PD-L1 Prevents the Development of Autoimmune Heart Disease in Graft-Versus-Host Disease

PD-L1 Prevents the Development of Autoimmune Heart Disease in Graft-versus-Host Disease

This information is current as Kathryn W. Juchem, Faruk Sacirbegovic, Cuiling Zhang, of October 1, 2021. Arlene H. Sharpe, Kerry Russell, Jennifer M. McNiff, Anthony J. Demetris, Mark J. Shlomchik and Warren D. Shlomchik J Immunol 2018; 200:834-846; Prepublished online 6 December 2017; doi: 10.4049/jimmunol.1701076 Downloaded from http://www.jimmunol.org/content/200/2/834

Supplementary http://www.jimmunol.org/content/suppl/2017/12/05/jimmunol.170107 http://www.jimmunol.org/ Material 6.DCSupplemental References This article cites 50 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/200/2/834.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

PD-L1 Prevents the Development of Autoimmune Heart Disease in Graft-versus-Host Disease

Kathryn W. Juchem,*,1 Faruk Sacirbegovic,† Cuiling Zhang,* Arlene H. Sharpe,‡ Kerry Russell,x,2 Jennifer M. McNiff,{,‖ Anthony J. Demetris,# Mark J. Shlomchik,*,**,††,3 and Warren D. Shlomchik*,†,x,††,4

Effector memory T cells (TEM) are less capable of inducing graft-versus-host disease (GVHD) compared with naive T cells (TN). Previously, in the TS1 TCR transgenic model of GVHD, wherein TS1 CD4 cells speciﬁc for a model minor histocompatibility Ag

(miHA) induce GVHD in miHA-positive recipients, we found that cell-intrinsic properties of TS1 TEM reduced their GVHD potency relative to TS1 TN. Posttransplant, TS1 TEM progeny expressed higher levels of PD-1 than did TS1 TN progeny, leading us to test the hypothesis that TEM induce less GVHD because of increased sensitivity to PD-ligands. In this study, we tested this

hypothesis and found that indeed TS1 TEM induced more severe skin and liver GVHD in the absence of PD-ligands. However, lack Downloaded from of PD-ligands did not result in early weight loss and colon GVHD comparable to that induced by TS1 TN, indicating that additional pathways restrain alloreactive TEM. TS1 TN also caused more severe GVHD without PD-ligands. The absence of PD-ligands on donor bone marrow was sufﬁcient to augment GVHD caused by either TEM or TN, indicating that donor PD- ligand–expressing APCs critically regulate GVHD. In the absence of PD-ligands, both TS1 TEM and TN induced late-onset myocarditis. Surprisingly, this was an autoimmune manifestation, because its development required non-TS1 polyclonal CD8+

T cells. Myocarditis development also required donor bone marrow to be PD-ligand deﬁcient, demonstrating the importance of http://www.jimmunol.org/ donor APC regulatory function. In summary, PD-ligands suppress both miHA-directed GVHD and the development of alloimmunity-induced autoimmunity after allogeneic hematopoietic transplantation. The Journal of Immunology, 2018, 200: 834–846.

llogeneic hematopoietic stem cell transplantation T cells (TEM) fail to induce sustained GVHD, but engraft and can (alloSCT) can cure hematological malignancies and mediate GVL (1–5). These data support the selective depletion of A nonmalignant inherited and acquired disorders of blood TN as a method of GVHD prevention. This has shown promise in cells. Mature allograft abT cells promote engraftment, contribute humans, wherein TN depletion reduced chronic GVHD, without by guest on October 1, 2021 to immune reconstitution, and can attack malignant cells, mediating an apparent increase in risk for relapse (6). Additional efforts to the graft-versus-leukemia (GVL) effect. However, alloreactive T cells understand the mechanisms behind this effect could help to further also attack recipient nonmalignant cells, causing graft-versus-host optimize this approach in humans. disease (GVHD). Because of GVHD, all patients receive some However, why memory T cells (TM) and, in particular, TEM, form of immunosuppression to diminish its incidence and severity. fail to induce GVHD is incompletely understood. Deciphering A primary goal of alloSCT research is to understand and differ- the mechanisms might provide strategies to similarly impair entiate mechanisms of GVHD and GVL to maximize the positive GVHD-inducing TN. We initially considered that TEM may be effects of donor T cells while minimizing GVHD. less capable of causing GVHD because they are relatively re- Toward this end, we and others discovered in mouse models that stricted from key sites of priming such as lymph nodes, but this naive T cells (TN) induce severe GVHD, whereas effector memory proved incorrect (7). To test whether TEM fail to induce GVHD

