Immune-checkpoint VISTA and PD-1 nonredundantly regulate murine T-cell responses

Jun Liua,b, Ying Yuana,1, Wenna Chena, Juan Putrac, Arief A. Suriawinatac, Austin D. Schenkd, Halli E. Millera, Indira Guleriae, Richard J. Barthd, Yina H. Huangc, and Li Wanga,2

aDepartment of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226; bJiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009, People’s Republic of China; cDepartment of Pathology and dDepartment of Surgery, Geisel School of Medicine at Dartmouth, Norris Cotton Center, Lebanon, NH 03756; and eBoston Children’s Hospital and Brigham and Women’s Hospital, Renal Division, Harvard Medical School, Boston, MA 02115

Edited by Michael J. Bevan, University of Washington, Seattle, WA, and approved April 2, 2015 (received for review October 23, 2014)

V-domain immunoglobulin suppressor of T-cell activation (VISTA) (mAb) enhances disease severity in the experimental autoimmune is a negative immune-checkpoint that suppresses T-cell encephalomyelitis (EAE) model, as well as boosts antitumor im- responses. To determine whether VISTA synergizes with another munity in multiple murine tumor models (7). immune-checkpoint, programmed death 1 (PD-1), this study charac- The regulatory functions of immune-checkpoints have been terizes the immune responses in VISTA-deficient, PD-1-deficient (KO) demonstrated using KO mice, which typically manifest loss of pe- mice and VISTA/PD-1 double KO mice. Chronic inflammation and ripheral tolerance and heightened T-cell responses. For example, spontaneous activation of T cells were observed in both single KO CTLA-4 KO mice die of young age because of overwhelming lym- mice, demonstrating their nonredundancy. However, the VISTA/PD-1 phoproliferative disease and inflammation (8, 9). PD-1 KO mice display genetic background-dependent late-onset autoimmune dis- double KO mice exhibited significantly higher levels of these pheno- ease of either dilated cardiomyopathy or arthritis (10, 11). PD-1 typesthanthesingleKOmice.Whenbredontothe2D2T-cell binds to two ligands, PD- 1 (L1) and PD-L2 (12, 13). Although transgenic mice, which are predisposed to development neither PD-L1 nor PD-L2 KO mice develop overt organ-specific of inflammatory autoimmune disease in the CNS, the level of autoimmune disease, PD-L1 genetic deficiency exacerbates disease disease penetrance was significantly enhanced in the double KO in the EAE model, as well as impairs fetomaternal tolerance (14, 15).

