM and MO wrote the initial draft of the editorial. AT and DF revised and approved the manuscript.

#### REFERENCES

1. Alexander T, Farge D, Badoglio M, Lindsay JO, Muraro PA, Snowden JA. Autoimmune Diseases Working Party (ADWP) of the European Society for Blood and Marrow Transplantation (EBMT). Hematopoietic stem cell therapy for autoimmune diseases - Clinical experience and mechanisms. J Autoimmun. (2018) 92:35–46. doi: 10.1016/j.jaut.2018. 06.002

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

Copyright © 2020 Malmegrim, Toubert, Farge and Oliveira. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# PD-1 controls Tonic signaling and lymphopenia-induced Proliferation of T lymphocytes

#### *Kristofor K. Ellestad1,2\*, Jiaxin Lin2,3, Louis Boon4 and Colin C. Anderson1,2,3,5\**

*1Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada, 2 Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada, 3Department of Surgery, University of Alberta, Edmonton, AB, Canada, 4Bioceros B.V., Utrecht, Netherlands, 5 Alberta Transplant Institute, University of Alberta, Edmonton, AB, Canada*

#### *Edited by:*

*Joanna Davies, San Diego Biomedical Research Institute, United States*

#### *Reviewed by:*

*Anne Cooke, University of Cambridge, United Kingdom Raymond John Steptoe, The University of Queensland, Australia Deborah J. Fowell, University of Rochester, United States*

#### *\*Correspondence:*

*Kristofor K. Ellestad<EMAIL_ADDRESS>Colin C. Anderson<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology*

*Received: 31 May 2017 Accepted: 26 September 2017 Published: 12 October 2017*

#### *Citation:*

*Ellestad KK, Lin J, Boon L and Anderson CC (2017) PD-1 Controls Tonic Signaling and Lymphopenia-Induced Proliferation of T Lymphocytes. Front. Immunol. 8:1289. doi: 10.3389/fimmu.2017.01289*

Recovery of the T lymphocyte compartment within a lymphopenic host by lymphopeniainduced proliferation (LIP) is regulated by inter- and intraclonal competition for limited resources, including homeostatic cytokines and peptide:MHC (pMHC) complexes with which the TCR can interact at least weakly to yield a tonic signal. Importantly, the process of LIP can synergize with other factors that promote T cell activation to drive inflammatory disease. While reconstitution of the lymphoid compartment of immune deficient Rag−/− mice by transfer of wild-type hematopoietic stem cells (HSC) does not generally result in an overt disease phenotype, transfer of HSC deficient in expression of the co-inhibitory molecule PD-1 results in severe systemic autoimmunity driven by newly generated T cells that emerge from the thymus into the periphery and undergo LIP. Importantly, autoimmunity does not appear to depend on a response to exogenous (i.e., gut flora-derived) antigens. PD-1 is well known to be upregulated during T cell activation in response to cognate antigens, but it is unclear whether PD-1 has a role in controlling LIP of T cells in the absence of cognate antigen, i.e., in response to tonic pMHC. We examined whether PD-1 controls LIP of newly generated T cells by controlling the response to tonic pMHC or the homeostatic cytokine IL-7. We found that PD-1-deficient T cells have a proliferative advantage over WT T cells during LIP and this effect is MHC-II dependent and independent of IL-7Rα signaling. Furthermore, our data suggest that signals through IL-7Rα can be dispensable for LIP and may instead be of increased importance for T cell survival in conditions of high competition for limited pMHC (e.g., post-LIP, in a lymphoreplete host). We hypothesize that autoimmunity post-PD-1−/− HSC transplant is the result of an overzealous T cell response to normally tonic self-pMHC precipitated by the synergy of LIP and PD-1 deficiency. Furthermore, potentiation of TCR signals in response to normally tonic self-pMHC may contribute to the success of PD-1 blockade in cancer immunotherapy.

Keywords: tolerance, autoimmunity, co-stimulation, co-inhibition, lymphopenia, PD-1, mouse models, IL-7

## INTRODUCTION

The number of cells in and diversity of the peripheral T lymphocyte pool is controlled by intraand interclonal competition for resources, which together define T cell "space" (1). Such resources include homeostatic cytokines like IL-7 and IL-15, but also peptide:MHC (pMHC) complexes; often self-pMHC, with which the TCR can interact and receive at least a weak "tonic" signal to promote

**8**

T cell survival. In lymphopenic hosts, recovery of the T cell compartment occurs *via* a process known as lymphopenia-induced proliferation (LIP), where resources are in excess and T cells expand to fill the available niche. While the term lymphopenia lacks a precise quantitative character, one can consider the extent to which LIP can occur, or "LIP potential" of a host, as a ratio of the available resources to the number of competitors for those resources (2). Thus, provision of competition for a particular pMHC can reduce LIP potential for other T cells that recognize the same pMHC (3–6). Similarly, Treg, which may be viewed as "super"-competitors for pMHC, can inhibit LIP (5, 7–14). Positive or negative regulation of the strength of the TCR signal a lymphocyte receives in response to a given pMHC, for example by blocking/reducing co-inhibition (15–19) or co-stimulation (9), respectively, can modulate LIP potential. Finally, a neonatal host might be viewed to have a low LIP potential due to small anatomic size (low absolute quantities of resources) as well as the recently described presence of an innate lymphoid cell population that may act as a sink for IL-7 (20).

Importantly, LIP is strongly associated with acquisition of an effector-memory phenotype in T cells, including the ability to rapidly mediate effector function (21), and in the context of concurrent infections or deficiencies of co-inhibitory pathways, LIP can result in overzealous T cell responses and autoimmune disease (2, 7, 14, 17, 19, 22–30). This association between LIP and inappropriate inflammation may be rationalized by considering that the conditions present in lymphopenia—including reduced competition for access to a given T cell's cognate or tonic pMHC, along with high concentrations of IL-7 and IL-15 [which have been suggested to potentiate TCR signals (31, 32)]—may conspire to increase the frequency with which T cells can productively interact with pMHC and generate an abnormally strong TCR signal when they do so. Systemic activation of sufficient numbers of T cells may result in immune pathology.

Reconstitution of the lymphoid compartment of an otherwise normal C57BL/6 (B6) Rag−/− mouse with B6 hematopoietic stem cells (HSC) does not typically result in the development of apparent autoimmune disease (19, 33). In stark contrast, we recently demonstrated that reconstitution of adult Rag−/− mice with HSC deficient in the co-inhibitory molecule PD-1 results in a rapid, severe, and frequently lethal systemic autoimmune disease soon after the first newly generated T cells emerge from the thymus (19). Severe and rapid autoimmunity can also result from transfer of PD-1−/− thymocytes to Rag−/− recipients, but not established peripheral cells from adult PD-1−/− mice [note, on the B6 background, PD-1−/− mice develop only a mild lupus-like autoimmune disease upon aging (34)]. The newly generated T cell population may have a higher average affinity for self-pMHC compared to established cells that developed in a lymphoreplete adult environment or an environment with low LIP potential (e.g., during early seeding of the lymphoid compartment in the neonate) and thus underwent more normal peripheral tolerance mechanisms (deletion, anergy, conversion to pTreg). One hypothesis to explain disease in our model is that in the "three-strikes" scenario of high LIP potential, PD-1 deficiency, and a periphery seeded with newly generated T cells, a stochastically greater number of the T cells on the higher end of the self-pMHC affinity spectrum receive a strong enough signal to acquire effector function giving rise to the observed systemic cytokinemia (19). On the other hand, when PD-1 is sufficient, its restraint on the magnitude of the TCR signal (35–37) limits the frequency of such spurious activation preventing a pathological systemic effect. Although multiple co-inhibitory molecules other than PD-1 have been shown to control LIP (15–18) and our data suggest that PD-1 does as well (19, 33), whether they do so *in vivo* by controlling TCR signals mediated by tonic self-pMHC is unknown. Indeed, PD-1 is known to be upregulated on *bona fide* activated T cells and it is unclear whether the generally weak, tonic signals provided by interaction with self-pMHC can drive its expression. Co-inhibitor deficient cells might instead come to predominate in LIP due to control of responses to homeostatic cytokines or completely independent of external signals. Furthermore, because of the polyclonal repertoire generated post-HSC transplant in our disease model, we cannot rule out that the response might be directed to foreign pMHC [although depletion of gut microbiota in PD-1−/− HSC recipients does not protect from disease (33)] or pMHC derived from tissuerestricted antigens against which developing PD-1−/− T cells failed to be appropriately negatively selected in the thymus. Herein, we examined whether PD-1 is able to control the response to tonic pMHC using transfer of monoclonal PD-1+/+ or PD-1−/− male antigen (HY)-specific Marilyn CD4+ TCR transgenic T cells (38) to MHC-II sufficient and deficient [MHC Class II transactivator (CiiTA) knockout (39)] lymphopenic hosts lacking the male antigen HY. We also examined whether PD-1 modulates signaling through IL-7Rα during LIP. We found that PD-1 controls pMHC-dependent tonic signals to T cells, independent of IL-7 signaling. Furthermore, our data suggest that IL-7 is particularly important for controlling T cell homeostasis during situations of high competition for limited pMHC but is not essential for LIP when available pMHC is abundant.

#### MATERIALS AND METHODS

#### Mice

B6.129S7-*Rag1*tm1Mom/J (Rag1−/−, Rag−/−), B6.Cg-*Foxp3*tm2(EGFP)Tch/J (FoxP3EGFP), B6.SJL-Ptprca Pepcb/BoyJ (CD45.1), and B6.129S2- *Ciita*tm1Ccum/J (CiiTA−/−) mice were originally purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and then bred at the University of Alberta. C57BL/6-*Pdcd1*−*/*− (backcrossed 11 generations to C57BL/6) were originally generated by Prof. T. Honjo and colleagues (34). FoxP3EGFP × *Pdcd1*−*/*− mice were generated by crossing the above FoxP3EGFP and B6-*Pdcd1*−*/*− mice and are referred to and used in the present manuscript simply as PD-1−/−. Rag−/− × CiiTA−/− mice were generated by crossing the above Rag−/− and CiiTA−/− mice. Marilyn Rag2−/− CD4+ anti-IAb - HY TCR transgenic mice (called Marilyn herein) were originally generated by Lantz and colleagues (38) and were originally obtained from the NIAID exchange program. Marilyn-CD45.1, referred to herein as Marilyn-WT, were generated by crossing the above Marilyn mice with CD45.1 × Rag2−/− mice (Taconic). Marilyn-PD-1−/− mice were generated by crossing the above Marilyn mice with PD-1−/− mice and were maintained as Rag2−/−. All Marilyn mice used as donors were female. Animals were cared for in accordance with the guidelines of the Canadian Council on Animal Care and housed under clean conventional housing conditions at the University of Alberta Health Sciences and Laboratory Animal Services facilities (HSLAS).

