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

Edited by Michael J. Bevan, University of Washington, Seattle, WA, and approved April 2, 2015 (received for review October 23, 2014)
May 11, 2015
112 (21) 6682-6687

Significance

Multiple immune-checkpoint proteins, such as programmed death 1 (PD-1), LAG3, and TIM3, are coexpressed on immune cells and functionally synergize with each other. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a recently identified immune-checkpoint molecule that suppresses T-cell activation. This study establishes that VISTA and PD-1 exert nonredundant immune regulatory functions and synergistically regulate T-cell responses. Combinatorial treatment using VISTA- and PD-ligand 1-specific monoclonal antibodies achieved synergistic therapeutic efficacy in murine tumor models. This study critically advances our knowledge of the immune regulatory function of VISTA and provides a rationale for targeting both VISTA and PD-1 to more effectively treat T-cell-regulated diseases such as cancer.

Abstract

V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a negative immune-checkpoint protein that suppresses T-cell responses. To determine whether VISTA synergizes with another immune-checkpoint, programmed death 1 (PD-1), this study characterizes the immune responses in VISTA-deficient, PD-1-deficient (KO) mice and VISTA/PD-1 double KO mice. Chronic inflammation and spontaneous activation of T cells were observed in both single KO mice, demonstrating their nonredundancy. However, the VISTA/PD-1 double KO mice exhibited significantly higher levels of these phenotypes than the single KO mice. When bred onto the 2D2 T-cell receptor transgenic mice, which are predisposed to development of inflammatory autoimmune disease in the CNS, the level of disease penetrance was significantly enhanced in the double KO mice compared with in the single KO mice. Consistently, the magnitude of T-cell response toward foreign antigens was synergistically higher in the VISTA/PD-1 double KO mice. A combinatorial blockade using monoclonal antibodies specific for VISTA and PD-L1 achieved optimal tumor-clearing therapeutic efficacy. In conclusion, our study demonstrates the nonredundant role of VISTA that is distinct from the PD-1/PD-L1 pathway in controlling T-cell activation. These findings provide the rationale to concurrently target VISTA and PD-1 pathways for treating T-cell-regulated diseases such as cancer.
T-cell activation requires engagement with antigen-presenting cells (APCs) that present the cognate peptide on MHC molecules. Antigen recognition is regulated by cosignaling ligands and receptors, whose integrated signaling determines the outcome of T-cell activation, differentiation, and function (1). The B7 family of coreceptors belongs to the Ig superfamily, consisting of stimulatory receptors such as CD28 and inducible T-cell costimulator (ICOS), cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) and programmed death 1 (PD-1) coinhibitory receptors, and B7-H3 and B7-H4 ligands, whose receptors have yet to be identified (1, 2). Together with additional inhibitory molecules such as T-cell immunoglobulin domain and mucin domain 3 (Tim-3), lymphocyte-activation gene 3 (LAG3), and B- and T-lymphocyte attenuator (BTLA), these immune-checkpoint proteins play critical roles in maintaining peripheral tolerance and controlling autoimmunity.
VISTA is a newly identified Ig domain-containing immune-checkpoint molecule that directly suppresses T-cell activation in vitro and in vivo (3, 4). The human and murine VISTA proteins share 90% identity and display similar expression patterns (5). VISTA is constitutively expressed within the hematopoietic compartment, with the highest expression level on myeloid cells and a lower level on CD4+, CD8+ T cells, and Foxp3+CD4+ regulatory T cells. A soluble VISTA-Ig protein that contains the extracellular domain fused with Ig-crystalizable fragment (Fc) or full-length VISTA expressed on APCs acts as a ligand to suppress T-cell proliferation and cytokine production (3). In addition, VISTA might function as an inhibitory receptor on T cells to suppress their activation (6). VISTA-specific monoclonal antibody (mAb) enhances disease severity in the experimental autoimmune encephalomyelitis (EAE) model, as well as boosts antitumor immunity in multiple murine tumor models (7).
The regulatory functions of immune-checkpoints have been demonstrated using KO mice, which typically manifest loss of peripheral tolerance and heightened T-cell responses. For example, CTLA-4 KO mice die of young age because of overwhelming lymphoproliferative disease and inflammation (8, 9). PD-1 KO mice display genetic background-dependent late-onset autoimmune disease of either dilated cardiomyopathy or arthritis (10, 11). PD-1 binds to two ligands, PD-ligand 1 (L1) and PD-L2 (12, 13). Although neither PD-L1 nor PD-L2 KO mice develop overt organ-specific autoimmune disease, PD-L1 genetic deficiency exacerbates disease in the EAE model, as well as impairs fetomaternal tolerance (14, 15). These data suggest a critical role for PD-L1 in maintaining T-cell peripheral tolerance at tissue sites. In contrast, genetic disruption of other immune checkpoint ligands such as B7-H4 fail to display any significant alterations of T-cell responses in vivo, suggesting there is a hierarchy and potential redundancy among various immune-checkpoint regulators (16). In this context, it is important to note that the expression pattern of VISTA overlaps with multiple other immune-checkpoint regulators (3). It is therefore critical to determine whether VISTA exerts nonredundant immune regulatory functions.
Studies have shown that several immune-checkpoints functionally synergize with each other (1719). For example, the combined disruption of LAG3 and PD-1 genes results in lethal autoimmune diseases, whereas loss of either gene alone leads to subtle phenotypes (17). In addition, PD-1 functionally synergizes with TIM3 (18, 19). A combinatorial antibody-mediated blockade of both PD-1 and TIM3 results in optimal T-cell responses against cancer, as well as during chronic viral infection (18, 19). In the current study, we use KO mice to address whether VISTA synergizes with PD-1 in regulating T-cell responses. Our data establish that VISTA and the PD-1/PD-L1 pathways nonredundantly regulate T-cell activation and demonstrate the feasibility of concurrently targeting these two immune-checkpoints to enhance tumor-specific immune responses.