*Department of Immunobiology, Yale University School of Medicine, New This work was supported by National Institutes of Health Grant R01 HL109599. This Haven, CT 06520; †Department of Medicine, University of Pittsburgh, Pitts- work benefited from the use of the special Becton Dickinson LSR Fortessa, funded by burgh, PA 15261; ‡Department of Microbiology and Immunology, Harvard National Institutes of Health Grant 1S10OD011925-01. Medical School, Boston, MA 02115; xDepartment of Medicine, Yale University { K.W.J. designed and performed experiments, analyzed data, and wrote the paper. F.S. School of Medicine, New Haven, CT 06520; Department of Dermatology, Yale ‖ designed and performed experiments and analyzed data. C.Z. performed experiments. University School of Medicine, New Haven, CT 06520; Department of Pathol- 2 2 K.R. interpreted echocardiography results. A.H.S. created the PD-L1/2 / mice. ogy, Yale University School of Medicine, New Haven, CT 06520; #Department J.M.M. and A.J.D. scored histopathology. W.D.S. and M.J.S. conceived the project, of Pathology, University of Pittsburgh, Pittsburgh, PA 15261; **Department of designed experiments, analyzed data, and wrote the paper. Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06520; and ††Department of Immunology, University of Pittsburgh, Pittsburgh, Address correspondence and reprint requests to Dr. Warren D. Shlomchik, University PA 15261 of Pittsburgh School of Medicine, Thomas E. Starzl Biomedical Science Tower, Room E1552, 200 Lothrop Street, Pittsburgh, PA 15261. E-mail address: [email protected] 1Current address: Boehringer Ingelheim, Ridgefield, CT. The online version of this article contains supplemental material. 2Current address: Novartis Institute for Biomedical Research, Cambridge, MA. Abbreviations used in this article: alloSCT, allogeneic hematopoietic stem cell trans- 3Current address: Department of Immunology, University of Pittsburgh, Pittsburgh, PA. plantation; BM, bone marrow; GVHD, graft-versus-host disease; GVL, graft-versus- 4 Current address: Department of Medicine, University of Pittsburgh, Pittsburgh, PA. leukemia; HA, hemagglutinin; miHA, minor histocompatibility Ag; TEM, effector memory T cell; TKO, triple knockout; T , memory T cell; T , naive T cell; Treg, ORCID: 0000-0002-1084-6240 (K.R.). M N regulatory T cell; WT, wild type. Received for publication July 28, 2017. Accepted for publication November 3, 2017. Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701076 The Journal of Immunology 835 solely because of their having a less alloreactive TCR repertoire Cell sorting and/or whether repertoire-independent properties also reduce + dim 2 TS1 TN (CD62L CD44 CD25 ) were sorted from unmanipulated TS1 2 + 2 their ability to cause GVHD, we developed a TCR-transgenic mice, and TS1 TEM (CD62L CD44 CD25 ) were sorted from memory mice. GVHD model that enabled direct comparison of TN and TEM To avoid staining cells with anti-CD4 Ab, which we found to reduce en- + + + with identical TCRs. In this model, BALB/c CD4+ TCR- graftment, TS1 cells were enriched via exclusion of CD11b , F4/80 ,CD8a , + + + transgenic T cells (TS1 T cells), specific for the hemagglutinin and CD25 cells. Sorted cells were 40–60% CD4 TS1 ,withnocontaminating CD8+ TS1+ cells. Purities of CD4+ T and T among TS1 cells were .99%. (HA)-derived S1 peptides 110–119 (SFERFEIFPK) presented by N EM I-Ed, were combined with BALB/c bone marrow (BM) and GVHD protocol transferred into irradiated BALB/c recipients that express HA at Seven- to twelve-week-old HA104 BALB/c hosts were irradiated with 750 6 a low level in all cells (HA104 mice) (8). TS1 TN induced severe cGy via a cesium irradiator and transplanted with 8 3 10 BM cells alone or acute GVHD, characterized by weight loss and typical GVHD in combination with 1000 sorted TS1 TN or TEM, except as otherwise pathology of skin, liver, and colon. In contrast, TS1 T induced noted. Recipients received wet food and Sulfatrim-treated water for 2 wk EM posttransplant. Weights were taken two to three times per week. Mice that only transient disease, demonstrating that TEM have TCR lost 20% of their body weight were euthanized and scored as dead. For repertoire-independent limitations. euthanized mice, the last recorded weight was carried forward to subse- Although TS1 TN and TEM caused very different degrees of quent time points. All transplants were performed in accordance with Yale GVHD, they nonetheless proliferated and accumulated to a similar University or the University of Pittsburgh Institutional Animal Care and extent in secondary lymphoid tissues early posttransplant (3). Use Committee regulations. However, compared with TN, TS1 TEM progeny produced less Histopathology IFN-g and accumulated to a lesser degree in the colon, a major site of GVHD in the model. In addition, although progeny of both Skin, liver, and colon histology were scored as described previously (3). Downloaded from Cardiac histology was scored as follows: 0 = no inflammation or necrosis; TN and TEM upregulated PD-1 posttransplant, PD1 expression was 0.5 = minimal inflammation and necrosis involving ,5% of the total cross- higher on the progeny of TS1 TEM relative to that of TS1 TN in sectional area of the left and right ventricles; 1 = mild inflammation and both secondary lymphoid tissues and colon (3). focal necrosis involving .5–33%; 2 = moderate inflammation and focal . In this study, we have investigated whether the higher level of necrosis involving 33–66%; 3 = severe inflammation and focal necrosis involving .66%. Tissues were taken for histopathological analysis when PD-1 expression on TS1 TEM progeny leads to greater inhibition, mice dropped ,80% of their original body weight (between 30 and 60 d which in turn would reduce their capacity to cause GVHD. We posttransplant). Histopathology for mice that maintained body weight http://www.jimmunol.org/ found that PD-ligands regulate GVHD directly mediated by TS1 .80% was taken at the end of each experiment. T and T . Surprisingly, and contrary to our expectations, PD- N EM Cell extractions ligands were also critical to prevent the emergence of autoimmune myocarditis triggered by alloimmune TS1. Together these data Livers were perfused with PBS, excised, minced, passed through a cell indicate that PD-ligands not only restrain alloreactive TS1 T , strainer, and incubated in 0.02% collagenase IV (Sigma) at 37˚C for 40 min. EM Lymphocytes were separated on a 25% Optiprep gradient (Accurate but are also critical for blocking subsequent GVHD-dependent Chemical and Scientific). To perfuse the heart, a cut was first made in the autoimmunity, which could masquerade clinically as GVHD and right atrium, and PBS with 1% heparin was then injected into the left be more difficult to cure. ventricle until flow of blood ran clear. The heart was then excised, minced, and incubated with 20 mg/ml DNAse (Sigma) and 0.1 mg/ml Liberase by guest on October 1, 2021 (Roche) at 37˚C for 30 min. The resulting cell suspension was passed Materials and Methods through a 40-mm cell strainer. Lymphocytes were isolated by centrifuga- Mice tion over a Percoll gradient (GE Healthcare Bio-Sciences AB). BALB/c mice were from the National Cancer Institute (Frederick, MD) Flow cytometry and intracellular cytokine staining or The Jackson Laboratory. After being obtained from the following sources, gene-modified mice were bred at Yale and the University of Flow cytometric and cell sorting were performed using an LSRII or Fortessa Pittsburgh: BALB/c RAG22/2 (Taconic); BALB/c PD-L1/22/2 and (BD) and a FACSAria II (BD), respectively. Data were analyzed using BALB/c PD-L12/2 (A.H. Sharpe); BALB/c TS1 (9) (A. Adler, Uni- FlowJo software (Tree Star). For intracellular cytokine staining, splenocytes versity of Connecticut); and BALB/c HA104 (10) (Wistar Institute). were cultured for 1 h in the presence of 20 ng/ml PMA (VWR Scientific) and These strains were intercrossed to produce the following strains: TS1 750 ng/ml ionomycin (VWR Scientific), and for an additional 2 h in the RAG22/2,PD-L1/22/2 HA104, PD-L12/2 HA104, PD-L1/22/2 presence of 10 mg/ml brefeldin A (BioLegend). Cells were treated with RAG2/2 HA104, and RAG2/2 HA104. For all experiments, mice were DNase (Sigma), surface stained for CD4 and TS1, and treated with heterozygous for the HA and TS1 alleles. All TS1 cells were RAG22/2 Cytofix/Cytoperm (BD). Perm/Wash (BD) was used for intracellular and are referred to as TS1. staining with IFN-g PE (XMG1.2; BioLegend). For FOXP3 staining, cells were surface stained for CD4 and TS1, treated with FOXP3 Fix/Perm TS1 memory cell generation buffer (BioLegend), and stained for FOXP3 in FOXP3 perm buffer (Bio- Legend). TS1 CD4+ T cells were enriched from splenocytes to .80% purity using a CD4+ T cell negative selection (STEMCELL Technologies). TS1 cells (106 In vivo CD4 and CD8 depletion cells/ml) were cultured with HA S1 peptides 110–119 (SFERFEIFPK, 10 mg/ml; Keck Facility, Yale University) and T cell–depleted, irradiated To deplete CD8+ T cells, mice received 200 mg TIB105 (Bio X Cell) i.p. on splenocytes (3 3 106 cells/ml) for 3 d. A total of 5 3 106 TS1 cells were days 26and24. Mice were transplanted on day 0 and injected with 100 mg 2/2 transferred i.v. into BALB/c RAG2 recipients. TS1 TEM were harvested of TIB105 on days 6, 13, 20, 27, 34, and 41. To deplete CD4 cells, mice from these “memory mice” for use 8–14 wk later. received 150 mg of GK1.5 (laboratory-made) on days 27, 31, 35, 39, 42, and 46 posttransplant. Additional groups received both anti-CD4 and anti- Abs CD8 or control rat IgG2a (Bio X Cell). Abs were from the following sources: CD4 Alexa 488 or Alexa 405 (GK1.5), In vivo measurements of cardiac function TS1 Alexa 647 (6.5), and F4/80 Alexa 488 (HB-198) were laboratory prepared. CD25 [R-PE (7D4)] was from Southern Biotech. FOXP3 (150D), CD62L Echocardiographic assessments of cardiac structure and function were PE-Cy7 (Mel-14), PD-1 PE (29F.1A12), CD8a PE (53-6.7), and CD44 performed 38 d posttransplant using a 40-MHz probe and a Vevo2100 allophycocyanin-Cy7 (1M7) were from BioLegend. Gr-1 PE (RB6-8C5) and Ultrasound system (FUJIFILM; VisualSonics, Toronto, ON, Canada). Mice CD11b PE (MI/70) were from eBioscience. CD4 PE (RM4-5; BD) and CD8 were lightly anesthetized with 0.5–1.0% isoflurane with continuous mon- PE (YTSI56.7.7; BioLegend), which could bind in the presence of the de- itoring of heart rate, body temperature (kept at normal by a heated plat- pleting Abs, were used for confirmation of in vivo T cell depletions. Ethidium form), and respiratory rate. Two-dimensionally guided M-mode images of monoazide (Invitrogen) or propidium iodide was used to exclude dead cells. the left ventricle were acquired in addition to two-dimensional evaluation 836 PD-L1 PREVENTS GVHD-INDUCED AUTOIMMUNITY