mice compared with in the single KO mice. Consistently, the magni- These data suggest a critical role for PD-L1 in maintaining T-cell INFLAMMATION tude of T-cell response toward foreign antigens was synergistically peripheral tolerance at tissue sites. In contrast, genetic disruption of IMMUNOLOGY AND higher in the VISTA/PD-1 double KO mice. A combinatorial blockade other ligands such as -H4 fail to display any using monoclonal antibodies specific for VISTA and PD-L1 achieved significant alterations of T-cell responses in vivo, suggesting there is a optimal tumor-clearing therapeutic efficacy. In conclusion, our study hierarchy and potential redundancy among various immune-check- demonstrates the nonredundant role of VISTA that is distinct from point regulators (16). In this context, it is important to note that the the PD-1/PD-L1 pathway in controlling T-cell activation. These find- expression pattern of VISTA overlaps with multiple other immune- ings provide the rationale to concurrently target VISTA and PD-1 checkpoint regulators (3). It is therefore critical to determine pathways for treating T-cell-regulated diseases such as cancer. whether VISTA exerts nonredundant immune regulatory functions. Studies have shown that several immune-checkpoints function- – immune-checkpoint | autoimmunity | tumor immunity | ally synergize with each other (17 19). For example, the combined | T-cell activation Significance -cell activation requires engagement with antigen-presenting Tcells (APCs) that present the cognate peptide on MHC mol- Multiple immune-checkpoint proteins, such as programmed ecules. Antigen recognition is regulated by cosignaling ligands and death 1 (PD-1), LAG3, and TIM3, are coexpressed on immune receptors, whose integrated signaling determines the outcome of cells and functionally synergize with each other. V-domain T-cell activation, differentiation, and function (1). The B7 family immunoglobulin suppressor of T-cell activation (VISTA) is a of coreceptors belongs to the Ig superfamily, consisting of stimu- recently identified immune-checkpoint molecule that sup- latory receptors such as CD28 and inducible T-cell costimulator presses T-cell activation. This study establishes that VISTA and (ICOS), cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) and PD-1 exert nonredundant immune regulatory functions and programmed death 1 (PD-1) coinhibitory receptors, and B7-H3 synergistically regulate T-cell responses. Combinatorial treat- and B7-H4 ligands, whose receptors have yet to be identified (1, 2). ment using VISTA- and PD-ligand 1-specific monoclonal anti- Together with additional inhibitory molecules such as T-cell im- bodies achieved synergistic therapeutic efficacy in murine munoglobulin domain and mucin domain 3 (Tim-3), lymphocyte- tumor models. This study critically advances our knowledge of activation 3 (LAG3), and B- and T-lymphocyte attenuator the immune regulatory function of VISTA and provides a ra- (BTLA), these immune-checkpoint proteins play critical roles in tionale for targeting both VISTA and PD-1 to more effectively maintaining peripheral tolerance and controlling autoimmunity. treat T-cell-regulated diseases such as cancer. VISTA is a newly identified Ig domain-containing immune- Author contributions: L.W. designed research; J.L., Y.Y., W.C., J.P., A.D.S., H.E.M., and L.W. checkpoint molecule that directly suppresses T-cell activation in performed research; J.L., I.G., Y.H.H., and L.W. contributed new reagents/analytic tools; vitro and in vivo (3, 4). The human and murine VISTA proteins J.L., Y.Y., W.C., J.P., A.A.S., A.D.S., R.J.B., Y.H.H., and L.W. analyzed data; and L.W. wrote share 90% identity and display similar expression patterns (5). the paper. VISTA is constitutively expressed within the hematopoietic com- Conflict of interest statement: L.W. is involved with the commercial development of partment, with the highest expression level on myeloid cells VISTA with ImmuNext Inc Corporation and received research support, salary, and/or + + + + and a lower level on CD4 ,CD8 T cells, and Foxp3 CD4 reg- consulting fees. ulatory T cells. A soluble VISTA-Ig protein that contains the This article is a PNAS Direct Submission. extracellular domain fused with Ig-crystalizable fragment (Fc) or 1Present address: College of Pharmacy, Shanghai University of Traditional Chinese Med- full-length VISTA expressed on APCs acts as a ligand to suppress icine, Shanghai 201203, People’s Republic of China. T-cell proliferation and cytokine production (3). In addition, 2To whom correspondence should be addressed. Email: [email protected]. VISTA might function as an inhibitory receptor on T cells to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. suppress their activation (6). VISTA-specific monoclonal antibody 1073/pnas.1420370112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1420370112 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 disruption of LAG3 and PD-1 results in lethal autoimmune blindly quantified on the basis of a semiquantitative scoring diseases, whereas loss of either gene alone leads to subtle pheno- method, and the VISTA/PD-1 double KO mice showed the types (17). In addition, PD-1 functionally synergizes with TIM3 highest scores compared with WT and single KO mice (Fig. 1B). (18, 19). A combinatorial antibody-mediated blockade of both Despite the significant accumulation of activated T cells, the PD-1 and TIM3 results in optimal T-cell responses against cancer, double KO mice did not develop overt autoimmune disease. as well as during chronic viral infection (18, 19). In the current study, Serum levels of IgM and IgA were moderately elevated in aged we use KO mice to address whether VISTA synergizes with PD-1 in double KO mice (Fig. S1). Our cohoused PD-1 KO mice also regulating T-cell responses. Our data establish that VISTA and the developed accumulation of spontaneously activated T cells and PD-1/PD-L1 pathways nonredundantly regulate T-cell activation chronic inflammation in multiple organs but failed to develop and demonstrate the feasibility of concurrently targeting these two arthritis or other previously reported autoimmune phenotypes immune-checkpoints to enhance tumor-specific immune responses. (10). This discrepancy might be a result of the different housing conditions or the age of the mice analyzed. Results Compared with VISTA KO and PD-1 KO mice, VISTA/PD-1 Loss of T-Cell Peripheral Tolerance on Combined Genetic Disruption of double KO mice at the age of 6–7 mo showed significantly in- hi lo + + VISTA and PD-1 or PD-L1. To determine whether VISTA and PD-1 creased frequencies of CD44 CD62L CD8 and CD4 T cells, regulate immune responses in a redundant or independent/syn- which is indicative of an activated or memory phenotype (Fig. 2 −/− −/− ergistic manner, Vista Pdcd1 mice (herein referred to as A and C). On in vitro restimulation, the double KO T cells VISTA/PD-1 double KO mice) were generated on the C57BL/6 produced significantly higher levels of cytokines, such as IFNγ, background and characterized. The double KO mice were born TNFα, and IL-17A, than WT and single KO cells (Fig. 2 B, D–F). fertile and produced normal litter sizes. Normal thymic de- PD-1 binds to ligands PD-L1 and PD-L2 (20). To corroborate velopment and lymphocyte populations (T and B lymphocytes, the results seen in the VISTA/PD-1 double KO mice, we bred natural killer cells, and natural killer T cells) in the bone marrow, VISTA KO onto the previously described PD-L1 KO (15) and – generated the VISTA/PD-L1 double KO mice. Our data dem- spleen, and lymph nodes were observed in 6 8-wk-old KO mice. + + Comprehensive multiorgan histological analyses were per- onstrated spontaneous activation of peripheral CD4 and CD8 formed in 12-mo-old WT, VISTA KO, PD-1 KO, and VISTA/ T cells in the VISTA/PD-L1 double KO mice, which was com- PD-1 double KO mice (Fig. 1). Hematoxylin and eosin (H&E) parable to that of VISTA/PD-1 double KO mice (Fig. S2 A and stained sections from heart, lung, liver, kidney, pancreas, salivary B). Together, these data support the conclusion that VISTA and gland, small and large intestines, and brain were examined. the PD-1/PD-L1 pathway nonredundantly control the peripheral Several organs, including lung, liver, and pancreas in the double tolerance of T cells. KO mice, were heavily infiltrated with leukocytes (Fig. 1A, Top) The phenotype of spontaneous T-cell activation in the double and showed significant tissue necrosis, presumably as a result of KO mice (Fig. 2 and Fig. S2) indicates that both VISTA and PD-1 immune cell-mediated destruction (Fig. 1A, Bottom). The levels regulate the threshold of TCR activation to autoantigens. We of leukocyte infiltration and tissue necrosis in the KO mice were hypothesize that disruption of both pathways will increase