Results

Loss of T-Cell Peripheral Tolerance on Combined Genetic Disruption of VISTA and PD-1 or PD-L1.

To determine whether VISTA and PD-1 regulate immune responses in a redundant or independent/synergistic manner, Vista−/−Pdcd1−/− mice (herein referred to as VISTA/PD-1 double KO mice) were generated on the C57BL/6 background and characterized. The double KO mice were born fertile and produced normal litter sizes. Normal thymic development and lymphocyte populations (T and B lymphocytes, natural killer cells, and natural killer T cells) in the bone marrow, spleen, and lymph nodes were observed in 6–8-wk-old KO mice.
Comprehensive multiorgan histological analyses were performed in 12-mo-old WT, VISTA KO, PD-1 KO, and VISTA/PD-1 double KO mice (Fig. 1). Hematoxylin and eosin (H&E) stained sections from heart, lung, liver, kidney, pancreas, salivary gland, small and large intestines, and brain were examined. Several organs, including lung, liver, and pancreas in the double KO mice, were heavily infiltrated with leukocytes (Fig. 1A, Top) and showed significant tissue necrosis, presumably as a result of immune cell-mediated destruction (Fig. 1A, Bottom). The levels of leukocyte infiltration and tissue necrosis in the KO mice were blindly quantified on the basis of a semiquantitative scoring method, and the VISTA/PD-1 double KO mice showed the highest scores compared with WT and single KO mice (Fig. 1B). Despite the significant accumulation of activated T cells, the double KO mice did not develop overt autoimmune disease. Serum levels of IgM and IgA were moderately elevated in aged double KO mice (Fig. S1). Our cohoused PD-1 KO mice also developed accumulation of spontaneously activated T cells and chronic inflammation in multiple organs but failed to develop arthritis or other previously reported autoimmune phenotypes (10). This discrepancy might be a result of the different housing conditions or the age of the mice analyzed.
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).
Compared with VISTA KO and PD-1 KO mice, VISTA/PD-1 double KO mice at the age of 6–7 mo showed significantly increased frequencies of CD44hiCD62Llo CD8+ and CD4+ T cells, which is indicative of an activated or memory phenotype (Fig. 2 A and C). On in vitro restimulation, the double KO T cells produced significantly higher levels of cytokines, such as IFNγ, TNFα, and IL-17A, than WT and single KO cells (Fig. 2 B, DF).
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.
PD-1 binds to ligands PD-L1 and PD-L2 (20). To corroborate the results seen in the VISTA/PD-1 double KO mice, we bred VISTA KO onto the previously described PD-L1 KO (15) and generated the VISTA/PD-L1 double KO mice. Our data demonstrated spontaneous activation of peripheral CD4+ and CD8+ T cells in the VISTA/PD-L1 double KO mice, which was comparable to that of VISTA/PD-1 double KO mice (Fig. S2 A and B). Together, these data support the conclusion that VISTA and the PD-1/PD-L1 pathway nonredundantly control the peripheral tolerance of T cells.
The phenotype of spontaneous T-cell activation in the double KO mice (Fig. 2 and Fig. S2) indicates that both VISTA and PD-1 regulate the threshold of TCR activation to autoantigens. We hypothesize that disruption of both pathways will increase predisposition to autoimmune disease on a susceptible background. To test this hypothesis, the VISTA/PD-1 double KO mice were bred with the 2D2 mice, which express a TCR transgene specific for the self-antigen, myelin oligodendrocyte glycoprotein (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 6 mo (Fig. 3A and Table S1). 2D2-PD-1 KO mice showed similar incidence of spontaneous disease as WT mice (2/40, 5%). In contrast, genetic deficiency of VISTA accelerated disease onset, such that ∼60% (25/42) of the 2D2-VISTA KO mice rapidly succumbed to complete hind limb paralysis within 3 mo. Combined deficiency of VISTA and PD-1 further increased disease incidence to ∼90% (35/37) (Fig. 3 A and B). Analysis of CNS tissue from paralyzed 2D2-VISTA/PD-1 double KO mice confirmed the accumulation of inflammatory cells and demyelination (Fig. 3 C and D). The disease onset age ranged between 4 and 16 wk in the 2D2-VISTA/PD-1 double KO mice, which was similar to the ages observed in the 2D2-VISTA KO mice (5–16 wk) (Fig. S3). Only one WT mouse developed disease around 12 wk of age. A small percentage of diseased mice from the 2D2-VISTA KO (2/40) and 2D2-VISTA/PD-1 double KO (3/37) mice developed atypical EAE, manifested as unilateral paralysis rather than bilateral paralysis. Detailed information regarding disease onset, severity, penetrance, and mortality is presented in Table S1.
Fig. 3.
Combined genetic deficiency of VISTA and PD-1 exacerbated autoimmune disease on the susceptible background. The CNS disease incidence (A) and mortality (B) were monitored in 2D2 TCR transgenic mice that were bred onto the VISTA KO, PD-1 KO, and double KO genetic background. Representative H&E stained spinal cord section from paralyzed double KO mice is shown (C). Enlarged images show areas of extensive lymphocyte infiltration. Luxol fast blue staining of spinal cord sections confirmed extensive demyelination (D). 2D2-WT (n = 30), 2D2-VISTA KO (n = 42), 2D2-PD-1 KO (n = 40), 2D2-VISTA/PD-1 double KO (n = 37). Only one 2D2-WT mouse developed disease.

Combined Disruption of VISTA and PD-1 Synergistically Augments T-Cell Responses on Antigen Challenge.