of left ventricular size and function. The data were collected in both short- have on TS1 development and TEM generation. As reported pre- and long-axis views at heart rates .400 beats/min. A total of three to five viously (3), transplantation of wild type (WT) BM and TS1 TEM cardiac cycles per measurement were averaged in each view to obtain functional and anatomical data. into WT HA104 recipients caused no death and only mild, transient weight loss (Fig. 1A, 1B). In contrast, the transfer of TS1 2/2 2/2 Statistics TEM and PD-L1/2 BM into PD-L1/2 HA104 hosts caused The significance of weight change was calculated by Student t test. The severe weight loss and death (Fig. 1A, 1B), but with delayed ki- significance of survival differences was calculated by log rank test. The netics compared with what is observed with TS1 TN with intact significance of all other comparisons (pathology, cell numbers, percentage PD-ligands (3) (Fig. 2A). Weight loss began 35 d posttransplant + of IFN-g cells, and IFN-g mean fluorescence intensity) was calculated by and rapidly progressed, with the majority of T recipients dying the Mann–Whitney U test. EM by day 50. Histological analysis revealed that TEM, in the absence Results of PD-ligands, induced more severe skin and liver GVHD relative to when PD-ligands were intact (Fig. 1C, 1D). In particular, liver PD-1 ligands inhibit TS1 T -dependent GVHD of skin, liver, EM GVHD was more severe than was induced by TS1 T in a PD- and heart N ligand–sufficient environment (p = 0.0064, Fig. 2). In contrast, the To investigate whether PD1 regulates TS1-induced GVHD, we absence of PD-ligands did not increase colon GVHD (Fig. 1E), modified the TS1 GVHD model to use BM donors and HA104 which is a dominant feature of TS1 TN-mediated disease (3) hosts that lacked both PD-L1 and PD-L2. We chose to create an (Fig. 2). Very mild liver disease was also observed in PD-L1/22/2 2/2 2/2 environment without PD-ligands instead of using PD-1 TS1 HA104 recipients of PD-L1/2 BM without TS1 TEM, indicating T to avoid the confounding effects the absence of PD-1 might that some aspects of liver disease are TS1 independent (Fig. 1D).

EM Downloaded from http://www.jimmunol.org/

FIGURE 1. PD-L1/2 limit TS1

TEM-mediated disease in the skin, liver, and heart, but not the colon. (A–F) WT or PD-L1/L22/2 BM was transferred into irradiated WT or PD-L1/22/2 HA104 mice, respectively, with or without 1000 TS1

TEM. Shown are survival (A), weight loss (B), and pathology scores for by guest on October 1, 2021 the skin (C), liver (D), colon (E), and heart (F). Data from (A)–(E) were combined from two independent experiments. Data from (F) were combined from two additional independent experiments, for which weight and survival curves are shown in Supplemental Fig. 1. The p values for survival and weight curves are relative to WT HA104 recipients of WT BM and TS1 TEM. (G–I) PD-L1/22/2 BM with or without TS1 TEM were transferred into irradiated HA104 PD-L1/22/2 hosts. Thirty-eight days posttransplant, mice were anesthetized and underwent echocardiography. (G) Weights are shown. (H) ECG tracing for one BM control and one TS1

TEM recipient is shown. The ECG tracing for the TS1 TEM recipient was representative of two out of ﬁve mice. (I) Heart rate, cardiac output, diastolic volume and systolic volume are shown. Data are from one experiment. The Journal of Immunology 837 Downloaded from

2/2 FIGURE 2. PD-L1/2 limit TS1 TN-mediated disease in the skin, liver, and heart, but not the colon. WT or PD-L1/2 BM was transferred into irradiated 2/2 WT or PD-L1/2 HA104 mice, respectively, with or without 1000 TS1 TN. Shown are survival (A), weight loss (B), and pathology scores for the skin (C), liver (D), colon (E), and heart (F). Data from (A)–(E) were combined from three independent experiments. Data from (F) were combined from two additional independent experiments, for which survival and weight curves are shown in Fig. 3A and 3B. The p values for survival and weight curves are relative to WT HA104 recipients of WT BM and TS1 TN. http://www.jimmunol.org/

To better pinpoint the cause of clinical disease, we performed repeat with TS1 TEM. In the absence of PD-L1/2, TS1 TN caused slightly experiments (weight loss and survival; Supplemental Fig. 1) in which increased skin disease and markedly more severe liver disease, but we examined kidney, lung, stomach, tongue, and small intestine and colon disease was similar to when PD-ligands were present found no significant pathology (data not shown). However, in the (Fig. 2C–E). Consistent with our prior report (3), in a PD-ligand– absence of PD-L1/2, TS1 TEM recipients developed cardiac pathol- sufficient environment, TS1 TN induced more severe histological ogy (Fig. 1F), manifested by a myocardial inflammatory infiltrate liver and colon GVHD than did TS1 TEM (p # 0.0024). However, with cardiomyocyte necrosis (Supplemental Fig. 2). Notably, severe in PD-ligand–deficient environments, TS1 TEM and TN caused 2/2