### Cell Preparations and Adoptive Transfer Experiments

For experiments involving transfer of thymocytes, recipient NK cells were depleted [to avoid potential NK-mediated killing of the input cells (40–42)] by treatment on days −4, −1, and +2 with 0.3 mg per mouse of anti-NK1.1 (PK136) injected intraperitoneally. In order to study the role of PD-1 in controlling LIP of newly generated T cells, which would not have been exposed to the periphery where they may have undergone peripheral tuning mechanisms, we chose to transfer minimally manipulated whole unfractionated thymocytes and monitor their proliferation *in vivo*. In mixed thymocyte transfer experiments, WT (CD45.1+) and PD-1−/− (CD45.1−) Marilyn unfractionated thymocytes were counted and mixed in equal proportions prior to labeling and transfer. Labeling of mixed thymocyte populations with Celltrace violet (CTV, ThermoFisher) was performed using 5 µM CTV according to the manufacturer's protocols except with a final cell concentration in the labeling reaction of up to ~40 × 106 cells per mL. For subsequent *in vivo* use cells were washed once with PBS and resuspended in PBS on ice for immediate intravenous tail vein injection into recipient mice. Mixed cells for infusion into mice were analyzed by flow cytometry to determine the starting (day 0) ratio of PD-1−/−: WT T cells calculated as %PD-1−/− T cells divided by % WT T cells. Initial ratios varied from 0.57 to 1.09. To facilitate combining data from multiple experiments, initial ratios were normalized to 1 by multiplying by a scaling factor which was applied to all subsequent PD-1−/−:WT T cell ratio measurements within a given experiment. Anti-IL-7Rα treated mixed thymocyte recipients received twice weekly intraperitoneal injections of antibody (as described below) for the full course of the experiment.

For *in vitro* restimulation assays, cells were resuspended in E-DMEM (high glucose DMEM + 2 mM l-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 50 µM 2-mercaptoethanol, 10% FBS) at 2 × 106 cells/mL and seeded 200 µL per well into a 96 well, round bottom plate. A final concentration of 16 nM of phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) and 1.4 µM ionomycin (Sigma-Aldrich) were added for 2 h, at which time brefeldin A (eBioscience, 3 µg/mL) and monensin (eBioscience, 2 µM final) were added for a further 2 h prior to surface staining, fixation, intracellular staining, and analysis.

#### Definition of Disease and Data Analysis

Macroscopic signs of disease in thymocyte recipients included cachexia/weight loss (>15%), kyphosis (hunched appearance), ruffled fur, dermatitis, ocular lesions, and diarrhea. Recipient mice were no longer considered disease free when two or more of the above symptoms were evident, or if mice lost ≥20% body weight. Kaplan–Meier survival curve analysis of disease onset/ incidence was performed using Graphpad Prism v5.0 software. Probability values reported for survival curve comparisons were calculated using the Mantel-Cox method. For thymocyte experiments, calculation of weight loss for disease determination was performed relative to weights at day 0 or day 1 relative to cell transfer. Unless otherwise indicated, animals were used between 6 and 20 weeks of age.

## Antibodies and Flow Cytometry

For flow cytometric staining, fluorophore-labeled antibodies against the following markers were obtained from eBioscience (San Diego, CA, USA) unless otherwise indicated: CD4 (RM4-5), TCRβ (H57-597), CD8 (53-6.7), PD-1 (J43), CD5 (53-7.3), IL-7Rα/CD127 (A7R34), IFN-γ (XMG1.2), Bcl2 (10C4). Antibodies were used at manufacturer's recommended concentrations. Flow cytometric staining always used an Fc block cocktail to block nonspecific staining. Fc block cocktail consisted of 3 mL each of normal mouse, rat, and hamster serum, with addition of 0.3 mg of anti-CD16/32 antibody (clone 2.4g2, BioXCell). For intracellular staining, cells were fixed and permeabilized using the eBioscience FoxP3 Fixation/permeabilization buffer kit according to supplied protocols. Standard flow cytometric analysis was performed using a BD LSR II instrument. Flow cytometric data analysis was performed using FlowJo (Treestar software, Portland, OR, USA).

Anti-IL-7Rα treatment *in vivo* was carried out by biweekly intraperitoneal injection of 0.5 mg of anti-IL-7Rα clone A7R34 (BioXcell for polyclonal PD-1−/− thymocyte disease model experiments; or generated by us for mixed Marilyn experiments) or Rat IgG2a isotype control (2A3, BioXcell or generated by us) beginning on the day of cell transfer.

#### Statistical Analyses

For statistical analyses, we used Graphpad Prism software. Unless otherwise noted Student's *t*-test was used for comparisons. In the case of unequal variances, *t*-test was performed using Welch's correction. For comparisons of multiple groups, one-way ANOVA with Tukey's multiple comparison test was used.

## RESULTS

## PD-1 Controls LIP in Response to Tonic Self-pMHC-II Independent of IL-7 Signals

We have previously demonstrated that transfer of a mixture of Treg-depleted polyclonal WT and PD-1−/− CD4+ T cells purified from the thymocyte or splenocyte population (including CD62Lhi selected cells) into lymphopenic Rag−/− hosts resulted in predominance of the PD-1−/− population in the periphery during LIP (33). In the same study, we also demonstrated that the proportion of WT and PD-1−/− cells could be equalized by treatment of hosts with anti-PD-L1, releasing the WT population from cell-intrinsic PD-1-dependent inhibition. These data indicated that the effect on population size was mediated through the PD-L1:PD-1 interaction. In order to examine whether PD-1 controls LIP in response to tonic self-pMHC signals in the absence of potential responses to conventional agonist cognate antigens by PD-1−/− cells within a polyclonal repertoire, we employed Marilyn male antigen-specific CD4+ TCR transgenic CD45.1 (CD45.1+, "Marilyn-WT") mice as well as Marilyn PD-1−/− mice (CD45.2+, Marilyn-PD-1−/−) (43). We also generated Rag−/− and MHC Class II transactivator deficient (CiiTA−/−) mice, which are largely MHC-II deficient (39) (CiiTA−/− × Rag−/− mice). We transferred a mixture (40 × 106 cells) of Marilyn-WT and Marilyn-PD-1−/− thymocytes labeled with CTV proliferation dye into NK-depleted Rag−/− females (tonic signals only) or males (systemic cognate HY antigen present) and CiiTA−/− × Rag−/− females or males, with or without biweekly anti-IL-7Rα treatment and monitored their relative proliferation and abundance over time (**Figure 1A**). NK depletion was carried out to maximize input cell survival as even syngeneic cells can be targets of NK cell killing (40–42).

Early after transfer (day 3), only barely detectable levels of proliferation had occurred within female Rag−/− recipients with none in the female Rag−/− anti-IL-7Rα-treated group (**Figure 1B**; Figure S1 in Supplementary Material), and no difference between the WT and PD-1−/− cells was apparent nor was there any change in the ratio of PD-1−/− to WT T cells in these recipients relative to the initial seeding ratio, regardless of anti-IL-7Rα treatment (**Figure 1D**). In contrast, in the presence of systemic cognate antigen all of the cells within the Rag−/− males, regardless of ant-IL-7Rα treatment, had proliferated extensively and had already almost completely diluted out their CTV dye (**Figure 1B**; Figure S1 in Supplementary Material). Proliferation had also clearly occurred albeit to a reduced extent within CiiTA−/− × Rag−/− male recipients, which may be due to small numbers of MHC-II sufficient thymic DC that were transferred along with the thymocytes or the known residual MHC-II expression in these knockouts (39, 44).

By day 5, PD-1−/− T cells had undergone significantly more proliferation than WT cells in Rag−/− female recipients (**Figure 1B**; Figure S1 in Supplementary Material) and the ratio of PD-1−/−:WT cells started to increase (**Figure 1D**). Furthermore, in these recipients, we detected an MHC-II-dependent upregulation of PD-1 expression within the WT cell population at this time point among the extensively proliferated cells (**Figure 2A**), indicating that tonic pMHC signals are sufficient to upregulate PD-1. We also explored expression of another co-inhibitory receptor, CD5, a commonly used marker of TCR affinity and that has co-inhibitory function (45–47), and found it was modulated during LIP in response to tonic pMHC signals. Upon extensive proliferation, CD5 became up or downregulated on a substantial proportion of WT cells, while PD-1−/− cells had less downregulation of CD5 (**Figure 2B**), suggesting CD5 expression may help compensate for the lack of PD-1.

By 7 days post-cell transfer, the majority of cells within the Rag−/− female recipients had proliferated extensively and diluted their CTV labeling beyond detection limits, although significantly more proliferation had occurred in the PD-1−/− cell population in these recipients as judged by comparison of CTV MFI (**Figures 1B,C**; Figure S1 in Supplementary Material). Furthermore, the ratio of PD-1−/−:WT cells in these animals increased approximately twofold relative to the input cell ratio which was significantly different from the female CiiTA−/− × Rag−/− recipients (2.03 ± 0.05 versus 1.18 ± 0.10, *p* < 0.0001, **Figure 1D**) in which the PD-1:WT ratio did not change and also in which the majority of the T cell population still had not proliferated (**Figures 1B,C**; Figure S1 in Supplementary Material). Importantly, this alteration in ratio was not affected by anti-IL-7Rα treatment of the hosts. During LIP 7 days post-mixed thymocyte transfer, the percentage of all cells that were TCRβ+CD4+ in blood of Rag−/− female recipients was not significantly affected by anti-IL-7Rα treatment; although, we did note a trend to reduced CD4 T cells with anti-IL-7Rα in the one experiment in which CD4 numbers were already quite high by day 7 (**Figure 2D**, green symbols). In contrast, in MHC-IIdeficient female CiiTA × Rag−/− recipients, blockade of anti-IL-7Rα significantly reduced the frequency of CD4+ T cells in the blood. Examination of the CTV dilution histograms suggested that anti-IL-7Rα treatment in the MHC-II sufficient female Rag−/− recipients appeared to primarily decrease the size of the unproliferated cell population (Figure S1 in Supplementary Material). In the presence of HY antigen in male Rag−/− recipients, by day 7 the ratio of PD-1−/−:WT cells had increased more than sixfold relative to the input cell proportions, and this was also not affected by anti-IL-7Rα treatment (**Figure 1D**). Interestingly, the ratio of PD-1−/−:WT cells did not change by day 7 in CiiTA−/− × Rag−/− male recipients despite robust expansion of input populations in these recipients; expansion that was potentially caused by residual MHC class II known to exist in the CiiTA knockout (39, 44). Male Rag−/− recipients had a significantly higher peripheral blood T cell abundance compared to females which was not significantly decreased by anti-IL-7Rα treatment (**Figure 2D**).

The increased proportion of PD-1−/− cells during tonic pMHC-stimulated LIP could have been due to increased proliferation or increased survival of proliferating cells. However, PD-1−/− cells did not have an increase of the pro-survival molecule Bcl2 (**Figure 2C**). In addition, Bcl2 expression, known to be stimulated by IL-7 signals (48), generally appeared to be depressed in response to anti-IL-7Rα treatment (**Figure 2C**) despite anti-IL-7Rα's lack of effect on both LIP (**Figure 1B**) and CD4 T cell numbers in MHC-II positive recipients (**Figure 2D**). However, IL-7Rα blockade together with a lack of MHC-II led to the strongest decrease in Bcl2 expression in both WT and PD-1−/− cells (**Figure 2C**), and this was associated with reduced CD4 T cell numbers (**Figure 2D**). Thus, IL-7 and Bcl2 appear important for CD4 T cells in the absence of tonic pMHC signals but not during tonic pMHC-stimulated LIP. Together, these data indicate that PD-1 controls signaling in response to tonic as well as cognate pMHC-II in a cell-intrinsic manner, which is independent of effects on IL-7 signaling.

Thus far, we have shown that PD-1 controls LIP to tonic pMHC signals. Whether tonic pMHC signals driven effector function (e.g., cytokine production) in addition to LIP is controlled by PD-1 is not known. We found no difference in the proportion of WT versus PD-1−/− T cells producing IFN-γ in response to *in vitro* restimulation at the d7 time point in the MHC-II sufficient female hosts, and in the absence of MHC-II IFN-γ was essentially undetectable (**Figure 2E**). Thus, while tonic pMHC signals are able to stimulate IFN-γ production, PD-1 appears important for controlling proliferation but not this effector activity triggered by tonic pMHC signals in LIP. However, it remains possible that PD-1 controls other effector functions of CD4 T cells during LIP.