Spontaneous T-cell activation and enhanced autoimmunity in aged VISTA/PD-1 double KO mice indicate that VISTA and PD-1 control T-cell tolerance toward autoantigens. We hypothesize that these pathways also critically regulate T-cell responses toward foreign antigens. To address this question, mice were immunized with soluble antigenic peptides together with poly (I:C) (TLR3 agonist) as adjuvant. 2W1S, an MHC class II-restricted peptide, and OVA257-264, an MHC class I-restricted peptide, were used (22). On day +7 postimmunization, splenic T cells were isolated and restimulated ex vivo with the respective peptides. Significantly higher numbers of IFNγ-producing T cells were present in the VISTA/PD-1 double KO mice compared with in WT or single KO mice, indicating that VISTA and PD-1 nonredundantly control T-cell responses (Fig. 4 A and B).
Fig. 4.
VISTA and the PD-1 collaboratively controlled antigen-specific T-cell 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 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 and restimulated with the respective peptides. IFNγ-producing cells were enumerated by the ELISPot assay. To stimulate T cells in vitro, CD11b+ CD11c+ DCs were sorted from WT, VISTA KO, PD-L1 KO, and VISTA/PD-L1 double KO mice and incubated with naive CD4+ OTII TCR transgenic T cells in the presence of cognate peptides OVA323–339 (10 ng/mL). [3H]-Thymidine was added to the culture for the last 8 h of the 72-h culture period for measuring T-cell proliferation (C). The production of IFNγ was quantified from the culture supernatants by ELISA (D).
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 expressed on CD11b+ myeloid APCs (2, 3), we hypothesize that a combined deficiency of VISTA and PD-L1 on APCs maximally enhances T-cell activation. To test this hypothesis, CD11b+ myeloid DCs were isolated from WT, VISTA KO, PD-L1 KO, and the VISTA/PD-L1 double KO mice (15, 23) and were used to stimulate naive CD4+ OTII TCR transgenic T cells in the presence of cognate peptide OVA323–339. Our data show that the combined deficiency of VISTA and PD-L1 on myeloid APCs synergistically enhanced T-cell proliferation and IFNγ production (Fig. 4 C and D).
Although the receptor for VISTA (VISTA-R) is unknown, we speculate that the engagement of VISTA-R on T cells suppresses TCR signaling independent of PD-1. In addition, we hypothesize that the coengagement of VISTA-R and PD-1 on T cells synergistically impairs TCR signaling. To test these hypotheses, proximal TCR signaling events were examined using immobilized fusion proteins VISTA-Ig and PD-L1-Ig, both of which suppressed T-cell proliferation and cytokine production in vitro (3, 12). LAT is a proximal signaling adaptor that is phosphorylated on TCR stimulation and forms a complex with multiple signaling molecules including SH2 domain containing leukocyte protein of 76kDa (SLP76) and phospholipase C (PLC)-γ1 (24). To determine whether VISTA functions by interfering with the phosphorylation of LAT, a solid phase immunoprecipitation assay was performed, in which the proximal signaling complexes could be recovered as the bound fraction to the plastic surface (25, 26). Our data show that in the presence of coimmobilized control-Ig or VISTA-Ig proteins, plate-bound anti-CD3ε mAb pulled-down comparable amounts of CD3ζ. This result excluded the possibility that the immobilized VISTA-Ig displaced anti-CD3ε mAb or impaired the binding of anti-CD3ε mAb to the TCR/CD3 complex (Fig. 5A). Despite the similar engagement of CD3ζ, immobilized VISTA-Ig significantly reduced the amount of LAT recruited to the CD3 complex, as well as its phosphorylation (Fig. 5A). When total cell lysates were examined, the phosphorylation of several proximal and downstream signaling molecules such as SLP76, PLC-γ1, Akt, and Erk1/2 were also impaired (Fig. 5B).