heart disease did not develop in PD-L1/2 BM controls. similar liver GVHD. by guest on October 1, 2021 This inflammation had apparent functional significance. Ap- Additional experiments were performed to evaluate heart disease proximately 5 wk posttransplant, in the absence of PD-ligands, TS1 (weight and survival are shown in Fig. 3A, 3B). TS1 TN caused recipients had a reduction in heart rate (Fig. 1G–I). Two of five such very mild heart disease when PD-L1/2 were intact; but, impor- mice had complete heart block and a third had an ectopic atrial tantly, they induced severe heart disease when PD-L1/2 were focus, consistent with conduction system damage. Echocardiograms absent (Fig. 2F; representative histology, Supplemental Fig. 3). demonstrated reduced cardiac output and diastolic and systolic These data indicate that PD-L1/2 limit TS1 TN-induced skin, liver, stroke volumes, even in mice without conduction abnormalities, and heart disease, similar to what was observed in TS1 TEM re- further evidence that the myocarditis was likely to be functionally cipients. However, unlike with TS1 TEM, PD-L1/2 also limit early significant and may have been a cause of death (Fig. 1G–I). TS1 TN-induced weight loss and death. Taken together, these data indicate that PD-ligands limit some, PD-ligands on recipient and donor cells restrict TS1- but not all, aspects of T function posttransplant, because in their EM dependent GVHD absence TS1 TEM induce skin, liver, and late-onset heart disease. However, in the absence of PD-ligands, TS1 TEM fail to recapit- We next dissected the individual roles of PD-ligands on recipient and ulate the severity of GVHD caused by TS1 TN, particularly colon donor-derived cells in modulating TS1-induced GVHD. We trans- 2/2 GVHD, indicating that pathways in addition to those downstream ferred TS1 TN or TEM and WT BM into PD-L1/2 HA104 hosts 2/2 of PD-1 distinguish TEM and TN function posttransplantation. and, reciprocally, TS1 TN or TEM and PD-L1/2 BM into WT HA104 hosts. Compared with WT HA104 recipients of TS1 TN and TS1 TN disease is inhibited by PD-ligands WT BM, the absence of host PD-L1/2 alone did not increase skin, To determine the role of PD-ligands in regulating GVHD mediated liver, or heart disease, but did increase mortality. In contrast, the 2/2 by TS1 TN, we reconstituted irradiated WT HA104 or PD-L1/2 absence of PD-L1/2 on donor BM alone did not increase mortality HA104 recipients with WT or PD-L1/22/2 BM (respectively), but exacerbated skin, liver, and heart disease, with scores as severe with or without TS1 TN. TS1 TN caused more early weight loss as those when PD-L1/2 were lacking on both donor BM and host and death in the absence of PD-L1/2 (Fig. 2A, 2B), consistent with cells (Fig. 3A–E). Notably, in this experiment, there was less early prior reports in GVHD models (11–13). This contrasts with what death than typically seen when donor and host lack PD-ligands. In was observed with TS1 TEM in a PD-ligand–deficient environment contrast to the case with TN-mediated GVHD, neither clinical nor wherein the increases in weight loss and death were delayed rel- histopathological TS1 TEM-induced GVHD was increased when ative to what was observed with TN (compare Fig. 1A, 1B with only the recipients lacked PD-ligands (Fig. 3F–J). However, the Fig. 2A, 2B; for weight loss, p # 0.003 from day +6 onward; for absence of PD-L1/2 on only donor BM increased weight loss and survival, p = 0.0002). Nonetheless, TS1 TN recipients developed a liver and heart pathology. Nonetheless, GVHD was less severe than second wave of weight loss and death that shared the kinetics seen when PD-ligands were absent on both the recipient and the donor 838 PD-L1 PREVENTS GVHD-INDUCED AUTOIMMUNITY

FIGURE 3. Role of PD-L1/2 on donor

BM and host cells. (A–E) TS1 TN and WT BM were transferred into irradiated PD-L1/ 2/2 2 HA104 hosts, and TS1 TN and PD-L1/ 22/2 BM were transplanted into irradiated WT HA104 hosts. As controls, WT BM into WT HA104 hosts and PD-L1/22/2 BM into PD-L1/22/2 HA104 hosts, with or without

TS1 TN, were also generated. Survival (A), weight loss (B), and pathology scores for skin (C), liver (D), and heart (E) are shown. Downloaded from Data for Fig. 2F and (E) were generated in the same experiment (two repetitions, data combined) to reduce mouse numbers. Data in (E) from groups in which donor and recipient were both PD-L1/2+/+ or PD-L1/22/2 are therefore the same as in Fig. 2F. (F–J) Mice were transplanted as in (A)–(E), except http://www.jimmunol.org/

TN were replaced with TEM.Survival(F), weight loss (G), and pathology scores for skin (H), liver (I), and heart (J) are shown. TN and TEM results were each combined from two independent experiments. The p values for survival and weight curves are relative to HA104 WT recipients of WT BM and TS1

TEM or TN. by guest on October 1, 2021

BM, indicating that PD-L1/2 on both donor BM and host cells PD-L1/22/2→WT chimeras (Fig. 4A–E). Therefore, PD-L1/2 on modulate TS1 TEM-induced GVHD. donor BM and host stromal cells, but not recipient hematopoietic cells, inhibits TS1 T -dependent cardiac disease. Roles of PD-L1/2 on host stromal and hematopoietic cells EM We next determined the contributions of PD-L1/2 on host hema- Roles of PD-L1 and PD-L2 topoietic and radioresistant, primarily nonhematopoietic, cells. To Whereas PD-L1 is widely expressed and can be induced by IFN-g, 2 2 do so, we created WT HA104→PD-L1/2 / HA104 and PD-L1/ PD-L2 is expressed primarily on hematopoietic cells (14). To test 2 2 2 / HA104→WT HA104 BM chimeras and used these as re- whether PD-L1 is sufﬁcient to modulate TS1-mediated GVHD, we 2/2 2/2 cipients in a second transplant in which GVHD was induced by transferred TS1 TEM and PD-L1 BM into PD-L1 HA104 2/2 TS1 TEM. As controls, WT HA104→WT HA104 and PD-L1/2 hosts. GVHD in the absence of PD-L1 alone phenocopied TS1 → 2/2 HA104 PD-L1/2 HA104 chimeras were also transplanted. TEM-mediated GVHD in the absence of both PD-L1 and PD-L2 Because the absence of PD-L1/2 on host cells alone does not (Fig. 5), indicating that PD-L1 is sufﬁcient for GVHD suppression. enable TS1 TEM-mediated GVHD (Fig. 3), all chimeras received PD-L1/22/2 BM in the second GVHD-inducing transplant. As Effect of PD-L1/2 on TS1 TEM accumulation and IFN-g 2/2 production expected, transfer of TS1 TEM and PD-L1/2 BM caused mild heart disease in WT→WT chimeras and severe weight loss, death, Despite increased TS1 TEM-induced GVHD in the absence of PD- and heart disease in PD-L1/22/2→PD-L1/22/2 chimeras. In L1/2, TS1 numbers in the spleen and the percentage of IFN-g+ comparison with PD-L1/22/2→PD-L1/22/2 chimeras, disease TS1 at days +14 or +42 were similar (Fig. 6). However, in was similar in WT→PD-L1/22/2 chimeras but was reduced in the absence of PD-L1/2, splenic TS1 at day 142 produced more The Journal of Immunology 839 Downloaded from

FIGURE 4. PD-1/2 expression on host stromal cells protects the heart from TS1 TEM-induced disease. The following BM chimeras were generated with WT HA104 and PD-L1/22/2 HA104 mice: (PD-L1/22/2→WT), (WT→PD-L1/22/2), (PD-L1/22/2→PD-L1/22/2), and (WT→WT). Eight weeks later, 2 2 / http://www.jimmunol.org/ chimeras were reirradiated and transplanted with PD-L1/2 BM and 1000 TS1 TEM. Survival (A), weight loss (B) and pathology for the skin (C), liver (D), and heart (E) are shown. Data were combined from two independent experiments. For weight and survival curves, p values are relative to WT→WT 2/2 recipients of PD-L1/2 BM and TS1 TEM.