7), female CiiTA−/− × Rag−/− + anti-IL-7Rα (two independent experiments, *n* = 3, 5, 5 at days 3, 5, 7), male Rag−/− (two independent experiments, *n* = 8), male Rag−/− + anti-IL-7Rα (two independent experiments, *n* = 7), male CiiTA−/− × Rag−/− (two independent experiments, *n* = 8, 7, 8 at days 3, 5, 7). \*\*\*\**p* < 0.0001, Student's *t*-test, Rag−/− day 3 versus day 7. The starting ratio was normalized and set to a value of one with subsequent ratio measurements scaled accordingly.

#### Figure 2 | Continued

Tonic peptide:MHC signals control expression of co-inhibitory receptors, drive IFNγ expression, and limit the need for IL-7 signals during LIP. (A) (Left and center panels) Representative flow cytometry overlaid contour plots showing PD-1 expression versus. Celltrace violet (CTV) staining within the indicated recipients of 40 × 106 mixed Marilyn-WT and PD-1−/− thymocytes at day 5 p.t. in blood. (Right panel) Mean fluorescence intensity (MFI) of PD-1 staining within the proliferated WT or PD-1−/− cell populations from individual recipients. No significant difference in PD-1 expression was noted in unproliferated WT or PD-1−/− cells in CiiTA−/− × Rag−/− hosts. \*\*\**p* < 0.001, Student's *t*-test. (B) (Left and center panels) Representative overlaid contour plots of CD5 expression versus CTV staining in the indicated recipients of mixed Marilyn-WT and PD-1−/− cells at day 7 p.t. in blood. (Right panel) MFI of CD5 staining within WT and PD-1−/− populations from individual recipients. \**p* < 0.05, Student's *t*-test. (C) Mean fluorescence intensity of Bcl2 staining in WT and PD-1−/− CD4+ T cell populations in the blood at day 5 p.t. within the indicated recipients. \*\*\*\**p* < 0.0001, One-way ANOVA with Tukey's Multiple comparison test. Data in (C) depicts individual biological replicates from one experiment. (D) Comparison of the % of all cells that were CD4+ T cells in blood of indicated recipient groups at day 7 p.t. \*\*\**p* < 0.001, Student's *t*-test. Two independent experiments are depicted with black (experiment 1) and green (experiment 2) symbols and means. (E) (Left and center panels) Representative overlaid contour plots of IFN-γ expression versus. CTV staining in indicated recipients at day 7 p.t. in *in vitro* restimulated splenocytes. (Right panel) % IFN-γ+ cells within WT and PD-1−/− CD4+ T cell populations from individual recipients. \*\*\*\**p* < 0.0001, Student's *t*-test. For panels (A,B,E), data presented are from individual recipients from one experiment that is representative of two independent experiments.

## IL-7 Receptor Blockade Decreases T Cell Compartment Size in Conditions of Limiting pMHC

The findings that IL-7 signals appeared not to be important early during pMHC-stimulated LIP but were important when pMHC was limited (CiiTA deficient recipients), suggested the possibility that IL-7 becomes important when there is more competition for pMHC, such as in the post-LIP period. In splenocyte populations harvested at 5 weeks post-Marilyn thymocyte transfer, CTV labeling was virtually undetectable in all cells within the Rag−/− female recipients (**Figure 1B**; Figure S1 in Supplementary Material). We reasoned that LIP was largely complete by this time given that we saw no further increase in absolute splenic CD4+ T cell numbers in mice harvested much later at day 76 post transfer (p.t.; data not shown). We, therefore, examined the phenotype and number of Marilyn cells late after cell transfer once T cell numbers had increased post-LIP and determined the effect of IL-7Rα blockade. A trend toward higher CD44 expression in PD-1−/− versus WT cells was noted at 5 weeks post-transfer, which reached statistical significance in the anti-IL-7Rα-treated MHC-II sufficient females (**Figure 3A**). Importantly IL-7Rα blockade itself did not significantly decrease CD44 expression among T cells in female or male recipients, but MHC-II deficiency in females significantly reduced memory phenotype acquisition (**Figure 3A**). We also noted that Marilyn T cells chronically exposed to agonist antigen present in male recipients had greatly reduced CD44 expression compared to female recipients where only tonic pMHC signals were present. In contrast to the day 7 time point (**Figure 2D**), at day 35 both MHC-II sufficient female and male recipients receiving anti-IL-7Rα treatment had markedly reduced absolute splenic T cell numbers compared to their untreated counterparts (**Figure 3B**). As expected, MHC-II deficient female hosts had significantly fewer splenic T cells compared to their MHC-II sufficient counterparts (**Figure 3B**), and this was further reduced by anti-IL-7Rα treatment with very few cells detectable overall in anti-IL-7Rα-treated MHC-II deficient female hosts. Although most cells in non-anti-IL-7Rα-treated MHC-deficient females appeared to have undergone proliferation by day 35 a significant proportion remained undivided (Figure S1 in Supplementary Material). In contrast, the very few detectable cells within anti-IL-7Rα-treated MHC-II-deficient females were almost completely proliferated cells, consistent with the previously noted decrease in the undivided cell population upon IL-7Rα blockade (Figure S1 in Supplementary Material). We did not note any differential expression of IL-7Rα expression between WT or PD-1−/− Marilyn T cells (**Figure 3C**) nor between cells in MHC-II sufficient or deficient female hosts although there was a trend toward lower IL-7Rα expression in the latter. Together, these data suggest that IL-7 signaling is dispensable for LIP in conditions of excess pMHC resources, but when the T cell compartment becomes more replete and competition for available pMHC resources increases or pMHC is otherwise limiting, IL-7 signals are critically important for survival of the T cell population.

## In a Competitive Environment Blockade of IL-7R**α** Ameliorates Morbidity Caused by PD-1**−**/**−** Thymocytes

TCR signals can downregulate IL-7Rα expression (e.g., **Figure 3C**) and have been reported to block, in an affinitydependent manner, the response to IL-7-mediated survival signals (49, 50). Because we noted that IL-7Rα blockade had a marked effect on the size of the T cell compartment 5 weeks post-mixed Marilyn T cell transfer (**Figure 3B**) but not after only 7 days in class II sufficient recipients (**Figure 2D**), we reasoned that IL-7 signals perhaps became more important to T cell survival as competition for available pMHC became more intense (i.e., as the T cell compartment became more replete, and fewer cells could sufficiently access pMHC and receive a TCR signal). In order to explore this concept, we examined whether constraints on IL-7 signaling in high versus low competition settings would influence the development of autoimmunity. For this we used our model of disease following transfer of polyclonal PD-1−/− thymocytes to Rag−/− animals, which results most commonly in weight loss, kyphosis, diarrhea, dermatitis, and ocular lesions. We transferred either a low amount (10 × 106 ) or high amount (30 × 106 ) of PD-1−/− thymocytes to NK-depleted recipient animals. The recipients were treated biweekly with monoclonal anti-IL-7Rα or isotype control antibodies and monitored for weight changes and disease symptoms. Here, we noted a striking effect of anti-IL-7Rα treatment on loss of weight in the high-dose thymocyte recipient group, which was statistically significant from day 20 through to the termination of the experiment even though anti-IL-7Rα treatment was withdrawn at day 36 (**Figure 4A**). On the

other hand, anti-IL-7Rα treatment of the low-dose thymocyte recipient group did not have any effect on the loss of weight (**Figure 4B**). Taken together, these data suggest that limitation of LIP potential by inhibiting the ability of T cells to respond to IL-7-mediated signals in situations of high but not low competition for finite pMHC can prevent the loss of weight associated with the pathology of LIP-driven autoimmunity.

## DISCUSSION

The potential for LIP in a host depends on the resources available for T cells (tonic or cognate pMHC, homeostatic cytokines) relative to the level of competition for those resources (1). Importantly, LIP can drive inflammatory and autoimmune disease (7, 14, 17, 19, 22–30), and thus modulation of LIP can increase or decrease its potential to result in pathologic T cell activation. Several molecules with co-inhibitory function, such as BTLA, LAG-3, and TGFβRII have been shown to modulate T cell homeostasis and LIP (15–18), although to our knowledge none have demonstrated that such control of LIP *in vivo* is *via* modulating signals in response to pMHC, particularly tonic pMHC. Recently we described a severe systemic autoimmune disease mediated by newly generated PD-1−/− T cells in lymphopenic animals which could be blocked by the addition of competitors or reducing lymph node stroma (19), and which was not due to a deficiency in the generation or function of PD-1−/− Treg (33), suggesting that the co-inhibitory molecule PD-1 also plays an important role in the establishment of immune tolerance through control of LIP. This gave rise to our hypothesis that the disease in lymphopenic recipients of PD-1−/− newly generated T cells is mediated by inappropriate activation of T cells in response to normally tonic self-pMHC in the context of LIP. However, the possibility existed that the T cell response in our disease model was directed toward tissue-restricted antigens that were not appropriately negatively selected against in the thymus in the context of PD-1 deficiency, or due to altered population dynamics because of changes in positive selection (51). Furthermore, it was unclear whether the relatively low-affinity interactions with tonic pMHC can promote

PD-1−/− unfractionated thymocytes were transferred to NK-depleted Rag−/− recipient animals with i.p. injection of 0.5 mg per mouse anti-IL-7Rα or isotype control twice weekly for the time periods indicated. Mice were monitored for disease symptoms and loss of weight. (A) Weight change in high-dose thymocyte recipients over the experimental period. (B) Weight change in low-dose thymocyte recipients over the experimental period. \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, Student's *t*-test. The data depict the mean ± SEM of individual biological replicates from one experiment.

upregulation of PD-1 on T cells for it to mediate its inhibitory function.

In female hosts, at least up to 7 days post transfer, PD-1−/− Marilyn T cells showed greater proliferation (**Figures 1B,C**; Figure S1 in Supplementary Material) and came to significantly outnumber the Marilyn-WT cells by ~2-fold and this was not affected by IL-7Rα blockade but was completely dependent on host MHC-II expression (**Figure 1D**). This supports our hypothesis that PD-1 deficiency enhances the TCR signaling response to tonic pMHC during LIP.

When we examined the expression of the anti-apoptotic molecule Bcl2 at 5 days post-cell transfer (a time during which rapid LIP was occurring), the PD-1−/− cells in female Rag−/− hosts showed a trend toward expressing less of this molecule (**Figure 2C**). Although we did not examine other IL-7/TCR signaling-associated anti-apoptotic molecules, such as Bcl-Xl and Mcl-1 (52), this suggests that any potential increased survival in the PD-1−/− population was not mediated by Bcl2. Importantly, examination of Bcl2 expression in T cells within all the recipient groups clearly indicated that Bcl2 expression was depressed in groups treated with anti-IL-7Rα and particularly in combination with pMHC deprivation in the CiiTA deficient hosts (**Figure 2C**). We also noted that at day 7, at which time cells were presumably still undergoing substantial LIP, IL-7Rα blockade did not reproducibly negatively impact the frequency of T cells in the blood in MHC-II sufficient hosts (**Figure 2D**). It is important to note that in contrast to studies that suggested that IL-7 signals were critical for the LIP of T cells (53, 54), anti-IL-7R treatment did not significantly inhibit proliferation in MHC-II sufficient hosts during the first week after thymocyte transfer when competition for pMHC would be expected to be low (**Figure 1B**; Figure S1 in Supplementary Material). The failure of T cells to undergo LIP in IL-7−/− × Rag−/− hosts (53, 54) is instead likely attributable to defects in lymph node structure and function as IL-7 is important for lymph node development (55). In contrast to our observation of no significant negative effect of anti-IL-7Rα treatment on the frequency of T cells in the blood of MHC-II sufficient recipients at day 7 post-transfer, IL-7Rα blockade led to significant decreases in absolute splenic T cell numbers by day 35 (**Figure 3B**). Together, these data suggest that by day 35, the recipients were more lymphoreplete and due to competition, tonic pMHC signals were no longer the most significant source for survival signals for the T cells and hence IL-7Rα blockade could now have a marked effect.

