VISTA is an inhibitory immune checkpoint that is increased after ipilimumab therapy in patients with prostate cancer

Abstract

To date, anti-CTLA-4 (ipilimumab) or anti-PD-1 (nivolumab) monotherapy has not been demonstrated to be of substantial clinical benefit in patients with prostate cancer. To identify additional immune-inhibitory pathways in the prostate-tumor microenvironment, we evaluated untreated and ipilimumab-treated tumors from patients in a presurgical clinical trial. Levels of the PD-L1 and VISTA inhibitory molecules increased on independent subsets of macrophages in treated tumors. Our data suggest that VISTA represents another compensatory inhibitory pathway in prostate tumors after ipilimumab therapy.

Figure 1. Treatment with ipilimumab increases immune cell infiltration, as well as expression of PD-L1 and VISTA in prostate tumors

(a) Frequency of CD4, CD8 and ICOS⁺ T cells in untreated (N=11) and treated (N=6) tumors. (b) IHC analyses of CD4, CD8, ICOS⁺, CD45RO⁺, and GrB⁺ T cells, as well as CD68⁺ macrophages. (c) IHC of PD-L1, PD-1, and VISTA in tumor cells and tumor-infiltrating immune cells, with b-c comprised of tumors from 3 different cohorts of stage-matched patients: untreated (N=18), ADT-treated (N=10), and ipilimumab + ADT (N=16). Asterisk indicates patients received high dose steroids with surgery delay. (d) Frequency of PD-L1 expression on CD4 T cells, CD8 T cells, CD68⁺ macrophages, and tumor cells. (e) Frequency of VISTA expression on CD4 T cells, CD8 T cells, and CD68⁺ macrophages, with d-e comprised of matched pre-treatment (N=10) and post-treatment tumors (N=10). (f) IHC staining of CD4 and CD8 T cells, and CD68⁺ macrophages in stage-matched untreated (N=18) and ipilimumab + ADT-treated (N=15) prostate tumors as compared to stage-matched untreated (N=18) and ipilimumab-treated (N=20) metastatic melanomas. Arrows indicate significant difference of CD8 T cells and CD68⁺ macrophages between untreated prostate tumors and untreated melanomas. (g) Frequency of PD-L1 expression on CD4 T cells, CD8 T cells, and CD68⁺ macrophages. (h) Frequency of VISTA expression on CD4 T cells, CD8 T cells, and CD68⁺ macrophages, with g-h comprised of matched pre-treatment (N=10) and post-treatment prostate tumors (N=10) as compared to matched pre-treatment (N=10) and post-treatment melanomas (N=10).

Figure 2. PD-L1⁺ and VISTA⁺ macrophages (CD68⁺) manifest an M2-phenotype and suppress T cell function

(a) Representative photographs from immunofluorescence (IF) multiplex staining of PD-L1 (purple), VISTA (white), CD68 (yellow), and tumor nuclei (blue) in post-treatment prostate tumors. (b) Frequency of PD-L1 and VISTA expression on CD68⁺ macrophages from stage-matched untreated (UnTx; N=9) and post-treatment prostate tumors (N=9); (c) Fold induction of M1-like genes in post-treatment prostate tumors (N=6) and post-treatment melanomas (N=20) as compared to respective untreated samples. (d) Quantitative IHC analysis of Arg1⁺ cells in untreated (N=10) and post-treatment (N=15) prostate tumors (left panel), as well as ratio of Arg1/iNOS in untreated and post-treatment tumors (right panel). (e) Representative photographs from multiplex IF staining of tumor (blue), CD68 (yellow) and CD163 (green) with PD-L1 (white) or VISTA (white) in post-treatment prostate tumors (upper panel), as well as quantitative analysis of CD163 expression by CD68⁺PD-L1⁺ or CD68⁺VISTA⁺ cells from pre- (N=5) and post-treatment (N=5) prostate tumors (lower panel). (f) IFN-γ (left panel) and TNF-α (right panel) production by patients’ (N=7) peripheral T cells after CD3 stimulation in the presence of plate-bound control Ig (Ctrl Ig), PD-L1-Ig, VISTA-Ig protein or a combination of both. (g) IFN-γ production by patient’s peripheral T cells after CD3 stimulation without co-culture of monocytes or in the presence of either untreated monocytes or monocytes pretreated with anti-VISTA antibody. Experiments were performed in triplicates.