Fig. 1. Histologic analysis of aged VISTA KO, PD-1 KO, and VISTA/PD-1 double KO mice. Necropsy was performed on 12-mo-old WT (n = 16), VISTA KO (n = 15), PD-1 KO (n = 28), and VISTA/PD-1 double KO (n = 25) mice. Organs were fixed, paraffin embedded, sectioned, and stained with H&E. Two representative H&E sections from lung, liver, and pancreas of the VISTA/PD-1 double KO mice are shown in A. Clusters of tissue-infiltrating leukocytes were marked with black arrows. (Top) Areas of necrotic tissues were marked with white arrows. (Bottom). All images are of 200× magnification. (Scale bar: 50 μ.) The inflammatory state of the tissues was evaluated on the basis of a semiquantitative method that scores the level of the leukocyte infiltration and tissue necrosis (B).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1420370112 Liu et al. Downloaded by guest on September 23, 2021 the 2D2-VISTA/PD-1 double KO mice, which was similar to the ABp< 0.0001 p< 0.0001 p< 0.0001 p: 0.01 – 100 p: 0.01 p: 0.01 20 p: 0.02 ns ages observed in the 2D2-VISTA KO mice (5 16 wk) (Fig. S3). % 80 p< 0.0001 p< 0.0001 ns ns Only one WT mouse developed disease around 12 wk of age. A low 15

% small percentage of diseased mice from the 2D2-VISTA KO (2/40)

60 + 10 CD62L 40 and 2D2-VISTA/PD-1 double KO (3/37) mice developed atypical hi γ IFN 5 EAE, manifested as unilateral paralysis rather than bilateral pa- 20 CD44 ralysis. Detailed information regarding disease onset, severity, 0 0 t t t t w O w O O w O O w O O penetrance, and mortality is presented in Table S1. -K -KO -K -K -K K -KO -K -K -KO D-KO V e D-KO V le D V- e D V e P P b P P bl doubl dou doubl dou Combined Disruption of VISTA and PD-1 Synergistically Augments Spleen Blood Spleen Blood T-Cell Responses on Antigen Challenge. Spontaneous T-cell activa- + tion and enhanced autoimmunity in aged VISTA/PD-1 double CD8 T cells KO mice indicate that VISTA and PD-1 control T-cell tolerance toward autoantigens. We hypothesize that these pathways also C p< 0.0001 p< 0.0001 D p: 0.0007 p: 0.001 critically regulate T-cell responses toward foreign antigens. To 80 p:0.002 p:0.01 30 p: 0.008 p: 0.04 address this question, mice were immunized with soluble anti- % p:0.0002 ns p: 0.001 p: 0.007

low 60 genic peptides together with poly (I:C) (TLR3 agonist) as adju- 20

% 257-264 + vant. 2W1S, an MHC class II-restricted peptide, and OVA 40 γ ,

CD62L + hi IFN 10 an MHC class I-restricted peptide, were used (22). On day 7 20 postimmunization, splenic T cells were isolated and restimulated CD44 0 0 ex vivo with the respective peptides. Significantly higher numbers t t t t w O O O w O w O O O w O O O γ -K -K -K KO -K -KO K -K -K -K -K -K of IFN -producing T cells were present in the VISTA/PD-1 D V e D- V e V le V e P bl P PD- PD oubl dou doubl doub d double KO mice compared with in WT or single KO mice, in- Spleen Blood Spleen Blood dicating that VISTA and PD-1 nonredundantly control T-cell responses (Fig. 4 A and B). + CD4 T cells We have previously reported that VISTA functions as a ligand that engages an unknown receptor on T cells and suppresses T-cell activation (3). Because both VISTA and PD-L1 are highly + E p: 0.002 p: 0.003 F p< 0.0001 p< 0.0001 expressed on CD11b myeloid APCs (2, 3), we hypothesize that INFLAMMATION 20 2.5 IMMUNOLOGY AND p: 0.01 ns a combined deficiency of VISTA and PD-L1 on APCs maximally p< 0.0001 p: 0.001 +