IFN-g/cell as measured by intracellular cytokine staining (Fig. 6C). BM alone controls. These data suggested a model wherein TS1 In the liver, TS1 numbers were increased in the absence of PD- cells are required for the initiation of a process that leads to heart ligands only on day +42 (Fig. 6A), consistent with the increased disease, but that non-TS1 T cells are directly pathogenic. These liver GVHD scores. In summary, these data indicate that PD-L1/2 polyclonal T cells could be donor or recipient BM-derived, radio- diminishes IFN-g production by splenic TS1 cells (15), which resistant recipient mature T cells or a combination of these. by guest on October 1, 2021 could result in fewer pathogenic TS1 cells inﬁltrating GVHD To test the hypothesis that polyclonal T cells contribute to heart target organs. Alternatively, or in addition, PD-L1/2 could sup- disease, we developed a system that lacked all non-TS1 T cells. PD- press TS1 cells locally. L1/22/2 HA104 or PD-L1/22/2 RAG22/2 (triple knockout 2/2 Non-TS1 T cells are required for cardiac disease [TKO]) HA104 mice were transplanted with PD-L1/2 or TKO BM, respectively, with no T cells or with TS1 TEM or TS1 TN. Unexpectedly, the absence of PD-L1/2 reduced the number of TS1 TKO HA104 recipients of TS1 T and TKO BM developed early + + EM cells in the heart (Fig. 6D). However, non-TS1 CD4 and CD8 weight loss and liver disease similar to controls; however, TKO T cells were increased 3- and 6-fold, respectively (Fig. 6E, 6F). This recipients suffered less late-onset weight loss and death and, 2/2 increase occurred only in TS1 TEM recipients and not in PD-L1/2 strikingly, no heart disease (Fig. 7A–D). Likewise, both RAG2-

FIGURE 5. The absence of only PD-L1 is sufﬁcient for the development of TS1 TEM-induced heart disease. WT or PD-L12/2 BM alone or in combination with TS1 TEM were transferred into irradiated HA104 WT or HA104 PD-L12/2 hosts, respectively. Survival (A), weight loss (B), and pathology scores for the skin (C), liver (D), and heart (E) are shown. Data were combined from two independent experiments. The p values for weight and survival curves are relative to HA104 recipients of WT BM and TS1 TEM. 840 PD-L1 PREVENTS GVHD-INDUCED AUTOIMMUNITY Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 6. Characterization of TS1 and polyclonal T cells in the absence of PD-L1/2. (A–D) Irradiated WT or HA104 mice were reconstituted with WT 2/2 or PD-L1/2 BM, respectively, with no T cells or with 1000 TS1 TEM.(A) Number of TS1 cells in spleen and liver on days +14 and +42 and the numbers of TS1 from heart on day +42 (D). Data were combined from two independent experiments for a total of 8–12 mice per group. The percentages of splenic TS1 cells on days +14 and +42 that were IFN-g+ and the IFN-g mean ﬂuorescence intensity are shown in (B) and (C). Data were combined from two independent experiments for a total of 19 mice per group for day +42 and from one experiment with ﬁve mice per group for day +14. (E and F) The number of non-TS1 CD4+ T cells and CD8+ T cells per heart are shown. Data are representative of two independent experiments, each with three to four mice per group.

2/2 sufficient and RAG2-deficient TS1 TN groups developed GVHD, again observed low-level liver histopathology in PD-L1/2 2 2 manifested by weight loss (Fig. 7E). Initial weight loss was more recipients of PD-L1/2 / BM without TS1, but not when donors 2/2 severe in RAG22/2 recipients. However, after regaining some and recipients were also RAG2 . This makes it likely that such disease is caused by polyclonal donor- or recipient-derived T cells. weight, the RAG2-sufficient TS1 TN group developed a second wave of weight loss beginning around day +40. We harvested Residual recipient polyclonal T cells are sufficient for hearts from both groups beginning on day +47. Strikingly, there autoimmune cardiomyositis was severe heart disease in RAG2-intact TS1 TN recipients, To determine whether recipient T cells were sufficient to cause whereas cardiac pathology scores in TKO TS1 TN recipients were cardiac disease, we performed GVHD experiments with TKO 2/2 +/+ indistinguishable from negative control PD-L1/2 RAG2 donor BM and RAG2+/+ PD-L1/22/2 HA104 recipients. BM alone mice (Fig. 7F; representative histology, Supplemental 2/2 Compared with recipients of TS1 TEM and PD-L1/2 +/+ Fig. 4). Consistent with a role for polyclonal CD8 cells in me- RAG2 BM, recipients of TS1 TEM and TKO BM developed diating heart disease, they were readily detectable in the spleen similar weight loss and liver disease while mortality and heart (Fig. 7G). Therefore, non-TS1 T cells are required for TS1- disease were increased (Fig. 8), indicating that recipient induced heart disease, but not for other aspects of GVHD. We polyclonal T cells are sufficient for myocarditis, whereas donor The Journal of Immunology 841 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 7. Polyclonal non-TS1 T cells are required for TS1-induced heart disease. (A–F) PD-L1/22/2 HA104 or PD-L1/22/2 RAG22/2 (TKO) HA104 2/2 mice were transplanted with PD-L1/2 or TKO BM, respectively, with no T cells or with 1000 TS1 TEM (A–D) or 300 TS1 TN (E and F). The TS1 TEM 2/2 2/2 experiments had an additional group wherein HA104 RAG2 mice were transplanted with RAG2 BM with no T cells or with TS1 TEM. Survival (A), weight loss (B), and pathology scores for the liver (C) and heart (D) from the TS1 TEM experiments are shown. Data were combined from two independent 2/2 2/2 experiments.The p values for weight and survival curves are relative to RAG2 HA104 recipients of TS1 TEM and RAG2 BM and are significant from + + 2 + + day +31 onward. Weight loss (E) and heart pathology scores (F) for the TS1 TN experiment are shown. Percentages of CD8 , CD4 TS1 , and CD4 TS1 cells in the spleen when mice were sacrificed for pathology are shown in (G). Data are from one experiment. p , 0.05, comparing weights of either TS1 group with its BM alone control from days 9 to 60 after BMT. TKO TS1 recipients had significantly more weight loss (p , 0.05) than PD-L1/22/2 TS1 recipients on days 2, 7–9, 12–21, and 23–58.