Fig. 5.
Engagement of both VISTA and PD-L1 during TCR activation maximally suppressed TCR signaling. To determine whether VISTA engagement impairs the recruitment of signaling adaptor protein LAT, DO11.10 hybridoma cells (100 × 106) were stimulated with plate-bound anti-CD3 mAb (2C11, 3 μg/mL), together with coimmobilized control-Ig (8 μg/mL) or VISTA-Ig fusion protein (8 μg/mL) for 10 min at 37 °C and lysed in situ. After removing the unbound cell lysates, plate-bound protein was eluted off the plate and examined by Western blotting (A). To examine the effect of VISTA on the phosphorylation of TCR signaling molecules, CD25CD4+ T cells were purified from naive splenocytes and stimulated with plate-bound 2C11 (3 μg/mL), together with control-Ig (8 μg/mL) or VISTA-Ig (8 μg/mL) for 5 min at 37 °C. Total cell lysates were prepared, and the phosphorylation status of LAT, SLP76, PLC-γ1, Akt, and Erk1/2 was examined (B). To determine whether coengagement of both VISTA and PD-L1 maximally suppresses LAT activation, DO11.10 cells were stimulated with plate-bound 2C11 (2.5 μg/mL), together with control-Ig (10 μg/mL), VISTA-Ig (5 μg/mL), PD-L1-Ig (5 μg/mL), or both Ig fusion proteins. Cells were lysed after 10 min stimulation, and plate-bound proteins were recovered and examined as described earlier (C). To determine the synergistic effects of engaging both VISTA and PD-L1, preactivated splenic CD4+ T cells were stimulated with plate-bound 2C11 (2.5 μg/mL), together with control-Ig (9 μg/mL), VISTA-Ig (3 μg/mL), PD-L1-Ig (6 μg/mL), or both Ig fusion proteins for 10 min at 37 °C. Total cell lysates were harvested for Western blotting analysis (D). Representative results from two to three independent experiments were shown.
Next, the effect of coengaging VISTA-Ig and PD-L1-Ig was examined (Fig. 5 C and D). Preactivated T cells were analyzed, as they expressed a high level of PD-1 (27). Consistent with our hypothesis, the combined engagement of VISTA-Ig and PD-L1-Ig maximally reduced the phosphorylation of LAT and its recruitment to the CD3 complex (Fig. 5C). Furthermore, VISTA-Ig and PD-L1-Ig coengagement maximally reduced the phosphorylation of SLP76, PLC-γ1, Akt, and Erk1/2 in total cell lysates (Fig. 5D). Together, these results support the conclusion that VISTA-R and PD-1 each impairs early TCR signaling and results in the most robust suppression when combined.
Enhanced T-cell responses in the VISTA/PD-1 double KO mice suggest that these two immune checkpoints could be targeted concurrently to optimize antitumor immunity. This hypothesis was tested in transplantable murine tumor models (Fig. 6). Our data show that in the CT26 colon cancer model, the combinatorial treatment of VISTA and PD-L1 mAbs on day +2 after tumor inoculation led to tumor regression and long-term survival (8/8), whereas either mAb alone was less effective (1/8 in the VISTA mAb-treated group and 3/8 in the PD-L1 mAb-treated group rejected tumor) (Fig. 6A). Analysis of tumor-specific T-cell activation showed synergistically enhanced