% p: 0.02 ns 2.0 ns ns + 15 enhances T-cell activation. To test this hypothesis, CD11b % + 1.5 myeloid DCs were isolated from WT, VISTA KO, PD-L1 KO, 10 TNFa

+ 1.0 and the VISTA/PD-L1 double KO mice (15, 23) and were used γ

IL-17A + 5 0.5

IFN to stimulate naive CD4 OTII TCR transgenic T cells in the 0 0.0 presence of cognate peptide OVA323–339. Our data show that the t t t wt O O O w O O w O O O w O -K -K -K -KO -K -K -K -K -K KO -KO -K combined deficiency of VISTA and PD-L1 on myeloid APCs D V le D V e D V D- V le P b P bl P P b u synergistically enhanced T-cell proliferation and IFNγ pro- dou do double dou C D Spleen Blood Spleen Blood duction (Fig. 4 and ). + CD4 T cells

Fig. 2. Spontaneous T-cell activation in the VISTA KO, PD-1 KO, and VISTA/ PD-1 double KO mice. Splenic T cells were collected from age- and sex- matched 6–7-mo-old WT (n = 6), VISTA KO (n = 4), PD-1 KO (n = 6), and + + VISTA/PD-1 double KO (n = 8) mice. The percentages of CD8 and CD4 T cells with activated phenotype (CD44hi CD62Llo) were quantified by flow cytometry. T cells were stimulated ex vivo overnight with soluble anti-CD3/ CD28 mAbs, and their cytokine production (i.e., IFNγ, TNFα, and IL-17A) was + examined by intracellular staining. CD8 T-cell phenotypes were shown in A and B. CD4+ T-cell phenotypes were shown in C–F. Representative results of at least three independent experiments were shown.

predisposition to autoimmune disease on a susceptible back- ground. To test this hypothesis, the VISTA/PD-1 double KO mice were bred with the 2D2 mice, which express a TCR trans- gene specific for the self-antigen, myelin oligodendrocyte gly- coprotein (MOG35–55) (21). Previous studies reported that 4% of 2D2 mice developed spontaneous EAE between the ages of 3 and 5 mo (21). A similar incidence of spontaneous EAE (1/30, ∼3%) was observed in our colony of 2D2-WT mice younger than 6mo(Fig.3A and Table S1). 2D2-PD-1 KO mice showed similar incidence of spontaneous disease as WT mice (2/40, 5%). In Fig. 3. Combined genetic deficiency of VISTA and PD-1 exacerbated auto- contrast, genetic deficiency of VISTA accelerated disease onset, immune disease on the susceptible background. The CNS disease incidence ∼ (A) and mortality (B) were monitored in 2D2 TCR transgenic mice that were such that 60% (25/42) of the 2D2-VISTA KO mice rapidly bred onto the VISTA KO, PD-1 KO, and double KO genetic background. succumbed to complete hind limb paralysis within 3 mo. Combined Representative H&E stained spinal cord section from paralyzed double KO deficiency of VISTA and PD-1 further increased disease in- C ∼ A B mice is shown ( ). Enlarged images show areas of extensive lymphocyte cidence to 90% (35/37) (Fig. 3 and ). Analysis of CNS tissue infiltration. Luxol fast blue staining of spinal cord sections confirmed ex- from paralyzed 2D2-VISTA/PD-1 double KO mice confirmed tensive demyelination (D). 2D2-WT (n = 30), 2D2-VISTA KO (n = 42), 2D2-PD-1 the accumulation of inflammatory cells and demyelination (Fig. KO (n = 40), 2D2-VISTA/PD-1 double KO (n = 37). Only one 2D2-WT mouse 3 C and D). The disease onset age ranged between 4 and 16 wk in developed disease.