BM-derived T cells are not required and might be protective. and CD8 cells were readily detected in spleens of PD-L1/22/2 Unexpectedly, heart disease also developed in a small but recipients of TKO BM, with or without TS1 TEM,atday60 signiﬁcant number of PD-L1/22/2 HA104 recipients of TKO (Fig. 8F), consistent with a role for host polyclonal T cells in 2/2 BM that did not receive TS1 TEM, but not in TKO HA104 mediating heart disease in both groups. Although PD-L1/2 2/2 recipients of TKO BM without TS1 TEM (Fig.7D).HostCD4 HA104 recipients of PD-L1/2 BM alone did not develop 842 PD-L1 PREVENTS GVHD-INDUCED AUTOIMMUNITY

FIGURE 8. Polyclonal recipient

T cells are sufﬁcient for TS1 TEM-induced myocarditis. Irradiated PD-L1/ L22/2 HA104 mice were reconstituted 2/2 with 1000 TS1 TEM and RAG2 PD-L1/22/2 BM or RAG2+/+ PD-L1/ 22/2 BM. The following control groups were also transplanted: 1) HA104 mice reconstituted with BALB/c BM and no T cells; 2) PD- L1/22/2 HA104 mice reconstituted with PD-L1/22/2RAG22/2 BM and no T cells; and 3) WT HA104 mice reconstituted with RAG22/2 BM and

1000 TS1 TEM.Survival(A), weight loss (B), and pathology scores for the skin (C), liver (D), and heart (E)are Downloaded from shown. The p values for weight and survival curves are relative to WT

HA104 recipients of TS1 TEM and RAG2/2 BM. Percentages of CD8+, CD4+ TS12,CD4+ FOXP3+ TS12, and CD4+ TS1+ cells in the spleen at day 60 posttransplant for two PD-L1/ http://www.jimmunol.org/ 22/2 recipients of TKO BM with and without TS1 TEM are shown in (F). Data for (A)–(E) were combined from two independent experiments. Data in (F) were from one experiment. by guest on October 1, 2021

severe heart disease, there were also occasional mice with mild Polyclonal CD8+ T cells contribute to myocarditis disease (see Figs. 2F, 3E, 3J). To determine the individual roles of polyclonal CD4+ and CD8+ Taken together, these results support the idea that the absence of T cells in TS1 TEM-induced cardiac disease, we used depleting PD-ligands during T cell reconstitution in the alloSCT setting is 2/2 2/2 Abs in PD-L1/2 HA104 recipients of TS1 TEM and PD-L1/2 permissive for myocarditis mediated by autoreactive polyclonal BM. CD8 cells were depleted with anti-CD8 twice in the week T cells. Alloreactive TS1 cells reproducibly trigger this autoimmunity, before transplant and then weekly thereafter. To avoid depleting whereas donor BM-derived polyclonal T cells may suppress it. TS1 cells during GVHD development, we began CD4 depletion on Regulatory T cell reconstitution in the presence and absence of day +27, before the onset of clinical cardiac disease (manifest by PD-ligands weight loss). CD4 depletion paradoxically increased mortality and weight loss If regulatory T cell (Treg) reconstitution in the absence of PD- but had no impact on heart disease (Fig. 10A–C). In contrast, CD8 ligands was diminished, this could have contributed to autoim- depletion alone and combined CD8 and CD4 depletion decreased mune myocarditis. We therefore enumerated Tregs in irradiated + 2/2 both mortality and heart disease. Enumeration of splenic CD4 WT or PD-L1/2 HA104 mice that were reconstituted with WT + 2 2 and CD8 T cells revealed that anti-CD4 treatment slightly de- or PD-L1/2 / BM, respectively, with or without TS1 T EM creased CD8+ T cell numbers but drastically reduced TS1 and (Fig. 9). On day 14 posttransplant, with or without TS1 TEM, PD- + 2 2 2 2 non-TS1 CD4 T cells numbers (Fig. 10D). Anti-CD8 treatment L1/2 / recipients of PD-L1/L2 / BM had higher percentages had no effect on CD4+ T cell numbers but reduced CD8+ T cells. and total numbers of Tregs; however, there was no difference in Taken together, these data indicate that polyclonal CD8 cells, and the ratios of TS1 to Tregs. On day 142 posttransplant, a higher not CD4 cells, are essential for heart disease. The persistence of percentage of splenocytes were Tregs in the absence of PD-L1/2, heart disease even after profound TS1 depletion suggests that once although neither the total numbers nor the Treg:TS1 ratios dif- 2 2 2 2 autoimmune cardiac disease is established, it is independent of fered. In the liver on day +42, PD-L1/2 / recipients of PDL1/2 / further actions by alloimmune cells. BM with and without TS1 TEM had a greater number and percentage of Tregs, but again without a signiﬁcant increase in the Treg:TS1 ratio. In summary, these data make it unlikely that au- Discussion toimmunity developed in the absence of PD-L1/L2 because of poor It has long been known that TEM cause less GVHD than do TN,a Treg reconstitution. phenomenon that we and others found to be independent of TCR The Journal of Immunology 843

FIGURE 9. The absence of PD- ligands does not diminish Treg reconstitution. Irradiated WT or HA104 mice were reconstituted with WT or PD-L1/22/2 BM, respectively, with or without 1000 TS1 Downloaded from

TEM. Percentages and numbers of Tregs (CD4+ FOXP3+) as well as Treg to TS1+ T cell ratios in the spleen and liver on day 14 and in the spleen and liver on day 42 are shown in (A)–(D). Day 14 data were combined from two independent exper- http://www.jimmunol.org/ iments, and day 42 data were combined from three independent experiments. TS1 cell numbers from these experiments are shown in Fig. 6A. by guest on October 1, 2021

repertoire differences (3, 16). The difference between TN and TM presence of polyclonal T cells, TS1 TN induced myocarditis and is clinically relevant because a recent human trial has suggested a late-onset weight loss and death. benefit with TN depletion (6). However, the mechanisms that re- The roles of PD-ligands in modifying HA-directed GVHD were strain the GVHD-inducing potential of TM have not been eluci- complex and context dependent, in part owing to their expression dated. In this work, we found that PD-ligands are one component on hematopoietic as well as parenchymal cells. Both TS1 TEM- and that limits alloreactive TEM. PD-ligands inhibited TS1 TEM- TN-induced liver GVHD were suppressed by PD-ligands. TS1 mediated GVHD through two main mechanisms. First, they TEM were particularly sensitive to PD-1 suppression because liver suppressed HA-directed GVHD in the liver and skin. However, nei- scores in the absence of PD-ligands were similar to scores in TS1 ther colon histopathology, a dominant feature of TS1 TN-induced TN recipients, with or without PD-ligands. In contrast, colon GVHD, nor early weight loss, were restored to that induced by GVHD was not increased in the absence of PD-ligands in TS1 TN 2/2 TS1 TN in a WT or PD-L1/2 environment. Therefore, addi- and TEM recipients, which suggests the possibility that PD-ligands tional properties of TS1 TEM must diminish their ability to cause do not regulate colon GVHD directly. For TN-mediated GVHD, GVHD relative to TS1 TN. Second, and contrary to expectations, the absence of PD-ligands on only donor BM-derived cells was PD-ligands maintained tolerance of polyclonal nonalloreactive necessary and sufficient for maximal acute GVHD intensification. T cells, which, in the absence of PD-ligands, was reproducibly These features were not modified when hosts also lacked PD-L1/ 2/2 broken by alloreactive TS1 TEM. TS1 TN-HA–directed GVHD 2 . The absence of PD-ligands on only donor BM was also was also more severe in the absence of PD-ligands. As we ob- sufficient to increase TS1 TEM-induced HA-directed liver GVHD; served with TS1 TEM, in the absence of PD-ligands and in the however, skin disease and maximal liver GVHD required that 844 PD-L1 PREVENTS GVHD-INDUCED AUTOIMMUNITY

FIGURE 10. In the absence of PD-L1/2, CD8+ T cells are required for TS1-induced heart disease. WT or PD-L1/22/2 BM, with or without

TS1 TEM, were transferred into WT HA104 or PD-L1/22/2 HA104 hosts, respectively. PD-L1/22/2 TS1 recipients were given isotype control, anti-CD8, anti-CD4, or both anti-CD4 and anti-CD8 Abs as follows: 1) isotype control and anti- CD8 were given on days 26, 24, 6, 13, 20, 27, 34, and 41; and 2) anti- CD4 was given on days 27, 31, 35, 39, 42, and 46. Survival (A), weight loss (B), and heart pathology (C) are shown. (D) Number of splenocytes that were CD4+ TS12, TS1+,or CD8+ are shown. Data were combined from two independent exper- Downloaded from iments. For weight and survival curves, p values are relative to isotype control recipients.