cytokine production (IFNγ and TNFα) and granzyme B production by tumor-specific CD8+ T cells from tumor-draining LN (Fig. 6B). Next, combinatorial treatment was tested on larger established tumors. When treatment was initiated on day +5 after CT26 inoculation, the average tumor size reached ∼4–5 mm in diameter (Fig. 6C). To facilitate tumor-specific T-cell activation in this therapeutic setting, mice were treated with anti-CD25 mAb on day +5 and day + 20 to transiently deplete CD25+Foxp3+CD4+ regulatory T cells. Synergistic efficacy was achieved with the combined treatment of VISTA and PD-L1 mAbs, which led to ∼80% tumor regression (8/10), whereas single mAb treatment failed to show any significant effect (Fig. 6C).
Fig. 6.
Optimal therapeutic efficacy on combined blockade of VISTA and PD-L1 in murine tumor models. CT26 colon carcinoma cells (100,000) were inoculated on the flank of naive mice on day 0 (n = 8). Day +3 after tumor inoculation, mice were treated with control Ig (300 μg), anti-VISTA mAb (300 μg), anti-PD-L1 mAb (200 μg), or combined anti-VISTA and anti-PD-L1 mab, every 2–3 d continuously for 3 wk. Tumor size was measured by a caliper and recorded as area (mm2) (A). The rate of tumor-free survival was also shown (A). To examine tumor-specific T-cell responses, lymphocytes were harvested from tumor-draining lymph nodes on day +14 after tumor inoculation (1 × 105), when average tumor size reached ∼8–10 mm. Expression of IFNγ, TNFα, and granzyme B by CD8+ T cells on stimulation with irradiated tumor cells was detected by flow cytometry (B). To evaluate the efficacy of antibody treatment on larger established tumors, mice were inoculated with a higher dose of CT26 cells (2.5 × 105). On day +5 after tumor inoculation, when tumor size reached ∼4–5 mm, mice were treated with VISTA or PD-L1 mAb or both, as described earlier. Mice were also treated with anti-CD25 specific antibody on 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). A less immunogenic B16BL6 melanoma tumor model was examined to further validate the efficacy of the combinatorial treatment. Mice were inoculated with B16BL6 (25,000) cells. On day +3 after tumor inoculation, mice were conditioned with low-dose irradiation (250 rads) and treated with four doses of GVAX before the treatment with either VISTA or PD-L1 mAb or both. Tumor growth and survival of tumor-bearing mice were monitored and shown (D). Representative results from two to three independent experiments were shown.
We next evaluated the therapeutic efficacy of the combinatorial treatment in a less immunogenic tumor model, such as the B16BL6 melanoma model, which is responsive to therapeutic blockade of CTLA-4 and PD-1 in previous studies (2831). A GM-CSF secreting cellular vaccine (GVAX) (28) was applied on day +3, +6, +9, and +12 after tumor inoculation to boost tumor-specific T-cell responses. A sublethal whole-body irradiation (250 rads) was applied on day +3, which was shown to facilitate T-cell-mediated antitumor immune responses (32). Consistent with our hypothesis, treatment with both VISTA and PD-L1 mAbs significantly suppressed tumor growth and conferred survival advantage, whereas single mAb treatment was largely ineffective (Fig. 6D). Together, these data indicate that VISTA and PD-L1/PD-1 pathways independently control tumor-specific T-cell responses, and combined therapeutic blockade synergistically enhances antitumor immunity.