Liu et al. PNAS Early Edition | 3of6 Downloaded by guest on September 23, 2021 as they expressed a high level of PD-1 (27). Consistent with A OVA257-264 B 2W1S p = 0.02 750 p < 0.0001 600 our hypothesis, the combined engagement of VISTA-Ig and PD- 500 p = 0.02 L1-Ig maximally reduced the phosphorylation of LAT and its 600 p = 0.005 C 400 p = 0.06 recruitment to the CD3 complex (Fig. 5 ). Furthermore, VISTA-Ig 450 p = 0.04 300 and PD-L1-Ig coengagement maximally reduced the phosphoryla- 300 tion of SLP76, PLC-γ1,Akt,andErk1/2intotalcelllysates(Fig.5D). 200 Spleen cells Spleen Spots / 500k Spleen cells 150 / 500k Spots Together, these results support the conclusion that VISTA-R 100 and PD-1 each impairs early TCR signaling and results in the 0 0 T O O O most robust suppression when combined. W K K T O O - -K - W K -K A 1 - -KO 1 Enhanced T-cell responses in the VISTA/PD-1 double KO T D1 D D IS P /P TA D1 V A IS P /P V mice suggest that these two immune checkpoints could be tar- IST TA V IS V geted concurrently to optimize antitumor immunity. This hy- WT pothesis was tested in transplantable murine tumor models (Fig. 6). VISTA-KO PD1-KO VISTA/PD1-KO C D p = 0.01 100 p = 0.003 p = 0.003 ns A DO11.10 cells C DO11.10 cells 200000 ns ns 80 ns p-LAT p-LAT 150000 ns ns 60 (pg/ml)

γ 40 Total-LAT Total-LAT 100000 CPM IFN 20 50000 CD3zeta CD3zeta 0 0 O O O WT K -K -K LI no-stim combo 1:3 1:6 A- -L1 VISTA-IgPD-L1-Ig T D- VISTA-Ig Control-Ig IS P D Control-Ig V /P Ratio (APC:T cells) A T IS V B CD4+CD25- T cells D CD4+CD25- T cells Fig. 4. VISTA and the PD-1 collaboratively controlled antigen-specific T-cell p-LAT p-LAT responses. Six- to 7-wk-old WT (n = 8), VISTA KO (n = 9), PD-1 KO (n = 7), and VISTA/PD-1 double KO (n = 6) mice were immunized with 50 μg soluble Total-LAT Total-LAT peptides OVA257-264 (A) or 2W1S (B), together with TLR3 agonist poly (I:C) (100 μg) as adjuvant. Splenocytes were harvested on day +7 postimmunization p-SLP76 p-SLP76 and restimulated with the respective peptides. IFNγ-producing cells were + + Total-SLP76 enumerated by the ELISPot assay. To stimulate T cells in vitro, CD11b CD11c Total-SLP76 DCs were sorted from WT, VISTA KO, PD-L1 KO, and VISTA/PD-L1 double KO + p-PLCγ-1 mice and incubated with naive CD4 OTII TCR transgenic T cells in the presence γ 3 p-PLC -1 of cognate peptides OVA323–339 (10 ng/mL). [ H]-Thymidine was added to the Total-PLCγ-1 culture for the last 8 h of the 72-h culture period for measuring T-cell pro- Total-PLCγ-1 liferation (C). The production of IFNγ was quantified from the culture super- p-Akt natants by ELISA (D). p-Akt Total-Akt Total-Akt Although the receptor for VISTA (VISTA-R) is unknown, we speculate that the engagement of VISTA-R on T cells suppresses p-Erk1/2 p-Erk1/2 TCR signaling independent of PD-1. In addition, we hypothesize Total-Erk1/2 Total-Erk1/2 that the coengagement of VISTA-R and PD-1 on T cells syn- ergistically impairs TCR signaling. To test these hypotheses, Rested no-stim combo VISTA-Ig VISTA-IgPD-L1-Ig proximal TCR signaling events were examined using immobi- Control-Ig Control-Ig lized fusion proteins VISTA-Ig and PD-L1-Ig, both of which suppressed T-cell proliferation and cytokine production in vitro Fig. 5. Engagement of both VISTA and PD-L1 during TCR activation maxi- mally suppressed TCR signaling. To determine whether VISTA engagement (3, 12). LAT is a proximal signaling adaptor that is phosphory- impairs the recruitment of signaling adaptor protein LAT, DO11.10 hybrid- lated on TCR stimulation and forms a complex with multiple sig- oma cells (100 × 106) were stimulated with plate-bound anti-CD3 mAb (2C11, naling molecules including SH2 domain containing leukocyte 3 μg/mL), together with coimmobilized control-Ig (8 μg/mL) or VISTA-Ig fu- protein of 76kDa (SLP76) and phospholipase C (PLC)-γ1 (24). To sion protein (8 μg/mL) for 10 min at 37 °C and lysed in situ. After removing determine whether VISTA functions by interfering with the the unbound cell lysates, plate-bound protein was eluted off the plate and phosphorylation of LAT, a solid phase immunoprecipitation assay examined by Western blotting (A). To examine the effect of VISTA on the was performed, in which the proximal signaling complexes could phosphorylation of TCR signaling molecules, CD25−CD4+ T cells were puri- be recovered as the bound fraction to the plastic surface (25, 26). fied from naive splenocytes and stimulated with plate-bound 2C11 (3 μg/mL), Our data show that in the presence of coimmobilized control-Ig or together with control-Ig (8 μg/mL) or VISTA-Ig (8 μg/mL) for 5 min at 37 °C. VISTA-Ig proteins, plate-bound anti-CD3e mAb pulled-down Total cell lysates were prepared, and the phosphorylation status of LAT, comparable amounts of CD3ζ. This result excluded the possibility SLP76, PLC-γ1, Akt, and Erk1/2 was examined (B). To determine whether that the immobilized VISTA-Ig displaced anti-CD3e mAb or coengagement of both VISTA and PD-L1 maximally suppresses LAT activa- e tion, DO11.10 cells were stimulated with plate-bound 2C11 (2.5 μg/mL), to- impaired the binding of anti-CD3 mAb to the TCR/CD3 complex μ μ μ (Fig. 5A). Despite the similar engagement of CD3ζ, immobilized gether with control-Ig (10 g/mL), VISTA-Ig (5 g/mL), PD-L1-Ig (5 g/mL), or both Ig fusion proteins. Cells were lysed after 10 min stimulation, and plate- VISTA-Ig significantly reduced the amount of LAT recruited to C A bound proteins were recovered and examined as described earlier ( ). To the CD3 complex, as well as its phosphorylation (Fig. 5 ). When determine the synergistic effects of engaging both VISTA and PD-L1, pre- + total cell lysates were examined, the phosphorylation of several activated splenic CD4 T cells were stimulated with plate-bound 2C11 proximal and downstream signaling molecules such as SLP76, (2.5 μg/mL), together with control-Ig (9 μg/mL), VISTA-Ig (3 μg/mL), PD- PLC-γ1, Akt, and Erk1/2 were also impaired (Fig. 5B). L1-Ig (6 μg/mL), or both Ig fusion proteins for 10 min at 37 °C. Total cell Next, the effect of coengaging VISTA-Ig and PD-L1-Ig was lysates were harvested for Western blotting analysis (D). Representative examined (Fig. 5 C and D). Preactivated T cells were analyzed, results from two to three independent experiments were shown.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1420370112 Liu et al. Downloaded by guest on September 23, 2021 the VISTA mAb-treated group and 3/8 in the PD-L1 mAb-treated A control-Ig (n=8) group rejected tumor) (Fig. 6A). Analysis of tumor-specific T-cell anti-VISTA (n=8) anti-PD-L1 (n=8) activation showed synergistically enhanced cytokine production 100 γ α 250 combo (n=8) (IFN and TNF ) and granzyme B production by tumor-specific ) control-Ig