PD-ligands be absent from both donor BM and recipient. The and recipients were RAG2+/+PD-L1/22/2. These data further dominant role of PD-ligands on donor BM-derived cells empha- support the notion that PD-ligands suppress the development of http://www.jimmunol.org/ sizes the importance of indirect presentation of minor histocom- autoreactive polyclonal T cells posttransplantation even without patibility Ags (miHAs; the HA-derived S1 peptide in this case) by GVHD, and that this autoreactivity is reproducibly triggered by donor APCs to donor CD4 cells (17, 18). Although indirect pre- alloreactive T cells. It is tempting to speculate that alloimmune sentation is commonly thought of as being stimulatory and attack, as in the system we studied, is just one example of an disease-promoting, our data show that indirect miHA presentation inflammatory condition that would trigger autoimmunity when by PD-ligand–expressing donor APCs suppresses GVHD. This PD-ligand engagement is suboptimal. Other examples might in- regulation could occur in secondary lymphoid tissues, peripheral clude chronic infection or toxic injury, both settings that are as- GVHD target organs, or both. PD-ligands on hematopoietic APCs sociated with autoimmune induction in general. by guest on October 1, 2021 may be especially important in the regulation of CD4-mediated How do PD-ligands regulate self-tolerance in the context of immunopathology, because CD4-mediated GVHD in miHA- alloSCT? A major clue is that the absence of PD-L1/2 on the donor disparate MHC-matched models can occur when non- BM is sufficient for myocarditis. Therefore, parenchymal ex- hematopoietic cells are MHCII2 (19). Therefore, hematopoietic pression of PD-L1–suppressing effector cells, as invoked in PD- cells could be the only cells, or at least the most common cell, ligand–dependent tumor immune resistance settings (20), cannot capable of engaging CD4 cells by their TCRs while presenting be the only mechanism by which PD-ligands regulate autoim- PD-ligands. munity. Instead, the absence of donor hematopoietic PD-L1/2 In contrast with our studies, prior work on PD-ligands in GVHD could lead to the peripheral activation of self-reactive polyclonal found a dominant role for host PD-ligands and, when examined, on T cells, be they mature T cells that survived radiation, donor al- host radioresistant nonhematopoietic cells (12). In these studies, lograft T cells, or T cells derived from hematopoietic progenitors. GVHD was in MHC-mismatched models. As such, direct recog- In this connection, presentation of self-antigen by APCs is thought nition of recipient MHC likely dominated the alloimmune re- to be a natural means of maintaining tolerance (21–23). In the sponse, which would dictate less of a role for indirect miHA absence of PD-ligands, such tolerogenic APCs could become presentation by donor APCs. Also, recipient nonhematopoietic stimulatory instead in a background of immune system activation PD-L1 would be expected to be dominant in models with CD8 by alloreactive CD4 cells. cells because CD8 cells must make TCR:MHCI contact with The absence of PD-L1/2 on recipient nonhematopoietic tissues nonhematopoietic cells to cause GVHD (19). promoted even more extensive TS1 TEM-triggered myocarditis. Surprisingly, genetic and depletion studies demonstrated that PD-L1/22/2 thymic parenchyma may have been permissive for TS1-induced cardiac disease in the absence of PD-ligands required the selection of self-reactive T cells. PD-L1 has been demon- polyclonal, CD8+ non-TS1 T cells. Commensurately, infiltrating strated on cardiac endothelia (24, 25), and therefore PD-ligands T cells were predominantly not TS1 clonotype, with ∼2-fold more could suppress both the generation of autoreactive CD8 cells and CD8+ than CD4+ T cells. That CD4 depletion, which also pro- their effector activities. Resolving the mechanisms that account foundly reduced TS1 TEM progeny, did not diminish myocarditis for TS1-induced autoimmunity would require both thymus and suggests that once TS1 cells break tolerance, they are no longer cardiac transplantation, which can be addressed in future work. required to maintain heart disease. Surprisingly, CD4 depletion, An unanswered question is why we observed only autoimmune but not dual CD4 and CD8 depletion, accelerated weight loss and heart disease and not autoimmune disease in other organs. One death. Anti-CD4 treatment likely depleted CD4+ Tregs, which possibility is that, for unknown reasons, autoreactive CD8 cells may suppress autoimmune CD8 cells. were reactive only against cardiac-specific Ags. In contrast, au- Of note, there was mild but significant heart disease in the toreactive CD8 cells could recognize Ags with a broader tissue absence of TS1 cells when donors were RAG22/2PDL-L1/22/2 distribution. However, we might not have readily been able to The Journal of Immunology 845 detect their contribution to disease in tissues wherein TS1 already some cases and in fact why chronic GVHD can be unremitting. cause significant immunopathology. For example, in the absence of Although we have used genetic knockouts to elicit strong pheno- PD-ligands, but without TS1, there was low-level liver histopa- types, differences in the strength of PD-ligand signals, perhaps thology, which was not present when donor and recipient were genetically encoded, could underlie susceptibility to chronic RAG22/2. Such mild changes could easily have been masked by GVHD, which itself could be partly caused by autoimmune in- HA-directed alloimmune damage. duction downstream of alloimmune-mediated tissue damage. The PD-1 pathway has not been implicated in protecting the heart in polyclonal models of GVHD. In most of these reports, Acknowledgments impairment of the PD-1 pathway led to early death such that later We acknowledge the animal technicians and the flow cytometry and cell onset myocarditis could not have been evaluated. It will be of sorting facilities at Yale University School of Medicine and the University interest to determine whether autoimmune myocarditis develops in of Pittsburgh School of Medicine. the absence of PD-ligands in polyclonal GVHD models, which will require establishing conditions such that PD-1 blockade does not result in early lethality, but still intensifies GVHD. Disclosures Outside the GVHD literature, there is evidence that PD-1 and its The authors have no financial conflicts of interest. ligands protect the heart against autoimmunity. PD-12/2 BALB/c mice develop fatal dilated cardiomyopathy, although this was References found to be Ab mediated (26, 27), whereas disease in our model 1. Anderson, B. E., J. McNiff, J. Yan, H. Doyle, M. Mamula, M. J. Shlomchik, and was caused by CD8+ T cells. PD-1– or PD-L1–deficient MRL-lpr W. D. Shlomchik. 2003. Memory CD4+ T cells do not induce graft-versus-host mice developed cardiomyositis with a mixed inflammatory infil- disease. J. Clin. Invest. 112: 101–108. Downloaded from 2. Zheng, H., C. Matte-Martone, H. Li, B. E. Anderson, S. Venketesan, H. Sheng trate along with anti-cardiac Abs (24, 28). PD-1 and PD-L1 also Tan, D. Jain, J. McNiff, and W. D. Shlomchik. 2008. Effector memory CD4+ suppressed experimental myocarditis directed against a model Ag T cells mediate graft-versus-leukemia without inducing graft-versus-host disease. Blood 111: 2476–2484. (25, 29), which differs substantially from the autoimmune disease 3. Juchem, K. W., B. E. Anderson, C. Zhang, J. M. McNiff, A. J. Demetris, we describe. D. L. Farber, A. J. Caton, W. D. Shlomchik, and M. J. Shlomchik. 2011. A Recent reports describe rare cases of myocarditis with check- repertoire-independent and cell-intrinsic defect in murine GVHD induction by Blood effector memory T cells. 118: 6209–6219. http://www.jimmunol.org/ point inhibitor therapies, especially when CTLA-4 and PD-1 were 4. Chen, B. J., X. Cui, G. D. Sempowski, C. Liu, and N. J. Chao. 2004. Transfer of combined (30–32). PD-1 or PD-L1 inhibition is being considered allogeneic CD62L- memory T cells without graft-versus-host disease. Blood in patients with relapsed hematologic malignancies after alloSCT 103: 1534–1541. 5. Chen, B. J., D. Deoliveira, X. Cui, N. T. Le, J. Son, J. F. Whitesides, and and has been applied in a small number of patients, mostly with N. J. Chao. 2007. Inability of memory T cells to induce graft-versus-host disease Hodgkin’s lymphoma relapsed after alloSCT (33, 34). Although is a result of an abortive alloresponse. Blood 109: 3115–3123. 6. Bleakley, M., S. Heimfeld, K. R. Loeb, L. A. Jones, C. Chaney, S. Seropian, prior mouse work clearly demonstrated the risk for increased T. Gooley, F. Sommermeyer, S. R. Riddell, and W. D. Shlomchik. 2015. Out- GVHD with such therapy (11–13, 35), our results raise additional comes of acute leukemia patients transplanted with naı¨ve T cells depleted stem concerns that autoimmune manifestations, including heart disease, cell grafts. J. Clin. Invest. 125: 2677–2689. 7. Anderson,B.E.,P.A.Taylor,J.M.McNiff,D.Jain,A.J.Demetris,A.Panoskaltsis- could be precipitated.

