VISTA Regulates the Development of Protective Antitumor Immunity

Abstract

V-domain Ig suppressor of T-cell activation (VISTA) is a novel negative checkpoint ligand that is homologous to PD-L1 and suppresses T-cell activation. This study demonstrates the multiple mechanisms whereby VISTA relieves negative regulation by hematopoietic cells and enhances protective antitumor immunity. VISTA is highly expressed on myeloid cells and Foxp3(+)CD4(+) regulatory cells, but not on tumor cells within the tumor microenvironment (TME). VISTA monoclonal antibody (mAb) treatment increased the number of tumor-specific T cells in the periphery and enhanced the infiltration, proliferation, and effector function of tumor-reactive T cells within the TME. VISTA blockade altered the suppressive feature of the TME by decreasing the presence of monocytic myeloid-derived suppressor cells and increasing the presence of activated dendritic cells within the tumor microenvironment. In addition, VISTA blockade impaired the suppressive function and reduced the emergence of tumor-specific Foxp3(+)CD4(+) regulatory T cells. Consequently, VISTA mAb administration as a monotherapy significantly suppressed the growth of both transplantable and inducible melanoma. Initial studies explored a combinatorial regimen using VISTA blockade and a peptide-based cancer vaccine with TLR agonists as adjuvants. VISTA blockade synergized with the vaccine to effectively impair the growth of established tumors. Our study therefore establishes a foundation for designing VISTA-targeted approaches either as a monotherapy or in combination with additional immune-targeted strategies for cancer immunotherapy.

Figure 1. VISTA monoclonal antibody (mab) treatment suppressed tumor growth and altered the tumor microenvironment in B16ova melanoma model

(A) B16OVA tumor-bearing mice (n=8 for control group, n=10 for 13F3 group) were treated with anti-VISTA mab (13F3) or control-Ig starting from day 0, every 2 days throughout the duration of the experiment. Tumor size was measured with a caliper. IFNγ ELISPOT was performed using tumor-draining lymph node cells on day 14. Cells were re-stimulated in vitro with irradiated tumor cells for 20 hrs. IFNγ-producing cells were visualized and counted. (B–C) Cells were harvested from peripheral lymph node, tumor-draining lymph node, and tumor tissues around day 20 when tumors reached ~8–10 mm diameter. VISTA expression on myeloid-derived suppressor cells (MDSC, CD11b^hi CD11c⁻ Gr1⁺) and myeloid DCs (CD11b⁺ CD11c⁺), and a comparison of VISTA and PD-L1 expression on tumor cells and tumor-infiltrating leukocytes (TILs) were shown by flow cytometry. (D) VISTA expression within B16 melanoma tumor tissue was examined by immunofluorescence. DAPI is in Blue, Cd11b is in Green, VISTA or control-IgG is in Red. (E–H) VISTA mab treatment altered the TME and improved the effector function of tumor-infiltrating CD8⁺ T cells. B16OVA-bearing mice were treated with 13F3 or control Ig from day 0 until tumors in the control group reached 9–10 mm diameter (~day+19). Tumors were harvested and CD45⁺ tumor-infiltrating leukocytes (TIL) were analyzed (E). Various TIL populations, including CD4⁺ and CD8⁺ T cells, and MDSC were identified by flow cytometry. Proliferation of tumor-infiltrating CD8⁺ T cells was analyzed based on Ki67 expression (F). (G–H) TIL CD8⁺ cells were stimulated with irradiated tumor cells for 20 hrs. Cytokine production (IFNγ), surface mobilization of CD107ab, and granzyme B expression were quantified. Data are representative of 2–3 independent experiments.

Figure 2. Effects of VISTA mab treatment on the growth and the tumor-microenvironment of MB49 bladder tumors

MB49 tumor-bearing mice (n=14 per group) were treated with control-Ig or 13F3 every 2 days starting from day0. Tumor size was measured with a caliper. (A) IFNγ ELISPOT was performed using tumor-draining lymph node cells on day 14 as described for the B16ova tumor model in Figure 1. (B) When MB49 tumors reached 9–10mm diameter (~day20), cells were harvested from peripheral lymph node, tumor-draining lymph node, and tumor tissues. VISTA expression on myeloid-derived suppressor cells (MDSC, CD11b^hi CD11c⁻ Gr1⁺) and myeloid DCs (CD11b⁺ CD11c⁺) was analyzed by flow cytometry. (C–E) VISTA mab treatment altered the TME and improved the effector function of tumor-infiltrating T cells. MB49-bearing mice were treated with 13F3 or control Ig from day 0 until tumors in the control group reached 9–10 mm diameter. Tumors were harvested and total CD45⁺ tumor-infiltrating leukocytes (TIL) were quantified. Tumor-infiltrating DCs were analyzed by flow cytometry for their surface expression of MHCII and CD80, and cytokine production (i.e. IL12p40 and TNFα). Tumor-infiltrating CD4⁺ and CD8⁺ T cells were stimulated with irradiated tumor cells for 20 hrs. Cytokine production (i.e. IFNγ and TNFα) was analyzed by flow cytometry and quantified. Data are representative of 2–3 independent experiments.