2 + anti-VISTA B 200 75 CD8 T cells from tumor-draining LN (Fig. 6 ). Next, combi- **** anti-PD-L1 combo natorial treatment was tested on larger established tumors. When 150 50 treatment was initiated on day +5 after CT26 inoculation, the 100 **** **** 25 average tumor size reached ∼4–5 mm in diameter (Fig. 6C). To Percent survival Percent 50 0 facilitate tumor-specific T-cell activation in this therapeutic set- ****

Tumor size ( mm size Tumor 0 20 40 60 80 100 0 ting, mice were treated with anti-CD25 mAb on day +5and 10 20 30 40 Time (Days) + + + + Time (Days) day 20 to transiently deplete CD25 Foxp3 CD4 regulatory p = 0.01 p = 0.04 p = 0.018 T cells. Synergistic efficacy was achieved with the combined

B + 50 + 20 25 ∼ p = 0.02 ns p = 0.018 treatment of VISTA and PD-L1 mAbs, which led to 80% tumor 40 20 cells P = 0.05 15 ns ns regression (8/10), whereas single mAb treatment failed to show % CD8 + + % CD8 % 30 + 15 any significant effect (Fig. 6C). α 10 20 10 We next evaluated the therapeutic efficacy of the combina- % CD8 + TNF γ GranzB

+ 5

+ 5 torial treatment in a less immunogenic tumor model, such as the 10 γ γ IFN

IFN B16BL6 model, which is responsive to therapeutic

0 0 IFN 0 Ig A Ig A Ig A o – - T -L1 - T -L1 bo T -L1 b blockade of CTLA-4 and PD-1 in previous studies (28 31). A ol IS D mbo ol IS D m ol- IS D r V P tr V P o tr V P om a a co a a c n a a c ont on o GM-CSF secreting cellular vaccine (GVAX) (28) was applied on c c c + + + + control-Ig (n= 9) day 3, 6, 9, and 12 after tumor inoculation to boost tumor- C aPD-L1 (n= 10) specific T-cell responses. A sublethal whole-body irradiation aVISTA (n= 10) 250 100 (250 rads) was applied on day +3, which was shown to facilitate ) combo (n= 10) 2 200 80 T-cell-mediated antitumor immune responses (32). Consistent control-Ig **** 150 60 with our hypothesis, treatment with both VISTA and PD-L1 ** aPD-L1 *** 40 aVISTA mAbs significantly suppressed tumor growth and conferred sur- 100 combo 20 vival advantage, whereas single mAb treatment was largely in- 50 survival Percent D tumor size (mm 0 effective (Fig. 6 ). Together, these data indicate that VISTA 0 0 20406080 and PD-L1/PD-1 pathways independently control tumor-specific 0 4 8 12162024 INFLAMMATION

