VEGF-A modulates expression of inhibitory checkpoints on CD8+ T cells in tumors

Abstract

Immune escape is a prerequisite for tumor development. To avoid the immune system, tumors develop different mechanisms, including T cell exhaustion, which is characterized by expression of immune inhibitory receptors, such as PD-1, CTLA-4, Tim-3, and a progressive loss of function. The recent development of therapies targeting PD-1 and CTLA-4 have raised great interest since they induced long-lasting objective responses in patients suffering from advanced metastatic tumors. However, the regulation of PD-1 expression, and thereby of exhaustion, is unclear. VEGF-A, a proangiogenic molecule produced by the tumors, plays a key role in the development of an immunosuppressive microenvironment. We report in the present work that VEGF-A produced in the tumor microenvironment enhances expression of PD-1 and other inhibitory checkpoints involved in CD8(+) T cell exhaustion, which could be reverted by anti-angiogenic agents targeting VEGF-A-VEGFR. In view of these results, association of anti-angiogenic molecules with immunomodulators of inhibitory checkpoints may be of particular interest in VEGF-A-producing tumors.

Figure 1.

VEGF-A–VEGFR inhibition decreases PD-1 expression on intratumoral CD8⁺ T cells. CT26 tumor-bearing mice were treated with sunitinib or DMSO (as a control; a), or anti–VEGF-A antibody or mouse serum (as a control; b), or masitinib or DMSO (as a control; c). Treatments were started when tumors reached 9–10 mm². Tumor growth (left) was shown. Mice were sacrificed at day 22 after 2 wk of treatment, and PD-1 expression on tumor-infiltrating CD8⁺ T cells was analyzed (right). Each dot represents an individual mouse, and histograms represent mean ± SEM of 2–4 pooled experiments with at least 4 mice/group. *, P = 0.0439; ***, P < 0.001. (d) Depletion of CD8⁺ T cells decreases anti-VEGFA–induced antitumor effects. (e) Correlation of VEGF-A concentration/gram of tumor tissue and number of PD-1-expressing CD8⁺ T cells/gram of tumor tissue (determined by flow cytometry). Each dot represents an individual tumor sample. (f) PD-1 expression on tumor-infiltrating CD8⁺ T cells was analyzed on wild-type MEF-tumor bearing mice (MEF WT) and VEGF-A–deleted MEF cells (MEF KO) after 14 d of tumor growth. Each dot represents an individual mouse. Histograms represent mean ± SEM of 2 pooled experiments with 4–5 mice/group. For tumor growths, one representative experiment out of 3 is shown with 5 mice/group. **, P < 0.01; ***, P < 0.001.

Figure 2.

VEGF-A enhances PD-1 expression on CD8⁺ T cells in vitro. (a) VEGF-R1 (left) and -R2 (right) expression is shown on CD8⁺ T cells from tumors and spleen of tumor-free (naive) and CT26 tumor-bearing mice. Each dot represents an individual mouse, histograms represent mean ± SEM of 2 pooled experiments with 3–5 mice/group. (b) VEGF-R1 (left) and -R2 (right) expression on purified CD8⁺ T cells after 24 and 48 h of culture with various doses of plate-bound anti-CD3 antibody. (c and d) Same experimental setting as in b, but showing a representative staining of VEGF-R2 by flow cytometry (c) and confocal microscopy (d). Isotype control of anti-VEGFR2 antibody is shown (blue line for c and top for d). (e) PD-1 expression on purified CD8⁺ T cells after 48 h of culture with plate-bound anti-CD3 with or without VEGF-A (50 ng/ml). (f) Same experiment as in e but with 10 µg/ml of plate-bound anti-CD3 and various doses of VEGF-A. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

VEGF-A enhances co-expression of inhibitory receptors involved in CD8⁺ T cell exhaustion in a VEGF-R2 and NFAT-dependent manner. Percentages of Tim-3 (a), CTLA-4 (b), and Lag-3 (c) expression on purified CD8⁺ T cells after 48 h of culture with plate-bound anti-CD3 (10 µg/ml) and various doses of VEGF-A. Histograms represent means ± SEM of 3 pooled experiments. (d) Same experimental settings as in (a) but mean fluorescence intensity (MFI) is shown. (e) The simultaneous expression of inhibitory receptors (PD-1, Tim-3, CTLA-4, and Lag-3) was examined on stimulated CD8⁺ T cells. (f) Same experimental setting as in (e) but in the presence of anti-VEGF-R1 or -R2 antibodies. (g) Transcriptional analyses of gene products linked to T cell exhaustion and VEGF-R2 signaling in CD8⁺ T cells stimulated or not with VEGF-A using a microfluidic card designed for qRT-PCR (TaqMan Low Density Mouse Immune Array from Applied Biosystems). Graph represents log fold changes (relative to nontreated controls, calculated with the ΔΔCT method (normalization with RNA18s as endogenous control) of transcripts. (h) Same experimental setting as in (e) in the presence of 11R-VIVIT. For simultaneous expression of inhibitory receptors, one representative experiment out of three is shown. *, P < 0.05.

Figure 4.

VEGF-A neutralization decreases expression of inhibitory receptors involved in exhaustion on CD8⁺ T cells in heterotopic and orthotopic mouse tumor models. Mice bearing subcutaneous CT26 tumors were treated with anti–VEGF-A antibody or mouse serum (as a control). Co-expressions of PD-1/Tim-3 (a), PD-1/CTLA-4 (b), or PD-1/Lag-3 (c) on intratumoral CD8⁺ T cells have been analyzed. 3 pooled experiments are shown with 5 mice/group. *, P < 0.05; **, P < 0.01. (d) Co-expression of PD-1, Tim-3, CTLA-4, and Lag-3 has been determined on intratumoral CD8⁺ T cells after 14 d of anti-VEGFA treatment (day 22). (e) Anti–VEGF-A was given to mice bearing CT26 hepatic metastases. Representative pictures of hepatic metastases (left panel) and the percentages of intratumoral CD8⁺ T cells expressing PD-1/Tim-3 (right panel) were shown. Two pooled experiments are shown with 3–4 mice/group. *, P < 0.05 (f). Same experimental setting as in d, but on CT26 hepatic metastases. (g) Co-expression of inhibitory receptors on CD8⁺ T from MC38 and MEF KO tumors. (h–i) VEGF-KO MEF (h) and wild-type MEF tumor-bearing mice (i) were treated with anti–PD-1 alone twice a week starting at day 7. (j) CT26 tumor-bearing mice were treated with anti-VEGFA or anti–PD-1 alone or both. Tumor growth was monitored twice a week. One representative experiment out of three is shown, with five mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.