Despite its now clear role in controlling LIP in response to tonic pMHC-II signals, it was unclear whether PD-1 would be upregulated during LIP stably and sufficiently such that it would be detectable by flow cytometric staining. Indeed, at day 5 post-transfer, PD-1 expression was detectably increased by tonic pMHC signals in the WT T cell population but only among the highly proliferated cells (**Figure 2A**). The lack of obvious PD-1 expression on non- or intermediately proliferated cells (i.e., CTV mean fluorescence intensity ≥103 , **Figure 2A**) might suggest that regulation of LIP (and even perhaps normal primary T cell activation in a lymphoreplete host) by PD-1 may not require its high-level surface expression on T cells, or might occur primarily after many rounds of division in the highly proliferated cells. PD-1 upregulation may vary depending on the strength of the TCR:pMHC interaction experienced by a given T cell. Thus, the extent of PD-1 upregulation evoked upon LIP of Marilyn T cells in response to tonic pMHC may differ significantly from that of a polyclonal population (19) owing to a greater diversity of interaction affinities in the latter. Consistent with this notion, in the experiments described herein we noted that PD-1 expression levels were markedly lower among highly proliferated cells in female compared to male Rag−/− recipients of Marilyn T cells (data not shown).

CD5 is a negative TCR signaling regulator commonly considered as a marker of TCR affinity set during thymic T cell selection processes as well as a reliable marker of TCR activation (45–47). CD5 expression has been correlated with the propensity for T cells from various TCR transgenic backgrounds to undergo LIP in a lymphopenic host (56–59). We found that highly proliferated cells stimulated by tonic pMHC in both the WT and PD-1−/− Marilyn T cell populations contained subpopulations that had either up- or downregulated CD5 expression – with significantly higher overall CD5 expression in the PD-1−/− group (**Figure 2B**). Potentially, CD5 is acting here as a readout of the extent of the TCR signal received by cells undergoing LIP. One could also speculate that the PD-1−/− T cells are under pressure to maintain or increase CD5 expression due to a lack of co-inhibition from PD-1. However, pragmatically speaking these findings suggest that CD5 expression can change considerably in a monoclonal T cell population during LIP, which raises important questions about the validity of its use as a marker of T cell affinity/avidity for thymic pMHC as commonly seen in the literature, particularly if the cells under analysis have been exposed to a lymphopenic environment and have undergone LIP.

Compared to its effects on LIP in response to tonic pMHC signals, the effect of PD-1 deficiency on LIP of Marilyn T cells was even more pronounced in response to cognate pMHC in male Rag−/− hosts (**Figure 1D**) and it was also IL-7 independent. In CiiTA−/− hosts, APC within the eye and brain can use a CiiTA-independent pathway to express MHC-II in response to IFN-γ and TNF-α (44), and it is possible that low numbers of MHC-II sufficient thymic APC were co-transferred with the Marilyn thymocytes. While robust proliferation occurred in male CiiTA−/− × Rag−/− hosts, interestingly no appreciable increase in the PD-1−/−:WT ratio was seen by day 7 post transfer. While further studies are needed to fully understand this difference, two possibilities seem likely. Either the residual MHC class II expression in the CiiTA−/− recipients is limited to specific APC types that present antigen but do not engage PD-1 or alternatively it is simply the quantity of antigen exposure that determines whether PD-1 controls the response. We have previously proposed that chronic antigen signaling, as would occur with widely distributed pMHC ligands, leads to upregulation of co-inhibitory signals (60). From this viewpoint, the intermittent (not chronic) exposure to sparse pMHC in female or male CiiTA−/− × Rag−/− recipients would not lead to co-inhibitory signals such as those through PD-1.

Although we did not note any difference in IL-7Rα expression between WT and PD-1−/− T cells (**Figure 3C**), we did note that compared to female hosts, the IL-7Rα-expressing T cell populations in male recipients were considerably less numerous which is consistent with these cells constantly receiving strong TCR signals from high-affinity cognate pMHC interactions and downregulating IL-7Rα. Chronic high-level stimulation may also underlie the decreased CD44 expression noted in male MHC-II sufficient hosts (**Figure 3A**), a phenomenon previously observed in chronic viral infection (61). Indeed, the much larger size of the T cell compartment within male versus female hosts (**Figure 3B**) stems from the presence of pMHC that can provide a higher affinity signal (i.e., cognate pMHC) and could be viewed as a richer source of resources. Thus a HY containing host was able to support a much larger population of T cells.

Consistent with our interpretation that increased competition for pMHC signals in more lymphoreplete conditions would create greater dependence on IL-7Rα signals, we found that IL-7Rα blockade had a robust effect on preventing and reversing weight loss only when high numbers of PD-1−/− thymocytes were transferred (**Figure 4A**). IL-7 has previously been associated with the promotion of IL-3 and GM-CSF expression in human T cells (62). Very recently, it was reported that IL-7 could promote the development of a unique subset of GM-CSF and IL-3-producing T cells ("Th-GM") in mice and this was associated with encephalitogenicity in the experimental autoimmune encephalomyelitis model (63). Based on these reports, we considered that the effects of IL-7Rα blockade might be attributable to inhibition of GM-CSF expression and, therefore, we explored this *in vivo via* administration of anti-GM-CSF antibody to lymphopenic recipients of PD-1−/− HSC. We found a small, statistically insignificant effect of anti-GM-CSF blockade in this experiment on disease incidence with no significant effect on loss of weight (Figure S2 in Supplementary Material). These data suggest that the effect of blockade of IL-7Rα is unlikely to be solely mediated through effects on GM-CSF. Our findings support the concept that in situations of high competition for pMHC, limiting LIP potential by blocking the response to homeostatic cytokines like IL-7 can help to ameliorate the wasting aspect of systemic autoimmunity.

Our finding that the co-inhibitory molecule PD-1 controls LIP by modulating the response to tonic self-pMHC signals *in vivo* lends support to our hypothesis that the disease in lymphopenic recipients of PD-1−/− HSC or newly generated T cells results from exaggerated responses to normally tonic self-pMHC signals. These data are consistent with the concept that establishment of peripheral tolerance involves a tuning process (64), a tuning that is regulated at least in part by PD-1. Furthermore, the effects of PD-1 deficiency on control of LIP were independent of IL-7Rα-mediated signaling, and IL-7-mediated signals were largely irrelevant to the size of the overall T cell compartment while LIP potential was still high and rapid LIP in response to abundant pMHC was occurring within the hosts. These data suggest that in LIP-associated inflammatory disorders, such as immune reconstitution inflammatory syndrome in HIV patients (25, 65), or graft-versus-host disease, therapies aimed at reducing TCR signaling during early phases of reconstitution may be more effective and should take priority over approaches that aim to limit homeostatic cytokine-mediated signals to T cells. Furthermore, the ability of PD-1 to control tonic signals in response to self-pMHC may partially underlie the effectiveness of PD-1 blockade in tumor immunotherapy—for example, by enhancing survival and activation of T cells in the context of limiting neoantigen-derived pMHC in the tumor microenvironment or by promoting tumor clearance through the enhancement of collateral damage (43).

#### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of the Canadian Council on Animal Care. The protocol was approved by the University of Alberta Health Sciences Animal Care and Use Committee.

## AUTHOR CONTRIBUTIONS

KE designed, performed research and data analysis, and wrote and critically edited the manuscript. JL performed research and data analysis and critically edited the manuscript. LB provided reagents and critically edited the manuscript. CA designed research, performed data analysis, and critically edited the manuscript.

## ACKNOWLEDGMENTS

The authors would like to thank Perveen Anwar and HSLAS staff for assistance with animal care. This research has been supported by operating funds to CCA from the Canadian Institutes of Health Research (PS148588, FRN79521), the Muttart Diabetes Research and Training Centre, the Women & Children's Health Research Institute (WCHRI) through the generous support of The Stollery Children's Hospital Foundation and by doctoral studentships from the Alberta Diabetes Institute and Alberta Innovates - Health Solutions (AIHS) to KE and a senior scholar award from AIHS to CA.

#### REFERENCES

This manuscript includes material previously included in the doctoral dissertation of KE (66).

#### SUPPLEMENTARY MATERIAL

The supplementary material for this article can be found online at http://www.frontiersin.org/article/10.3389/fimmu.2017.01289/ full#supplementary-material.

and availability. *Proc Natl Acad Sci U S A* (2007) 104:17765–70. doi:10.1073/ pnas.0708767104

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

*Copyright © 2017 Ellestad, Lin, Boon and Anderson. 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.*

# Prior to Peripheral Tolerance, newly generated cD4 T cells Maintain Dangerous autoimmune Potential: Fas- and Perforin-independent autoimmunity controlled by Programmed Death-1

*Kristofor K. Ellestad1,2\*, Govindarajan Thangavelu2,3, Yohannes Haile2 , Jiaxin Lin2,3,4, Louis Boon5 and Colin C. Anderson1,2,3,4\**

*1Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada, 2Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada, 3Department of Surgery, University of Alberta, Edmonton, AB, Canada, 4Alberta Transplant Institute, University of Alberta, Edmonton, AB, Canada, 5Bioceros B.V., Utrecht, Netherlands*

#### *Edited by:*

*Kelen Cristina Ribeiro Malmegrim, University of São Paulo, Brazil*

#### *Reviewed by:*

*Philippe Saas, INSERM UMR1098 Interactions Hôte-Greffon-Tumeur & Ingénierie Cellulaire et Génique, France Federica Casiraghi, Istituto Di Ricerche Farmacologiche Mario Negri, Italy*

#### *\*Correspondence:*

*Kristofor K. Ellestad<EMAIL_ADDRESS>Colin C. Anderson<EMAIL_ADDRESS>
#### *Specialty section:*

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

> *Received: 21 October 2017 Accepted: 04 January 2018 Published: 24 January 2018*

#### *Citation:*

*Ellestad KK, Thangavelu G, Haile Y, Lin J, Boon L and Anderson CC (2018) Prior to Peripheral Tolerance, Newly Generated CD4 T Cells Maintain Dangerous Autoimmune Potential: Fas- and Perforin-Independent Autoimmunity Controlled by Programmed Death-1. Front. Immunol. 9:12. doi: 10.3389/fimmu.2018.00012*

Lymphopenia can result from various factors, including viral infections, clinical interventions, or as a normal property of the fetal/neonatal period. T cells in a lymphopenic environment undergo lymphopenia-induced proliferation (LIP) to fill the available "niche" as defined by peptide–MHC and homeostatic cytokine resources. We recently reported systemic autoimmunity following reconstitution of the lymphoid compartment of Rag1−/− mice with PD-1−/− hematopoietic stem cells or by transfer of thymocytes, but not splenocytes, suggesting that programmed death-1 (PD-1) plays a crucial role in controlling recent thymic emigrants (RTE) and preventing autoimmunity upon their LIP. However, it is unclear whether RTE residing within the periphery of a lymphoreplete host maintain enhanced autoimmune generating potential or if this property only manifests if RTE experience a lymphopenic periphery immediately after export from the thymus. Furthermore, it is unclear which of a variety of T cell effector mechanisms generate pathology when control of RTE by PD-1 is lacking. Herein, we determined that PD-1 is upregulated on CD4 T cells undergoing the natural LIP characteristic of the neonatal period. Newly generated T cells lacking PD-1 maintained an enhanced autoimmune potential even after residence in a lymphoreplete periphery, emphasizing the importance of PD-1 in the establishment of peripheral tolerance. Neither Fas nor perforin-dependent killing mechanisms were required for autoimmunity, while host MHC-II expression was critical, suggesting that LIP-driven autoimmunity in the absence of PD-1 may primarily result from a CD4 T cell-mediated systemic cytokinemia, a feature potentially shared by other autoimmune or inflammatory syndromes associated with immune reconstitution and LIP.