Time (Days) IMMUNOLOGY AND Time (Days) T-cell responses, and combined therapeutic blockade synergis- tically enhances antitumor immunity. D notreat notreat Gvax/RT + control-Ig Gvax/RT + control-Ig Gvax/RT + anti-PD-L1 Gvax/RT+ anti-PD-L1 Discussion 200 Gvax/RT + anti-VISTA Gvax/RT + anti-VISTA ) Gvax/RT + combo 100 2 Gvax/RT + combo VISTA and PD-1 both function as immune checkpoint proteins (n=10/group) 150 75 that suppress T-cell activation. They share overlapping expression

ns patterns within the hematopoietic compartment. It is therefore 100 *** 50 important to define their independent immune-regulatory roles.

50 **** **** 25 Evidence based on studies of the KO mice indicates that VISTA Percent survival tumor size (mm size tumor 0 0 and PD-1 nonredundantly regulate immune responses. Genetic 912151821 0204060 disruption of VISTA accelerated autoimmune disease on a sus- Time (Days) Time (Days) ceptible background, as well as resulted in multiorgan chronic in- Fig. 6. Optimal therapeutic efficacy on combined blockade of VISTA and flammation because of spontaneous T-cell activation (22). Aged PD-L1 in murine tumor models. CT26 colon carcinoma cells (100,000) were PD-1 KO mice were reported to develop late-onset autoimmunity inoculated on the flank of naive mice on day 0 (n = 8). Day +3 after tumor in the C57BL/6 background (10). Our study of PD-1 KO mice inoculation, mice were treated with control Ig (300 μg), anti-VISTA mAb also showed accumulation of spontaneously activated T cells (300 μg), anti-PD-L1 mAb (200 μg), or combined anti-VISTA and anti-PD-L1 and chronic inflammation in multiple organs. Furthermore, in the mab, every 2–3 d continuously for 3 wk. Tumor size was measured by a caliper current study of the VISTA/PD-1 double KO mice, we provided and recorded as area (mm2)(A). The rate of tumor-free survival was also strong evidence for independent control of T-cell responses by shown (A). To examine tumor-specific T-cell responses, lymphocytes were these two checkpoints. Synergistic or additive T-cell activation harvested from tumor-draining lymph nodes on day +14 after tumor in- was observed from aged double KO mice compared with the oculation (1 × 105), when average tumor size reached ∼8–10 mm. Expression of “ ” + single KO mice, which might reflect the lack of brakes in TCR IFNγ,TNFα,andgranzymeBbyCD8 T cells on stimulation with irradiated tumor signaling, resulting in lower threshold of T-cell activation and loss cells was detected by flow cytometry (B). To evaluate the efficacy of antibody of peripheral tolerance to autoantigens. Similarly, synergistic treatment on larger established tumors, mice were inoculated with a higher T-cell responses were observed in double KO mice in response to dose of CT26 cells (2.5 × 105). On day +5 after tumor inoculation, when tumor ∼ – foreign antigens. size reached 4 5 mm, mice were treated with VISTA or PD-L1 mAb or both, as We hypothesize that VISTA and PD-1 both function as brakes described earlier. Mice were also treated with anti-CD25 specific antibody on + + for T-cell activation. Because APCs lacking both VISTA and day +5 and day +20 to transiently deplete Foxp3 CD4 Tregs. Tumor growth and survival of the CT26 tumor-bearing mice were monitored and shown (C). PD-L1 stimulated T cells better than single KO APCs or WT A less immunogenic B16BL6 melanoma tumor model was examined to further APCs in vitro, these data indicate that VISTA and PD-L1 en- validate the efficacy of the combinatorial treatment. Mice were inoculated with gage independent inhibitory receptors on T cells. To determine B16BL6 (25,000) cells. On day +3 after tumor inoculation, mice were conditioned the effects of VISTA-R and PD-1 on TCR signaling, immobi- with low-dose irradiation (250 rads) and treated with four doses of GVAX before lized VISTA-Ig and PD-L1-Ig were used to engage VISTA-R the treatment with either VISTA or PD-L1 mAb or both. Tumor growth and and PD-1, respectively. Our data show that VISTA-Ig and PD- survival of tumor-bearing mice were monitored and shown (D). Representative L1-Ig fusion proteins impaired the activation of LAT, as well as results from two to three independent experiments were shown. the phosphorylation of proximal signaling molecules (SLP76 and PLC-γ1) and downstream molecules (Akt and Erk1/2). The coengagement of both Ig fusion proteins to their receptors Our data show that in the CT26 colon cancer model, the resulted in additive effects. On the basis of these data, we con- combinatorial treatment of VISTA and PD-L1 mAbs on day +2 clude that similar to PD-1, VISTA-R impairs early TCR sig- after tumor inoculation led to tumor regression and long-term naling. PD-1 has been shown to accumulate at the immune survival (8/8), whereas either mAb alone was less effective (1/8 in synapse and recruits Src homology region 2 domain-containing