Figure 3. Effects of VISTA mab treatment on the growth and the tumor-microenvironment of B16-BL6 melanoma

(A) Mice (n=12 per group) were inoculated with B16BL6 tumor cells (18,000) on the right flank. Mice were treated with anti-VISTA mab every 2 days, starting from day +2 for the entire duration of the experiment. Tumor growth was measured by a caliper every 2 days. (B) Tumors were harvested when control tumors reached ~9–10 mm diameter, which typically corresponded to day19–20 post tumor inoculation. Tumor-infiltrating leukocyte (TIL) populations, including CD4⁺ and CD8⁺ T cells, and MDSCs (CD11b⁺ CD11C⁻ Gr1⁺) were analyzed by flow cytometry. (C) Purified TRP1 transgenic CD4⁺ T cells (25,000) were adoptively transferred into congenic B16-tumor bearing mice that were treated with anti-VISTA mab or control Ig. The number of tumor-infiltrating TRP1 cells was analyzed day+10 post transfer. (D) The proliferative marker (Ki67 expression) and activation status (CD44 and CD62L expression) of tumor-infiltrating polyclonal CD4⁺ and CD8⁺ T cells were analyzed. (E) The expression level of effector molecules (i.e. IFNγ and granzyme B) by TIL CD8⁺ T cells was analyzed after overnight stimulation by antigen-loaded BMDCs. (F) Expression levels of surface markers (i.e. MHCII and CD80) and cytokine production (i.e. IL12p40 and TNFα) of tumor-infiltrating CD11C⁺ DCs were analyzed. Data are representative of 2–3 independent experiments.

Figure 4. VISTA regulates the suppressive function of Foxp3⁺CD4⁺ nTregs, as well as the de novo induction of iTregs

(A) VISTA expression on nTreg subsets. nTreg subsets from spleens of 7–8 weeks old naïve mice were distinguished based on markers CD62L and ICOS. (B) Higher levels of VISTA expression on Tregs within the TME, when compared with Tregs from the peripheral LN. (C) VISTA-Ig promotes the de novo induction of Foxp3⁺CD4⁺ iTregs. Naïve murine CD4⁺ T cells were stimulated with plate-bound αCD3 (2.5 μg/mL) together with either control-Ig or VISTA-Ig (5 μg/mL). Exogenous TGFβ (2.5 μg/mL) was added as indicated. Cells were examined for the induction of Foxp3 after 72hrs. Foxp3GFP⁺ iTregs were sorted on day+5 and their suppressive activity was examined as described in methods. (D). VISTA mab treatment diminished tumor-mediated induction of iTregs. Congenic naïve OTII CD4⁺ T cells were adoptively transferred into B16OVA tumor-bearing mice (n=14 for control, n=17 for 13F3 group), which were treated with control-Ig or VISTA mab 13F3 every 2 days starting from Day0. Mice were analyzed around day 20 when tumor size in control group reached 9–10mm diameter. When necessary, small size tumors (diameter less than 5mm) were pooled within the group to obtain sufficient cell number for analysis. Conversion of naïve OTII to Foxp3⁺ iTregs in tumor-draining LN and within the tumor tissue were quantified and shown as the percentages of Foxp3⁺ cells among gated OTII cells. (E) The suppressive activity of Treg subsets is partially impaired by VISTA mab. Foxp3⁺CD4⁺ nTreg subsets were sorted from spleen based on expression level of ICOS and CD62L. These nTregs were co-cultured with naïve CD4⁺ T cells in indicated ratios, in the presence of VISTA mab or control IgG. The proliferation of T cells was measured by tritium incorporation. Data are representative of 2–3 independent experiments.

Figure 5. VISTA monoclonal antibody treatment suppressed tumor growth and altered the tumor microenvironment in the PTEN/BRAF inducible melanoma model

Tumors were induced by application of tamoxifen on the back skin. Mice were treated with VISTA mab 13F3 (n=10) or control hamster Ig (n=8) every 2 days throughout the duration of the experiment. (A) Representative pictures of melanomas grown on mice, as well as dissected tumors were shown. Since these inducible tumors grew with irregular and diverse height, which is in contrast to the relatively uniform shape and height of transplantable B16 tumors, tumor size was measured with a caliper and shown as mm³ (length × width × height). (B) High levels of VISTA expression on tumor-infiltrating myeloid cells (C) Expression of VISTA and PD-L1 on CD45⁺ tumor-infiltrating leukocytes (TILs) and CD45⁻ tumor cells is shown. (D–F) Analysis of tumor-infiltrating CD8⁺ T cells (D), MDSCs (CD11b^hi CD11c⁻ Gr1⁺) (E), and Cytokine (IFNγ) production of tumor-infiltrating CD8⁺ T cells upon in vitro stimulation with anti-CD3 (F) were shown. Data are representative of 2–3 independent experiments.

Figure 6. VISTA monoclonal antibody synergized with tumor vaccine to impair the growth of established B16-BL6 tumors

Naïve mice (n=13 per group for A, and n=8–10 per group for B) were inoculated with B16-BL6 tumor cells (18,000) on the right flank. On day+2 (A) or day+7 (B) post inoculations, mice were treated with peptide vaccine complex (1 dose), or anti-VISTA mab 13F3 (continuous treatment for the entire duration of the experiment), or the combination as indicated. (C) To determine whether the combination therapy induced optimal T cell responses when compared to single treatment, tumor-bearing mice were treated on day+7 with vaccine, or 13F3, or the combination as indicated. Tumor-infiltrating lymphocytes were harvested ~day+21, restimulated with the immunizing peptides TRP1/deltaV-TRP2, and analyzed for their cytokine production (i.e. IFNγ) by flow cytometry as described in methods. (D) Mice that survived the first B16BL6 tumor challenge (n=12) after combination treatment were re-challenged with B16BL6 cells (18,000) on the same flank on day+60 post primary tumor rejection. Naïve mice were used as the control group. Tumor size was measured every 2–3 days with a caliper. Data are representative of 2–3 independent experiments.