Keywords: tolerance, autoimmunity, co-stimulation, co-inhibition, lymphopenia, checkpoint inhibitor, cytokine storm, graft versus host

**Abbreviations:** LIP, lymphopenia-induced homeostatic proliferation; HSC, hematopoietic stem cells; pTreg, peripherally generated regulatory T cell; Treg, regulatory T cell; SP, single positive; RTE, recent thymic emigrants; pMHC, peptide–MHC; Tcon, conventional T cell; PD-1, programmed death-1; MFI, mean fluorescence intensity; WT, wild type.

## INTRODUCTION

Thymic selection processes can be viewed as the first "filtration" step on the developing T cell repertoire. Although these processes serve to remove the majority of strongly self-reactive T cells from the developing repertoire or convert them to thymic Treg, some self-reactive conventional T cell (Tcon) clones escape (1). This is, for example, evidenced by the ability to induce autoimmune diseases, such as myelin-oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in mice (2). Peripheral tolerance mechanisms, such as tuning, anergy, deletion, or conversion to peripherally generated Treg (pTreg), are thus important for the establishment and maintenance of immune tolerance and can be viewed as a second filter on the peripheral T cell repertoire.

Aside from the naturally lymphopenic neonatal period associated with immune system development (3), lymphopenia also occurs in a variety of clinical settings. Hematopoietic cell transplantation, T cell depleting therapy for solid organ transplantation, cancer chemotherapy, as well as HIV infection are all associated with lymphopenia of varying severity. In the lymphopenic state, cells undergo a process known as lymphopeniainduced proliferation (LIP), which is facilitated and regulated in its extent by the availability of "resources" for T cells that together define T cell "space" (4). These include peptide–MHC complexes (derived from self or otherwise) that can mediate at least a weak, "tonic" signal through the TCR, as well as homeostatic cytokines, such as IL-7 and IL-15. Regulatory T cells (Treg) (5–13), costimulatory molecules such as CD28 (7, 14), as well as molecules with known co-inhibitory activity [e.g., BTLA (15), LAG-3 (16), TGFβRII (17, 18)] can modulate the kinetics of LIP and the maximum size of the T cell compartment. In addition, LIP can promote autoimmune disease and LIP of TGFβRII−/− T cells results in autoimmunity (17).

Programmed death-1 (PD-1), a co-inhibitory receptor expressed on activated T cells, enters the immune synapse upon T cell:APC interaction and is known to recruit the phosphatase SHP2 upon receptor ligation which can dampen proximal TCR signaling cascades (19–21) or co-stimulatory signals through CD28 (22). In contrast to the C57BL/6 PD-1−/− mouse, which displays a relatively mild phenotype characterized by development of a lupus-like disease with spontaneous arthritis and glomerulonephritis upon aging (23), we have shown that reconstitution of the lymphoid compartment of lymphopenic adult Rag1−/− animals *via* transfer of PD-1−/− hematopoietic stem cells (HSC) leads to a rapid, severe, and lethal systemic autoimmune disease soon after the first newly generated T cells, or recent thymic emigrants (RTE), emerge into the periphery (24). The disease is associated with infiltration of CD4 and CD8 T cells into multiple organs, including heart, liver, and kidney, although Rag1−/− Kb−/−Db−/− animals remain fully susceptible suggesting that MHC-I-restricted CD8 T cells are dispensable for disease. Significantly elevated levels of several pro-inflammatory cytokines and chemokines in serum (24) as well as elevated pro-inflammatory cytokine transcripts in infiltrated organs (25) are also associated with disease. Macroscopically the disease is characterized by kyphosis, cachexia, diarrhea, and skin and ocular lesions. Interestingly, PD-1−/− HSC reconstitution of day 1 Rag−/− neonates results in a drastically reduced incidence of disease (24), suggesting that limited T cell "space" due to small anatomic size (e.g., of lymph nodes) or other factors can limit the aberrant activation of T cells promoted by LIP. Indeed lymph node-deficient Rag−/−γc−/− or irradiated LTα−/− hosts were also resistant to disease after PD-1−/− HSC transfer (24). Transfer of PD-1-deficient thymocytes to adult Rag1−/− mice likewise results in autoimmunity; however, transfer of splenocytes from mature PD-1−/− mice does not result in disease. These data suggest that the RTE/newly generated T cell population, which has not yet been subject to peripheral tolerance mechanisms, has greater autoimmune potential than established peripheral T cells and that PD-1 is critically important for controlling their activity during LIP. However, several lines of evidence suggest that newly generated T cells have properties that promote tolerance (26). It is not clear whether newly generated T cells in an adult retain a heightened potential for the generation of autoimmunity after their emergence into the periphery or whether exposure of newly generated T cells to a lymphoreplete environment leads to their rapid tolerization. Herein, we have taken advantage of the Rag2pGFP transgenic (Tg) mouse strain in which GFP is expressed during early T cell development and remains detectable as a marker of newly generated lymphocytes after their emergence into the periphery (27, 28). PD-1−/− peripheral newly generated T cells or established T cells were purified from adult mice and tested for their ability to drive autoimmunity upon transfer into lymphopenic hosts. We found that purified peripheral PD-1−/− newly generated T cells are similar to thymocytes in their ability to drive systemic autoimmunity upon transfer to lymphopenic hosts. Using lymphopenic hosts lacking Fas or MHC-II expression, or PD-1−/− donors lacking perforin expression, we also show that host MHC-II expression is required for disease after PD-1−/− HSC transfer, and that Fas- and perforindependent killing mechanisms are dispensable for disease. Taken together, our data suggest that even in a lymphoreplete adult host, peripheral newly generated T cells retain a heightened potential for LIP-driven autoimmunity in the absence of PD-1, which is mediated by CD4 T cells.

#### MATERIALS AND METHODS

#### Mice

B6.129S7-*Rag1*tm1Mom/J (Rag1−/−, Rag−/−), B6.Cg-*Foxp3*tm2(EGFP)Tch/J (FoxP3EGFP, used in the present manuscript as WT), B6.MRL-*Fas*lpr/J (Faslpr), C57BL/6-*Prf1*tm1Sdz/J (Prf1−/−), and B6.129S2- *Ciita*tm1Ccum/J (CiiTA−/−) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Days 13–14 gestation pregnant C57BL/6-CD45.1 (B6-CD45.1) mice were purchased from NCI (Frederick, MD, USA). C57BL/6-*Pdcd1*−/− (backcrossed 11 generations to C57BL/6) were originally generated by Prof. T. Honjo and colleagues (23). FoxP3EGFP × *Pdcd1*−/− mice were generated by crossing the above FoxP3EGFP and B6-*Pdcd1*−/− mice and are referred to in the present manuscript simply as PD-1−/−. PD-1−/− × Prf1−/− mice were generated by crossing the above PD-1−/− and Prf1−/− mice without any selection for the FoxP3EGFP transgene. Rag−/− × CiiTA−/− mice were generated by crossing the above Rag−/− and CiiTA−/− mice. Faslpr × Rag−/− mice were generated by crossing the above Faslpr and Rag−/− mice. B6.Rag2pGFP (Rag2pGFP) mice (27, 28) were kindly provided by Pamela Fink (University of Washington, Seattle, WA, USA). Rag2pGFP × PD-1−/− mice were generated by crossing Rag2pGFP mice with the above B6-*Pdcd1*−/−, with screening and selection of breeders for high GFP expression. Marilyn Rag2−/− CD4+ anti-HY/I-Ab TCR Tg mice (called Marilyn herein) were generated by Lantz and colleagues (29) and were originally obtained from the NIAID exchange program. Cells from Marilyn mice were tracked based on their expression of CD4, CD45.2, and a Vβ6 TCR and lack of CD45.1. Animals were cared for in accordance with the guidelines of the Canadian Council on Animal Care and housed under clean conventional housing conditions at the University of Alberta Health Sciences and Laboratory Animal Services facilities (HSLAS).

#### Cell Preparations and Adoptive Transfer Experiments

For experiments involving transfer of thymocytes or peripheral T cells, recipient NK cells were depleted [to avoid potential NK-mediated killing of the input cells (30–32)] by treatment on days −4, −1, and +2 with 0.3 mg per mouse of anti-NK1.1 (PK136) injected intraperitoneally. Thymocytes or splenocytes for injection were prepared by disruption in HBSS (Gibco) + 2% fetal bovine serum (FBS, Sigma-Aldrich) through a 70-µm nylon cell strainer. Cells were centrifuged at ~300 × *g* for 5–10 min at room temperature, and red blood cell lysis was performed by resuspending cells in ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA) with incubation for 5 min, followed by addition of ~10 volumes of HBSS + 2% FBS, and two cycles of centrifugation at ~300 × *g* for 5–10 min, and resuspension in PBS with no additives. If cells were prepared for further manipulation (e.g., staining and sorting), they were instead washed and resuspended in HBSS + 2% FBS. For experiments involving adoptive transfer of HSC, fetal liver cells (FLC; embryonic days 14–15) were used as a source of HSC. On ice, fetal livers were disrupted by repeated trituration through a 5-mL serological pipet, followed by filtration through a 70-µm nylon mesh filter basket. Cells were then centrifuged at ~300 × *g* for 10 min at 4°C, and resuspended according to their intended further use. FLC were used either fresh or frozen. For immediate use for *in vivo* transfers, FLC were resuspended at 50 × 106 cells/mL in PBS. For freezing, cells were resuspended at 2 × 108 cells/mL in 90% FBS + 10% DMSO, frozen in a −1°C per minute rate-controlled cell freezing apparatus in a −80°C freezer and transferred to the vapor phase of a liquid nitrogen tank for long-term storage. 1.5 × 107 fresh or frozen FLC were transferred intravenously to the indicated recipients. In islet transplantation experiments, female Rag−/− recipients with an established islet transplant received a mixture of 4:1 female B6-CD45.1 and female Marilyn FLC (total of 10–15 × 106 cells). Sex of fetuses was determined by PCR as described (33).

#### Definition of Disease and Data Analysis

Macroscopic signs of disease in HSC/thymocyte/peripheral cell recipients included cachexia/weight loss (>15%), kyphosis (hunched appearance), ruffled fur, dermatitis, ocular lesions, and diarrhea. Recipient mice were no longer considered disease free when two or more of the above symptoms were evident, or if mice lost ≥20% body weight. For thymocyte experiments, calculation of weight loss for disease determination was performed relative to weights at day 0 or day 1 relative to cell transfer. For HSC experiments, calculation of weight loss was determined relative to initial weight measurements taken prior to day 30. Unless otherwise indicated, animals were used at 8–16 weeks of age.