Discussion

VISTA and PD-1 both function as immune checkpoint proteins that suppress T-cell activation. They share overlapping expression patterns within the hematopoietic compartment. It is therefore important to define their independent immune-regulatory roles.
Evidence based on studies of the KO mice indicates that VISTA and PD-1 nonredundantly regulate immune responses. Genetic disruption of VISTA accelerated autoimmune disease on a susceptible background, as well as resulted in multiorgan chronic inflammation because of spontaneous T-cell activation (22). Aged PD-1 KO mice were reported to develop late-onset autoimmunity in the C57BL/6 background (10). Our study of PD-1 KO mice also showed accumulation of spontaneously activated T cells and chronic inflammation in multiple organs. Furthermore, in the current study of the VISTA/PD-1 double KO mice, we provided strong evidence for independent control of T-cell responses by these two checkpoints. Synergistic or additive T-cell activation was observed from aged double KO mice compared with the single KO mice, which might reflect the lack of “brakes” in TCR signaling, resulting in lower threshold of T-cell activation and loss of peripheral tolerance to autoantigens. Similarly, synergistic T-cell responses were observed in double KO mice in response to foreign antigens.
We hypothesize that VISTA and PD-1 both function as brakes for T-cell activation. Because APCs lacking both VISTA and PD-L1 stimulated T cells better than single KO APCs or WT APCs in vitro, these data indicate that VISTA and PD-L1 engage independent inhibitory receptors on T cells. To determine the effects of VISTA-R and PD-1 on TCR signaling, immobilized VISTA-Ig and PD-L1-Ig were used to engage VISTA-R and PD-1, respectively. Our data show that VISTA-Ig and PD-L1-Ig fusion proteins impaired the activation of LAT, as well as 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 resulted in additive effects. On the basis of these data, we conclude that similar to PD-1, VISTA-R impairs early TCR signaling. PD-1 has been shown to accumulate at the immune synapse and recruits Src homology region 2 domain-containing phosphatase (SHP)-1/2 to down-regulate TCR signaling (33, 34). Whether or not similar phosphatases might be involved in VISTA-R-mediated effects remains to be determined.
We speculate that multiple mechanisms underlie the synergistic T-cell activation when both VISTA and PD-1 are blocked in vivo. In addition to being a ligand, VISTA acts as a receptor that transduces inhibitory signals during T-cell activation (6). This function likely contributes to the synergistic T-cell activation seen in the VISTA/PD-1 double KO mice. Furthermore, VISTA might exert T-cell extrinsic functions. VISTA is highly expressed on myeloid cells such as Cd11b+ DCs and macrophages (3). Our future studies will dissect lineage-specific roles of VISTA in controlling both innate and adaptive immune responses.
Additional immune regulatory molecules, such as LAG3 and Tim3, have been shown to synergize with PD-1 to control T-cell responses (19, 35). Our current study shows that VISTA and PD-1 synergistically regulate T-cell responses against self- and foreign antigens, and concurrently targeting both molecules leads to optimal therapeutic efficacy in murine tumor models. The absence of overt autoimmune disease in the double KO mice suggests that the combinatorial blockade of VISTA and PD-1 might achieve optimal therapeutic efficacy with less severe immune-related adverse events, and therefore might be more amenable for the treatment of cancer.

Materials and Methods

Mice.

C57BL/6 mice were purchased from Charles River Laboratories. VISTA KO mice on a fully backcrossed C57BL/6 background were obtained from the Mutant Mouse Regional Resource Centers (University of California–Davis) (36). 2D2 TCR transgenic mice were purchased from the Jackson Laboratory. PD-1 KO mice were provided by Dr. Honjo (10). PD-L1 KO mice were as described (15). All animals were maintained in a pathogen-free facility at the Medical College of Wisconsin. All animal protocols were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.

Mice Necropsy and Semiquantitative Pathological Analysis.

