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

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.

Keywords: T-cell activation; autoimmunity; cancer immunotherapy; immune-checkpoint; tumor immunity.

Conflict of interest statement

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.

Figures

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).

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.

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.

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).

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, CD25−CD4+ 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.

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.