#### Islet Transplantation

Diabetes was induced in recipient mice *via* treatment with streptozotocin (Sigma-Aldrich) at 185–190 mg/kg. Recipients were considered to be diabetic after two consecutive blood glucose measurements of >20 mM using a OneTouch Ultra glucometer (Lifescan Canada, Burnaby, BC, Canada). Pancreatic islets were isolated from male or female Rag−/− mice as previously described (34). Diabetic recipients were transplanted with 500 islets placed under the kidney capsule and thereafter monitored for return to normoglycemia. All recipients returned to normoglycemia within 48 h of islet transplantation and remained normoglycemic until they were given FLC between 3 and 6 months after islet transplantation; post fetal liver injection some mice with male islets became hyperglycemic (rejected the islet transplant) as described in the Section "Results."

### Antibodies, Flow Cytometry, and Fluorescence-Activated Cell Sorting (FACS)

For flow cytometric staining and sorting, fluorophore-labeled antibodies against the following markers were obtained from eBioscience (San Diego, CA, USA) unless otherwise indicated: CD4 (RM4-5), TCRβ (H57-597), CD8α (53-6.7), PD-1 (J43), Ki-67 (SolA15), CD44 (IM7), CD19 (1D3), Granzyme B (NGZB), CD45.1 (A20), CD45.2 (104), Vβ6 (RR4-7). Antibodies were used at manufacturer's recommended concentrations. Flow cytometric staining always used an Fc block cocktail to block nonspecific staining. Fc block cocktail consisted of 3 mL each of normal mouse, rat, and hamster serum, with addition of 0.3 mg of anti-CD16/32 antibody (clone 2.4g2, BioXCell). Fixation and permeabilization were performed using the eBioscience FoxP3 Fixation/Permeabilization buffer kit (Thermo Fisher) according to the manufacturer's protocols. For cell sorting, a BD Influx cell sorter was used controlled with Spigot software (Beckton Dickinson, Franklin Lakes, NJ, USA). Briefly, for sorting source cells were stained and resuspended in HBSS + 20% FBS + 10 mM HEPES and sorted directly into FBS supplemented with 10 mM HEPES. Standard flow cytometric analysis was performed using a BD LSR II instrument. Flow cytometric data analysis was performed using FlowJo (Treestar software, Portland, OR, USA).

## Statistical Analysis

Statistical analysis was performed using Graphpad Prism software. Details of statistical tests used are provided in figure legends. However, in general, for comparisons of two groups, Student's *t*-test was used. In cases of comparison of groups with unequal variances, Welch's correction was applied. Unless otherwise noted, for multiple group comparisons, one-way ANOVA with Tukey's multiple comparison test was used. Disease onset/incidence was compared by the Kaplan–Meier method. Probability values reported for survival curve comparisons were calculated using the Mantel–Cox method.

## RESULTS

## PD-1 Expression Is Tied to Lymphopenia-Induced Proliferation in Newly Generated T Cells

Programmed death-1 expression appears to be critical for the generation of immune tolerance during LIP of newly generated T cells. We previously showed that newly generated T cells in a lymphopenic setting (i.e., in a lymphopenic recipient of WT HSC) had a high proportion of PD-1 expressing T cells that diminished over time (24). Whether the higher PD-1 expression on newly generated T cells was dependent on exposure to the lymphopenic environment or is an intrinsic property of newly generated T cells even in lymphoreplete mice has not been fully assessed. We, therefore, examined PD-1 expression in the steady state in both TCRβ+ thymocytes and peripheral splenocytes from 10- to 12-week-old adult Rag2pGFP animals (**Figures 1A,B**). Unexpectedly, peripheral newly generated CD4 single positive (SP) splenocytes expressed very little PD-1 while approximately 15% of established or "mature" GFP− CD4 SP T cells were found to be PD-1 positive (**Figure 1A**). Likewise, mean fluorescence intensity of PD-1 staining was significantly higher in the CD4 SP GFP− cells compared to the GFP+ population (**Figure 1B**). Neither established nor newly generated T cells within the splenic CD8 SP population expressed appreciable levels of this co-inhibitor. Within the thymocyte population, approximately 90% of the CD4 SP and 85% of the CD8 SP were GFP+ (data not shown), with the remainder presumably representing mature cells that had recirculated from the periphery back to the thymus, although it is conceivable that at least a subset could represent cells that failed to exit the thymus for longer than 3 weeks post-VDJ recombination (28). GFP+ thymic CD4 and CD8 SP cells were slightly enriched for PD-1 positivity (3.1 and 2.6% positive, respectively, **Figure 1A**) compared to their splenic counterparts (both 1.3%), although this difference was not statistically significant. Similar to what was seen in splenocytes, the thymic CD4 SP GFP− population contained a sizeable population of PD-1+ cells (26%), and thus, these more established cells had higher overall PD-1 expression compared to the GFP+ newly generated T cell population (**Figures 1A,B**). Unlike their splenic counterparts, there was a trend toward increased PD-1 positivity and overall PD-1 expression in the thymic CD8 SP GFP− cells compared to the GFP+ cells although this difference was not statistically significant (**Figures 1A,B**).

Thus far, our data indicated that in the steady state, RTE of adult lymphoreplete mice do not express high levels of PD-1 (**Figure 1**) while adoptive transfer of RTE-generating HSC to lymphopenic mice did lead to high PD-1 expression (24). We, therefore, asked whether heightened PD-1 expression is peculiar to LIP in adoptive transfer or whether it might occur naturally (without adoptive transfer) during the LIP triggered by the lymphopenic state of the neonatal period (3). We examined PD-1 expression on peripheral lymphocytes of 10-day-old Rag2pGFP neonatal mice. More than 97% of thymic CD4 SP cells are GFP+ in young Rag2pGFP mice (Figure S1B in Supplementary Material), and all splenic T cells in 10-day-old neonates would be considered to be newly generated based on elapsed time from initial development and, therefore, might also be expected to be GFP+. However, a significant percentage (~30% of CD4, ~65% of CD8) of the splenic T cells had lost detectable GFP fluorescence in 10-day-old neonates (**Figure 1C**). The association between loss of GFP in CD4 T cells and acquisition of a CD44high memory phenotype, as well as an association of CD44 with Ki-67 expression, a marker of cycling cells (**Figure 1D**, upper panels) suggests that this was due to these cells having undergone multiple rounds of LIP, diluting GFP. Importantly, loss of GFP expression, high CD44 expression, and high Ki-67 expression were all associated with elevated PD-1 expression (**Figure 1D**, bottom panels): more than 90% of PD-1-expressing CD4 T cells were Ki-67 high. Very few CD8 T cells expressed PD-1 in the neonates at this age and PD-1 expression was not associated with heightened Ki-67 expression in these cells (Figure S1A in Supplementary Material).

Together, these data indicate that while PD-1 is upregulated on T cells following LIP or a period of residency in the periphery (possibly due to encounter with antigen), only very low/barely detectable levels of PD-1 are expressed on newly generated adult T cells. Thus, the heightened PD-1 expression on newly generated T cells seen in lymphopenic HSC recipients is not an intrinsic characteristic of these cells but is LIP-induced. The naturally occurring LIP of the neonatal period also upregulated PD-1 on newly generated CD4 T cells, suggesting PD-1 is involved in establishing tolerance under physiologic conditions (i.e., not just under conditions of cell transfer).

### Steady State Peripheral Newly Generated T Cells in Adult Mice Maintain Heightened Autoimmune Potential Relative to Established T Cells

Our previous studies showed that newly generated T cells exported from the thymus directly into a lymphopenic environment are critically dependent on PD-1 to establish tolerance and prevent autoimmunity. In contrast, PD-1 was not needed to maintain tolerance after transfer of peripheral T cells from PD-1−/− adult animals, which would largely be comprised of established cells. However, it is unknown whether steady-state newly generated T cells in the periphery of immunocompetent adult mice retain this heightened potential for autoimmunity or if instead the peripheral tolerance process is rapid and newly generated T cells are immediately tolerized upon export to a lymphoreplete environment. In order to test the hypothesis that steady-state peripheral RTE/newly generated T cells had

Figure 1 | Newly generated T cells in adults lack programmed death-1 (PD-1) expression while in neonates their expression of PD-1 is linked to LIP*.* (A) Representative flow cytometric analysis of splenocytes and thymocytes from 10- to 12-week-old adult Rag2pGFP mice for PD-1 expression among TCRβ+ CD4 or CD8 single positive (SP) cells that are GFP+ (newly generated T cells) or GFP− (mature cells). T cells from PD-1−/− mice were also stained as a control for background. Values above gates are the group average %PD-1 positive cells within the mature GFP− population (black text) or GFP+ population (green text), ±SEM. (B) Mean fluorescence intensity (MFI) of PD-1 staining in CD4 or CD8 SP, GFP+ or GFP− T cells from Rag2pGFP splenocytes or thymocytes from the experiment depicted in (A). \*\**p* < 0.01, one-way ANOVA with Tukey's multiple comparison test. (C) Comparison of percent GFP+ cells among peripheral splenocytes in the indicated cell populations in adult vs. neonatal mice ± SEM. *n* = 8 per group. (D) Representative (*n* = 3) flow cytometry plots of the indicated markers in splenic TCRβ+ CD4 SP T cells from 10-day-old Rag2pGFP neonates. Histogram (right) analyses of Ki-67 on CD44 high or PD-1 high cells and CD44 low/negative cells or PD-1 low/negative cells employed gates as shown in the red and blue rectangles (middle dot plots), respectively. Data presented are from samples that were either permeabilized and stained with antibodies to TCRβ, CD4, CD8, PD-1, CD44, and Ki-67, or non-permeabilized and stained with antibodies to TCRβ, CD4, CD8, PD-1, and CD44 (to allow analysis of GFP).

increased ability to drive autoimmunity in a lymphopenic host, we generated B6 Rag2pGFP x PD-1−/− mice and purified the GFP+ or GFP− T cell populations from splenocytes of adult animals by FACS. Purified cells or thymocytes containing an equivalent number of SP T cells were transferred to Rag−/− recipient animals and disease was monitored. Approximately 20 days after transfer, mice that received either PD-1−/− thymocytes or purified GFP+ newly generated T cells began to develop autoimmune disease while the recipients of established cells were relatively spared (**Figure 2A**, left panel). Similarly, while all recipient mice lost some weight beginning almost immediately after cell transfer until around 2 weeks posttransfer, after this point PD-1−/− established T cell recipients began to regain their lost weight while the thymocyte and newly generated T cell recipients clearly and significantly diverged from the established cell recipients and continued to lose weight (**Figure 2A**, right panel). Despite the clearly higher propensity of newly generated vs. established cells to drive autoimmunity, it should be mentioned that a low proportion of the GFP− established T cell recipients also met the criteria for disease albeit at a much later time point relative to the other groups (**Figure 2A**). Approximately, 40% of the purified GFP+ newly generated cells were CD4 SP and 56% were CD8 SP (a CD4:CD8 ratio of ~0.7:1), whereas the purified GFP− population contained approximately 80% CD4 SP and 17% CD8 SP (CD4:CD8 ratio ~4.7:1, **Figure 2B**). While our experiments were designed to test whether the newly generated T cells as a whole have heightened autoimmune potential, the differences in the proportions of CD4 vs. CD8 SP cells in newly generated vs. established T cells could potentially contribute to the differences in their ability to drive disease.