Age- and sex-matched WT and VISTA KO mice were killed by CO2 asphyxiation. Organs were harvested and fixed in 10% (vol/vol) buffered formalin. H&E stain was performed on tissue sections. Tissue inflammatory status was scored in a blind manner by a pathologist using the following semiquantitative scoring criteria: 0 = normal; 1 = mild/small foci of dense lymphocytic infiltrate; 2 = moderate/multiple foci of dense/large activated lymphocytic infiltrate with/without germinal center; and 3 = marked reactive/activated or atypical lymphocytic infiltrate.

Flow Cytometry and Data Analysis.

Flow cytometry analysis was performed either on FACScalibur or LSRII, using CellQuest software (BD Bioscience). Data analyses were performed using FlowJo software (Treestar).

Graphs and Statistical Analysis.

All graphs and statistical analysis were generated using Prism 4 (GraphPad Software, Inc.). Student’s t test (two tailed) or two-way ANOVA were used for data analyses. ***P < 0.005, **P < 0.025, *P < 0.05.
Additional materials and methods are provided in the SI Materials and Methods.

Acknowledgments

We thank Dr. Miyuki Azuma of Tokyo Medical and Dental University for providing MIH5 hybridoma (anti-PD-L1 mAb), Dr. Tasuku Honjo (Kyoto University) for PD-1 KO mice, and Dr. Philippa Marrack (National Jewish Health) for DO11.10 T-cell hybridoma. We appreciate the experimental protocols, discussions, and manuscript editing provided by Dr. Subramaniam Malarkannan during manuscript preparation. 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 a Healthier Wisconsin Research and Education Program fund (L.W.), and the Melanoma Research Foundation Career Development Award (to L.W.). This work was also supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Cancer Research Program under Award No. W81XWH-14-1-0587 (to L.W.).

Supporting Information

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Supporting Information

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Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 112 | No. 21
May 26, 2015
PubMed: 25964334

Classifications

Submission history

Published online: May 11, 2015
Published in issue: May 26, 2015

Keywords

  1. immune-checkpoint
  2. autoimmunity
  3. tumor immunity
  4. cancer immunotherapy
  5. T-cell activation

Acknowledgments

We thank Dr. Miyuki Azuma of Tokyo Medical and Dental University for providing MIH5 hybridoma (anti-PD-L1 mAb), Dr. Tasuku Honjo (Kyoto University) for PD-1 KO mice, and Dr. Philippa Marrack (National Jewish Health) for DO11.10 T-cell hybridoma. We appreciate the experimental protocols, discussions, and manuscript editing provided by Dr. Subramaniam Malarkannan during manuscript preparation. 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 a Healthier Wisconsin Research and Education Program fund (L.W.), and the Melanoma Research Foundation Career Development Award (to L.W.). This work was also supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Cancer Research Program under Award No. W81XWH-14-1-0587 (to L.W.).

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

Jun Liu
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226;
Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009, People's Republic of China;
Ying Yuan
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226;
Present address: College of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, People's Republic of China.
Wenna Chen
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226;
Juan Putra
Department of Pathology and
Arief A. Suriawinata
Department of Pathology and
Austin D. Schenk
Department of Surgery, Geisel School of Medicine at Dartmouth, Norris Cotton Cancer Center, Lebanon, NH 03756; and
Halli E. Miller
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226;
Indira Guleria
Boston Children’s Hospital and Brigham and Women’s Hospital, Renal Division, Harvard Medical School, Boston, MA 02115
Richard J. Barth
Department of Surgery, Geisel School of Medicine at Dartmouth, Norris Cotton Cancer Center, Lebanon, NH 03756; and
Yina H. Huang
Department of Pathology and
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226;

Notes

2
To whom correspondence should be addressed. Email: [email protected].
Author contributions: L.W. designed research; J.L., Y.Y., W.C., J.P., A.D.S., H.E.M., and L.W. performed research; J.L., I.G., Y.H.H., and L.W. contributed new reagents/analytic tools; 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 the paper.

Competing Interests

Conflict of interest statement: L.W. is involved with the commercial development of VISTA with ImmuNext Inc Corporation and received research support, salary, and/or consulting fees.

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    Immune-checkpoint proteins VISTA and PD-1 nonredundantly regulate murine T-cell responses
    Proceedings of the National Academy of Sciences
    • Vol. 112
    • No. 21
    • pp. 6519-E2848

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