Mortari, A. Ager, B. R. Blazar, W. D. Shlomchik, and M. J. Shlomchik. 2008. Effects by guest on October 1, 2021 When cardiac dysfunction occurs after alloSCT, it is frequently in of donor T-cell trafficking and priming site on graft-versus-host disease induction by the setting of complex medical problems that can include GVHD. naive and memory phenotype CD4 T cells. Blood 111: 5242–5251. 8. Shih, F. F., D. M. Cerasoli, and A. J. Caton. 1997. A major T cell determinant Heart pathology is rarely examined, such that cardiac GVHD could from the influenza virus hemagglutinin (HA) can be a cryptic self peptide in HA be underappreciated. Nonetheless, cardiac GVHD has been reported. transgenic mice. Int. Immunol. 9: 249–261. 9. Kirberg, J., A. Baron, S. Jakob, A. Rolink, K. Karjalainen, and H. von Boehmer. Diagnoses included heart block (as we observed), myocarditis, 1994. Thymic selection of CD8+ single positive cells with a class II major cardiomyopathy, and coronary artery disease, with instances of histocompatibility complex-restricted receptor. J. Exp. Med. 180: 25–34. patients improving with immunosuppressants (36, 37). 10. Caton, A. J., J. R. Swartzentruber, A. L. Kuhl, S. R. Carding, and S. E. Stark. 1996. Activation and negative selection of functionally distinct subsets of Our results bear on the long-standing hypothesis that chronic antibody-secreting cells by influenza hemagglutinin as a viral and a neo-self GVHD has an autoimmune component (38–41). Acute GVHD is antigen. J. Exp. Med. 183: 13–26. the single greatest risk factor for chronic GVHD, which suggests 11. Blazar, B. R., B. M. Carreno, A. Panoskaltsis-Mortari, L. Carter, Y. Iwai, H. Yagita, H. Nishimura, and P. A. Taylor. 2003. Blockade of programmed that alloimmunity can beget autoimmunity. Our data directly death-1 engagement accelerates graft-versus-host disease lethality by an IFN- demonstrate that this can occur and that it is important for path- gamma-dependent mechanism. J. Immunol. 171: 1272–1277. 12. Saha, A., K. Aoyama, P. A. Taylor, B. H. Koehn, R. G. Veenstra, A. Panoskaltsis- ogenesis. It is of a chronic nature in that it occurs only later in the Mortari, D. H. Munn, W. J. Murphy, M. Azuma, H. Yagita, et al. 2013. Host course, after the initial wave of TS1-mediated allorecognition that programmed death ligand 1 is dominant over programmed death ligand 2 ex- leads to acute disease. Multiple nonexclusive mechanisms have pression in regulating graft-versus-host disease lethality. Blood 122: 3062–3073. 13. Deng, R., K. Cassady, X. Li, S. Yao, M. Zhang, J. Racine, J. Lin, L. Chen, and been suggested for GVHD-associated autoimmunity in other D. Zeng. 2015. B7H1/CD80 interaction augments PD-1-dependent T cell apo- contexts (41–44). Our data suggest that if chronic GVHD is at ptosis and ameliorates graft-versus-host disease. J. Immunol. 194: 560–574. least in part autoimmune, PD-ligands may be an additional 14. Keir, M. E., M. J. Butte, G. J. Freeman, and A. H. Sharpe. 2008. PD-1 and its ligands in tolerance and immunity. Annu. Rev. Immunol. 26: 677–704. mechanism that promotes self-tolerance. In our experiments, re- 15. Wherry, E. J. 2011. T cell exhaustion. Nat. Immunol. 12: 492–499. sidual recipient T cells were sufficient to cause myocarditis. Even 16. Zhang, P., J. Wu, D. Deoliveira, N. J. Chao, and B. J. Chen. 2012. Allospecific CD4(+) effector memory T cells do not induce graft-versus-host disease in mice. with ablative conditioning, a substantial number of patients have Biol. Blood Marrow Transplant. 18: 1488–1499. residual circulating host-derived T cells posttransplant (45–51). 17. Markey, K. A., T. Banovic, R. D. Kuns, S. D. Olver, A. L. Don, N. C. 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CD8+ but not CD4+ T cells require cognate interactions with target tissues to mediate PD-ligand–dependent regulation of autoimmunity that is triggered GVHD across only minor H antigens, whereas both CD4+ and CD8+ T cells by initial alloimmunity. This could explain chronicity of GVHD in require direct leukemic contact to mediate GVL. Blood 111: 3884–3892. 846 PD-L1 PREVENTS GVHD-INDUCED AUTOIMMUNITY

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