## T Cells with Specificity for a Neo-Self Antigen Can Persist in the Periphery with a Slow Decline in Frequency Over time

The finding that newly generated T cells have a greater potential for autoimmunity than established peripheral T cells appears incongruent with several reported aspects of RTE biology (26) such as reduced effector functions or an increased propensity to convert to pTreg (25, 35, 36) compared with established T cells. These studies raise the question of how newly generated T cells could have a greater capacity to cause autoimmune disease. There are at least two "filters" against self-reactivity, namely central and peripheral tolerance. We surmised that newly generated T cells may have a greater potential for generating autoimmunity because they have yet to pass through the second filter, peripheral tolerance. Autoimmunity caused by newly generated T cells during LIP might be due in part to a greater frequency of T cells with self-specific receptors than are present in the established T cells that have undergone peripheral tolerance mechanisms, such as deletion or conversion to pTreg. In order to explore the concept that potentially dangerous self-reactive cells could persist for a significant length of time in a lymphoreplete periphery post immune reconstitution of lymphopenic mice and be held in check by peripheral tolerance mechanisms without resulting in overt immune pathology, we used the HY antigen expressed by an established graft as a model neo-self antigen (33). We gave mixed Marilyn (anti-male antigen, HY/ IAb ) and WT B6 female HSC to Rag−/− female mice bearing an established islet graft from male or control female Rag−/− donors and monitored subsequent graft rejection and the percentage of cells that were Marilyn over time post immune reconstitution (**Figure 2C**). We found that reconstitution with a mixture of Marilyn and WT HSC (1:4 Marilyn to WT) led to a small but significant fraction (<30%; 5 of 17) of recipients rejecting the transplant. This rejection appeared to be associated with the appearance in the periphery of a high ratio of Marilyn to WT T cells and earlier export of Marilyn T cells into the periphery compared to WT T cells (data not shown). Nevertheless, the vast majority of recipients tolerated the peripheral male islet transplant. In these male islet graft recipients, we compared the frequency of Marilyn T cells (CD45.2+, CD45.1−, CD4+Vβ6+) to recipients of a control female islet graft. The data in **Figure 2D** show that the male islet transplant, given 3–4 months prior to a 1:4 (Marilyn:WT) mixture of HSC, causes a slow reduction in frequency of neo-self antigen-specific Marilyn T cells. The mice with male but not female islets were tolerant to the male antigen, as they accepted a male skin graft given 4–6 months post HSC (**Figure 2E**). Thus, peripheral CD4 tolerance was associated with a decreased frequency of neo-self antigen-specific T cells over time, with newly generated T cells having a higher frequency of CD4 T cells specific to the neo-self antigen.

## Host MHC Class II but Not Fas Is Required for Disease upon Transfer of PD-1**−**/**−** HSC

We previously showed that adult Rag1−/− Kb−/−Db−/− mice were fully permissive for the development of autoimmune disease following transfer of PD-1−/− HSC (24); importantly, no perturbations in the time to disease onset or severity were reported compared to Rag−/− hosts, as would be reasonably expected if the disease were CD8 T cell independent. To delineate the importance of CD4 T cells in this disease model, we generated Rag−/− and MHC Class II transactivator-deficient (CiiTA−/−) mice, which are largely deficient in MHC-II expression (37). We transferred PD-1−/− HSC to Rag−/− or Rag−/− × CiiTA−/− hosts and monitored the mice for signs of disease. In addition, we examined potential effector mechanisms of disease. Two canonical pathways of T cell killing, namely the FasL-Fas and perforin-dependent pathways, have been described (38). In order to begin to elucidate which, if any, of these pathways are involved in mediating autoimmunity in the PD-1−/− HSC model, we generated a double mutant Faslpr × Rag−/− mouse, which lacks functional Fas expression (39). While Rag−/− mice developed systemic autoimmune disease around day 44 after transfer, Rag−/− × CiiTA−/− were completely spared (**Figure 3A**). Similarly, the pronounced weight loss encountered in the Rag−/− recipient group was not seen in the Rag−/− × CiiTA−/− recipients and indeed the latter group actually gained weight for much of the experiment (**Figure 3B**) despite CD8 T cell and B cell development by day 46 post-transfer (**Figure 3C**). These data indicate that MHC-II expression in the host is required for disease after PD-1−/− HSC transfer, suggesting disease is dependent on CD4 T cells. Transfer of

symptoms. (A) *Left panel:* disease incidence in recipients of thymocytes, GFP+ newly generated T cells or GFP− established T cells. Survival curve comparison demonstrated a significant difference between the three groups with *p* = 0.003; PD-1−/− GFP+ newly generated cell recipient vs. established cell recipient curves *p* = 0.02; PD-1−/− thymocyte recipient vs. PD-1−/− GFP+ newly generated cell recipient curves, *p* = NS. Data are combined from two independent experiments, starting *n* values are indicated in the legend. *Right panel:* weight changes in recipients of indicated cells from two independent experiments ± SEM. Weight observations for which similar time points were available between experiments (±2 days) were combined and used for this analysis. \**p* < 0.05, GFP+ newly generated T cells vs. GFP− established T cells, one-way ANOVA with Tukey's multiple comparison test. (B) Representative GFP expression (left panel) and CD4 and CD8 SP proportions (right 2 panels) in purified cell populations used in (A). (C) Diagram of experimental approach using an established minor mismatched transplant as a model peripheral neo-self antigen and anti-donor (HY) TCR transgenic (Tg) Marilyn T cells to track the frequency of CD4 T cells specific to the "neo-self" antigen over time. Male (or control female) islet transplants were allowed to heal ≥3 months into streptozotocin (STZ)-induced diabetic female Rag−/− recipients prior to immune reconstitution *via* transfer of female hematopoietic stem cells (HSC) from CD45 congenic wild-type (B6-CD45.1) and monoclonal TCR Tg mice. (D) Frequency of Marilyn T cells (CD4+ CD45.2+ CD45.1− Vβ6+) within total CD4 T cells in the peripheral blood of recipients with male vs. female islets that maintained normoglycemia is shown (mean, SEM, and *n* for each time point is shown). \**p* < 0.05, Student's *t*-test. (E) Six months post fetal liver cell injection, male skin grafts were transplanted to normoglycemic Rag−/− recipients that had either a male (*n* = 5) or female (*n* = 4) islet transplant and grafts were monitored for rejection. Survival curves are significantly different with *p* = 0.018, Mantel–Cox test.

disease incidence among HSC recipients—groups are significantly different with *p* = 0.0002. Data are combined from two independent experiments, with starting numbers per group indicated in the legend. (B) Weight changes in recipients relative to day 27 post-transfer, combined from two independent experiments, ±SEM. Weight observations for which similar time points were available between experiments (±2 days) were combined. For each timepoint, only data from groups with *n* > 2 are presented. \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, Faslpr × Rag−/− vs. CiiTA−/− × Rag−/−, one-way ANOVA with Tukey or Kruskal–Wallis with Dunn's multiple comparison test (days 27–49) or Student's *t*-test (day > 51). (C) % of all peripheral blood cells that were CD4+ or CD8+ T cells or CD19+ B cells in Faslpr × Rag−/− and CiiTA−/− × Rag−/− recipients from one experiment in (A,B) above, measured at day 46 post-HSC transfer.

PD-1−/− HSC to Faslpr × Rag−/− double mutants revealed that host Fas expression was completely dispensable for the generation of autoimmunity in this model (**Figures 3A,B**). No statistically significant difference in the survival curves of the Rag−/− and Faslpr × Rag−/− recipient groups was detected. Similarly, weight loss in Faslpr × Rag−/− hosts was indistinguishable from that seen in the Rag−/− hosts (**Figure 3B**). Thus, host Fas expression is not required for disease post PD-1−/− HSC transfer.

## Perforin Expression in the Lymphoid Compartment Is Not Required for LIP-Driven Autoimmunity after Transfer of PD-1**−**/**−** Thymocytes

Next, we examined whether perforin expression in lymphocytes was required for disease following transfer of PD-1−/− thymocytes to a lymphopenic host. We generated perforin knockout (Prf1−/−) and PD-1−/− mice. The double knockout animals we were able to generate were the F1 progeny of heterozygous crosses and never successfully bred in our facility as homozygous double knockouts (unpublished observations). We transferred thymocytes from the Prf1−/− x PD-1−/− or PD-1−/− mice to Rag−/− recipients and monitored the animals for disease and weight loss. Prf1−/− × PD-1−/− thymocyte recipients all developed autoimmune disease between days 13 and 21 post-transfer, while the PD-1−/− thymocyte recipients had a relatively slightly delayed course of disease (**Figure 4A**, *p* = 0.01). Furthermore, weight loss (**Figure 4B**) was significantly greater in the Prf1−/− × PD-1−/− thymocyte recipients, and in general the severity of symptoms, particularly diarrhea, appeared greater in this group compared to PD-1−/− thymocyte recipients. In addition to these findings, we also performed two experiments to question whether Prf1−/− was required for disease in the HSC transfer model. In the first experiment, Prf1−/− × PD-1−/− T cell depleted bone marrow cells were transferred to Rag−/− hosts. In the second, Prf1−/− HSC were transferred to Rag−/− hosts treated intraperitoneally with 200 μg/mouse monoclonal blocking anti-PD-1 antibody (clone J43) every 2 days from day 25 until termination. In both of these experiments, recipients developed autoimmunity (data not shown) further supporting the notion that perforin expression in T cells is not required for disease in this model.

Because of the potential for functional redundancy between the Fas–FasL and perforin-dependent killing pathways, we also tested whether adoptive transfer of cells from diseased Rag−/− recipients of Prf1−/−PD-1−/− thymocytes to Faslpr × Rag−/− hosts would result in the development of autoimmunity. Indeed, splenocytes + lymph node cells from the diseased Prf1−/−PD-1−/− thymocyte recipients generated robust development of autoimmunity when transferred either to Rag−/− or Faslpr × Rag−/− recipients (disease onset: day 18, 22, >22 vs. 13, 18 × 3, 22, respectively; *p* = NS). Although both recipient groups lost weight, by day 22 post-adoptive transfer the Faslpr × Rag−/− hosts had lost significantly more weight than the Rag−/− recipients (25 vs. 15%, **Figure 4C**), suggesting that Fas in the host may actually play an immunoregulatory role. Taken together, these data show that neither perforin expression in T cells nor Fas expression in the host are required for LIP- and newly generated T cell-driven autoimmunity in the context of PD-1 deficiency.