Liu et al. PNAS Early Edition | 5of6 Downloaded by guest on September 23, 2021 phosphatase (SHP)-1/2 to down-regulate TCR signaling (33, 34). College of Wisconsin. All animal protocols were approved by the Institutional Whether or not similar phosphatases might be involved in Animal Care and Use Committee of the Medical College of Wisconsin. VISTA-R-mediated effects remains to be determined. We speculate that multiple mechanisms underlie the syner- Mice Necropsy and Semiquantitative Pathological Analysis. Age- and sex-matched gistic T-cell activation when both VISTA and PD-1 are WT and VISTA KO mice were killed by CO2 asphyxiation. Organs were harvested blocked in vivo. In addition to being a ligand, VISTA acts as a and fixed in 10% (vol/vol) buffered formalin. H&E stain was performed on receptor that transduces inhibitory signals during T-cell acti- tissue sections. Tissue inflammatory status was scored in a blind manner by a vation (6). This function likely contributes to the synergistic pathologist using the following semiquantitative scoring criteria: 0 = normal; T-cell activation seen in the VISTA/PD-1 double KO mice. 1 = mild/small foci of dense lymphocytic infiltrate; 2 = moderate/multiple foci Furthermore, VISTA might exert T-cell extrinsic functions. of dense/large activated lymphocytic infiltrate with/without germinal center; + VISTA is highly expressed on myeloid cells such as Cd11b and 3 = marked reactive/activated or atypical lymphocytic infiltrate. DCs and macrophages (3). Our future studies will dissect lineage-specific roles of VISTA in controlling both innate and Flow Cytometry and Data Analysis. Flow cytometry analysis was performed adaptive immune responses. either on FACScalibur or LSRII, using CellQuest software (BD Bioscience). Data Additional immune regulatory molecules, such as LAG3 and analyses were performed using FlowJo software (Treestar). Tim3, have been shown to synergize with PD-1 to control T-cell responses (19, 35). Our current study shows that VISTA and Graphs and Statistical Analysis. All graphs and statistical analysis were generated PD-1 synergistically regulate T-cell responses against self- and using Prism 4 (GraphPad Software, Inc.). Student’s t test (two tailed) or two-way foreign antigens, and concurrently targeting both molecules ANOVA were used for data analyses. ***P < 0.005, **P < 0.025, *P < 0.05. leads to optimal therapeutic efficacy in murine tumor models. Additional materials and methods are provided in the SI Materials The absence of overt autoimmune disease in the double KO and Methods. mice suggests that the combinatorial blockade of VISTA and PD-1 might achieve optimal therapeutic efficacy with less severe ACKNOWLEDGMENTS. We thank Dr. Miyuki Azuma of Tokyo Medical and Dental University for providing MIH5 hybridoma (anti-PD-L1 mAb), immune-related adverse events, and therefore might be more Dr. Tasuku Honjo (Kyoto University) for PD-1 KO mice, and Dr. Philippa amenable for the treatment of cancer. Marrack (National Jewish Health) for DO11.10 T-cell hybridoma. We appreciate the experimental protocols, discussions, and manuscript editing Materials and Methods provided by Dr. Subramaniam Malarkannan during manuscript preparation. Mice. C57BL/6 mice were purchased from Charles River Laboratories. VISTA This study was supported by research funding from National Cancer Institute Grant CA164225 (to L.W.), NIH Grant R01 AI089805 (to Y.H.H.), the Advancing KO mice on a fully backcrossed C57BL/6 background were obtained from the – a Healthier Wisconsin Research and Education Program fund (L.W.), and the Mutant Mouse Regional Resource Centers (University of California Davis) Melanoma Research Foundation Career Development Award (to L.W.). This (36). 2D2 TCR transgenic mice were purchased from the Jackson Laboratory. work was also supported by the Office of the Assistant Secretary of Defense PD-1 KO mice were provided by Dr. Honjo (10). PD-L1 KO mice were as described for Health Affairs through the Peer Reviewed Cancer Research Program under (15). All animals were maintained in a pathogen-free facility at the Medical Award No. W81XWH-14-1-0587 (to L.W.).

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