## DISCUSSION

Mechanisms of T cell homeostasis function to maintain a diverse repertoire of sufficient size for effective immune surveillance of the host. Reconstitution of the T lymphocyte compartment of a lymphopenic host by HSC transplant or transfer of T cells results in LIP as the cells expand to fill the available niche defined by

Figure 4 | Systemic autoimmunity after PD-1−/− thymocyte transfer to lymphopenic recipients is independent of both perforin and Fas-mediated killing. 10 × 106 Thymocytes from PD-1−/− or Perforin−/− × PD-1−/− (Prf1−/− × PD-1−/−) animals were transferred i.v. to NK depleted adult Rag−/− recipients and mice were monitored for disease symptoms. (A) Kaplan–Meier survival curve analysis of disease incidence among individual recipients of the indicated thymocytes. Survival curve comparison demonstrated a significant difference between the two groups with *p* = 0.01. (B) Weight change among the recipients in (A), ±SEM. Data are from one experiment, starting *n* values are indicated in the legend. (C) Splenocytes and inguinal lymph node cells were harvested from three diseased Prf1−/− × PD-1−/− thymocyte recipients from the experiment depicted in (A) at day 28 post-transfer, and 20 × 106 cells were transferred i.v. into NK depleted, adult Rag−/− or Faslpr × Rag−/− recipients. One Faslpr × Rag−/− recipient received 10 × 106 cells. Weight change among cell recipients, ±SEM. \**p* < 0.05, Student's *t*-test. Data are from one experiment with starting *n* values in the legend.

available peptide–MHC (pMHC) and cytokine "resources." The relative abundance of resources in a host with respect to the numbers of competitors for those resources can be considered as a way to define the "severity" of lymphopenia in that host or its "LIP-potential" (40). Importantly, LIP is strongly associated with the promotion of autoimmune or inflammatory disease (5, 13, 17, 24, 40–49). Such promotion of autoimmunity may result from potentiation of TCR signaling by high concentrations of homeostatic cytokines like IL-7 and IL-15 (50, 51) and/ or unimpeded access to self-pMHC due to decreased competition from other Tcon or Treg. Thus, perturbations which would be expected to increase the LIP potential of a host, such as decreased Treg, increased homeostatic cytokines, or decreases in co-inhibitory signals would be expected to increase the potential for such autoimmunity, and *vice versa*. Newly generated T cells during LIP appear particularly predisposed to the generation of autoimmunity when PD-1 mediated co-inhibitory signals are compromised (24). Newly generated T cells have not been subjected to peripheral tolerance mechanisms and thus may contain an increased proportion of strongly self-reactive cells compared to established T cells that arose in a host with low LIP potential upon initial seeding of its peripheral T cell compartment (i.e., during the neonatal period) and were tolerized. Indeed, in the current studies, we showed that a high frequency of CD4 T cells specific to a peripheral neo-self antigen can persist for an extended period in a lymphoreplete adult host, declining slowly with time without overt immune-mediated pathology (i.e., lack of rejection of the neoantigen expressing graft; **Figures 2C–E**). In addition, we have recently shown that PD-1 contributes to control of tonic pMHC signaling, which may tune signaling thresholds to establish a general T cell homeostasis needed for peripheral tolerance (52). Altogether our data suggests that PD-1 is more critical for control of newly generated CD4 T cells because of increased self-specific T cells in this population as well as a need to set response thresholds to tonic pMHC signals in all CD4 T cells. Although this view is consistent with recent findings from Jiang and colleagues, in which they showed PD-1 was important in reducing the number of high-affinity self-specific CD4 T cells (53), their conclusion differs substantially from our own. While they concluded that PD-1 is needed to maintain tolerance, both their data and ours are instead consistent with the view that PD-1 is needed to establish tolerance, with PD-1 playing relatively little or no role in the maintenance of tolerance once it is established. On the other hand, much of the published literature describing newly generated T cells/RTE suggests that they have functional properties geared toward promotion of tolerance (26). However, we found that newly generated PD-1−/− T cells isolated from the periphery of adult animals were indeed capable of generating autoimmune disease (particularly cachexia, kyphosis, and diarrhea) similar to PD-1−/− thymocytes, and much more efficiently than established T cells which contained approximately 1.3% contaminating GFP+ cells (**Figure 2A**). These data indicate that steady state newly generated T cells have a dangerous autoimmune potential even when their initial export from the thymus is into a lymphoreplete environment.

Tolerance first becomes established in mice during the neonatal period. Compared to adult Rag−/− mice, we consider that prior to/during initial seeding of the lymphoid compartment neonatal mice would, by virtue of anatomic size (small lymph nodes, reduced overall resources) and competition by specialized populations of innate lymphoid cells for IL-7 (54) have significantly lower LIP potential. This is supported by our finding that neonatal Rag−/− hosts rarely developed severe autoimmunity after reconstitution with PD-1−/− HSC (24). A lymphoreplete adult WT host on the other hand would be considered to have lower LIP potential than either an adult Rag−/− or a WT neonate (i.e., the ranking of LIP potential would be adult Rag−/− > neonatal WT > adult WT) (40). We hypothesize that the mild but significant LIP potential in neonates creates a situation in which establishment of tolerance in the nascent T cell population by PD-1 is important, and the absence of PD-1-mediated control of neonatal LIP may set the stage for the lupus-like autoimmunity that manifests later in life in PD-1−/− animals.

Although PD-1 is critical for tolerance in newly generated T cells, and PD-1 was expressed on neonatal T cells undergoing LIP, we did not, in the steady state, detect PD-1 expression above background on the peripheral newly generated T cell population of adult Rag2pGFP mice and only low expression in thymic GFP+ T cells (**Figure 1**). In contrast, both the peripheral CD4 SP and thymic CD4 and CD8 SP established (GFP−) T cell population contained sizeable populations that robustly expressed PD-1. Previous data from our lab assessing PD-1 expression in steady state RTE (i.e., in WT lymphoreplete mice) used CD24 as the marker to define RTE and the data suggested RTE have higher PD-1 (24). However, based on our current data with the Rag2pGFP mouse and the fact that only a small fraction of newly generated T cells expressed CD24 and established T cells can also express CD24, we conclude that steady-state adult newly generated T cells do not have increased PD-1. Together with our finding that PD-1 can regulate LIP and the response to tonic pMHC signals (25, 52), these observations suggest that PD-1 is upregulated during LIP as a negative feedback mechanism. Finally, it is worth noting that because a significant portion (10–15%) of thymic T cells are established cells based on lack of GFP expression, consistent with the previously described ability of peripheral T cells to recirculate back to the thymus (55), the use of the term "recent thymic emigrants" to describe GFP+ cells in the Rag2pGFP Tg model as is common in the literature is somewhat imprecise as presumably some cells emerging from the thymus in an adult are also GFP−. Therefore, although all peripheral GFP+ cells are RTE, all RTE are not necessarily GFP+.

One potential explanation for our finding that purified peripheral newly generated T cells efficiently caused disease upon transfer to lymphopenic hosts while established cells did not is that the relative proportions of CD4 and CD8 T cells differed significantly between these populations. Based on the present data including the lack of disease in CiiTA−/− hosts despite generation of CD8 T cells and B cells (**Figure 3C**) and our previous findings that MHC Class I-deficient lymphopenic hosts were fully disease permissive, we conclude that CD4 T cells in this setting are the key effectors of autoimmune pathology. Given that the purified established T cell population contained a greater frequency of CD4 vs. CD8 T cells, their inability to drive disease (and conversely the ability of GFP+ PD-1−/− newly generated cells to do so) cannot be explained by insufficient numbers of CD4 T cells.

While CD4 T cells are most commonly considered as "helpers" of the immune response, numerous studies have suggested that they can in some circumstances acquire cytolytic effector function *via* upregulation of killing mechanisms typically associated with CD8 cytotoxic T lymphocytes such as the perforin and Fas-ligand (FasL) pathways (56). Our finding that neither Faslpr × Rag−/− recipients of PD-1−/− HSC, nor recipients of Prf1−/− × PD-1−/− thymocytes were spared from disease (**Figures 3** and **4A,B**) suggests that both of these canonical T cell effector pathways are dispensable for LIP-driven autoimmunity in the setting of PD-1 deficiency. Furthermore, our finding that disease in Prf1−/− × PD-1−/− thymocyte recipients was exacerbated compared to PD-1−/− thymocyte recipients (**Figures 4A,B**) suggests that perforin-dependent effector pathways may actually play an immunoregulatory role during LIP. This is perhaps not surprising given that perforin has been reported to be important for contraction of the CD8 population following infection (57, 58), and as a mediator of suppression by Tr1 cells (59). Similarly, weight loss of increased severity in the Faslpr × Rag−/− compared to Rag−/− recipients of adoptively transferred Prf1−/− PD-1−/− cells (**Figure 4C**) suggested that Fas may also play an immunoregulatory role during LIP. This might also be anticipated given the lymphoproliferative disease characteristic of the Fas and Fas-ligand-deficient *lpr* and *gld* mice (60) which is partially attributable to defects in Fas-mediated killing of antigen-presenting cells (61). The latter experiment also ruled out functional redundancy between Fas–FasL and perforindependent killing pathways. We did consider the possibility that Granzyme B produced by T cells activated during LIP might act independently of perforin, for example through the mannose-6-phosphate receptor (62). However, examination of Granzyme B expression in WT vs. PD-1−/− HSC recipients at the peak of disease (day 45) demonstrated no significant difference in splenic CD4 or CD8 T cell populations although a small trend toward increased expression in PD-1−/− HSC recipient CD8 T cells was noted (Figure S2 in Supplementary Material).

The broad upregulation of a number of pro-inflammatory cytokines in PD-1−/− HSC recipients (IFN-γ, IL-13, TNF-α, IP-10, MIG, MCP-1, VEGF) (24) along with the lack of requirement for either perforin- or Fas-dependent killing pathways for autoimmunity in this model suggest that the immune pathology seen is primarily a CD4 T cell and "cytokine-storm" dependent phenomenon. Several similarities exist between the LIP-driven autoimmunity described herein and certain clinical syndromes, such as immune reconstitution inflammatory syndrome (IRIS) in HIV patients experiencing a rebound of the CD4 T cell compartment after treatment with antiviral drugs (44, 63) or in chronic GVHD post-allogeneic bone marrow transplantation (64). Notably a mouse model of IRIS was recently described in which transfer of purified CD4 T cells to *Cryoptococcus*-infected lymphopenic mice yields weight loss and systemic inflammatory disease associated with cytokine dysregulation (45). In addition, the striking increases in the chemokines IP-10 and MIG described previously in our model (24) are also observed in clinical chronic GVHD (65). Indeed, the term "cytokine-storm" was originally used to describe the syndrome of cytokine dysregulation (particularly IL-6, IL-1, and TNF-α) which is associated with and greatly contributes to GVHD pathology (66, 67). One could make the argument that our model of LIP-driven autoimmunity after transfer of PD-1−/− newly generated T cells to a lymphopenic host might actually be viewed as a model of syngeneic GVHD, similar to the autoimmunity that occurs when cyclosporine A (CsA) is discontinued post bone marrow transplantation. However, broad-spectrum antibiotics prevented CsA-induced syngeneic GVHD (68) but did not prevent disease in our model (25). Instead, the stimulus for PD-1-deficient RTE appears to involve autoantigens, potentially including the low-affinity interactions with self-peptide MHC that otherwise generate only tonic survival signals (40). Thus, beyond the insights it offers into the role of co-inhibitory molecules in establishing tolerance, further characterization of this model may lead to translatable insights to treat cytokine-driven systemic autoimmunity and inflammatory disease including after therapies involving immune reconstitution.

## ETHICS STATEMENT

This study was carried out in accordance with the recommendations of the Canadian Council on Animal Care. The protocol was approved by the University of Alberta Health Sciences Animal Care and Use Committee.

## AUTHOR CONTRIBUTIONS

KE designed, performed research and data analysis, and wrote and critically edited the manuscript. GT, YH, and JL designed and performed research and data analysis and critically edited the manuscript. LB provided reagents and critically edited the manuscript. CA designed research, performed data analysis, and critically edited the manuscript.

## ACKNOWLEDGMENTS

The authors would like to thank Perveen Anwar and HSLAS staff for assistance with animal care and Deb Dixon for islet transplantation. This manuscript includes material previously included in the doctoral dissertation of KE (69).

## FUNDING

This research has been supported by operating funds to CA from the Canadian Institutes of Health Research (PS148588, FRN79521), the Muttart Diabetes Research and Training Centre, the Women and Children's Health Research Institute (WCHRI) through the generous support of The Stollery Children's Hospital Foundation and by doctoral studentships from the Alberta Diabetes Institute and Alberta Innovates Health Solutions (AIHS) to KE and a senior scholar award from AIHS to CA.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://www.frontiersin.org/articles/10.3389/fimmu.2018.00012/ full#supplementary-material.

## REFERENCES

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

*Copyright © 2018 Ellestad, Thangavelu, Haile, Lin, Boon and Anderson. 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.*

*Kristina M. Harris1 \*, Tingting Lu1 , Noha Lim1 and Laurence A. Turka1,2*

*1 Immune Tolerance Network, Bethesda, MD, United States, 2Center for Transplantation Sciences, Massachusetts General Hospital, Boston, MA, United States*