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

Voron T, Colussi O, Marcheteau E, Pernot S, Nizard M, Pointet AL, Latreche S, Bergaya S, Benhamouda N, Tanchot C, Stockmann C, Combe P, Berger A, Zinzindohoue F, Yagita H, Tartour E, Taieb J, Terme M - J. Exp. Med. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U970, Paris Cardiovascular Research Center, Université Paris-Descartes, Sorbonne Paris Cité, 75015 Paris, France Service d'immunologie biologique, Service d'oncologie médicale, Service de chirurgie digestive, Service d'hépatogastroentérologie et d'oncologie digestive, Hôpital Européen Georges Pompidou, 75015 Paris, France.

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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.
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fig4: 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.

Mentions: To document if VEGF-A neutralization could revert expression of PD-1 and other inhibitory receptors in vivo, we tested anti–VEGF-A treatment on CT26-bearing mice. CT26 cells were implanted subcutaneously but also directly to the liver by subcapsular injection of tumor cells in the left lobe of the liver resulting in the development of liver metastases within 7–10 d, which is more relevant than our subcutaneous tumor model. Consistent with our in vitro results, VEGF-A inhibition decreased the proportion of tumor-infiltrating CD8+ T cells expressing PD-1/Tim-3, PD-1/CTLA-4, and PD-1/Lag-3 (Fig. 4, a and c). Analysis of simultaneous expression of these receptors on CD8+ T cells revealed that anti–VEGF-A treatment decreased the proportion of tumor-infiltrating CD8+ T cells expressing 3–4 receptors when treatment was started when tumors reached ∼10 mm2 (Fig. 4 d). It is of note that anti–VEGF-A treatment has no effect on the expression of 3–4 inhibitory receptors on tumor-infiltrating CD8+ T cells when very large tumors were treated (>90 mm2 at the beginning of the treatment). Mice bearing hepatic metastases (Fig. 4 e, left) were treated with anti–VEGF-A from day 10–20. Analysis of inhibitory receptors revealed a decrease of CD8+ T cells bearing 3–4 inhibitory receptors and mainly of PD-1/Tim-3 expression confirming our results in this more relevant tumor site (Fig. 4, e and f). Interestingly, in two tumor models where intratumoral VEGF-A level is low, VEGF- tumors (MEF-KO) and the MC38 colorectal tumor model (133.3 ± 15.35 pg/ml), the proportion of tumor-infiltrating CD8+ T cells expressing 3–4 inhibitory receptors was limited (<25%; Fig. 4 g). Together, these results showed that targeting VEGF-A can decrease the VEGF-induced expression of inhibitory receptors mediating CD8+ T cell exhaustion, not only in subcutaneously established tumors, but also in hepatic metastases from colorectal cancer.


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

Voron T, Colussi O, Marcheteau E, Pernot S, Nizard M, Pointet AL, Latreche S, Bergaya S, Benhamouda N, Tanchot C, Stockmann C, Combe P, Berger A, Zinzindohoue F, Yagita H, Tartour E, Taieb J, Terme M - J. Exp. Med. (2015)

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.
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fig4: 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.
Mentions: To document if VEGF-A neutralization could revert expression of PD-1 and other inhibitory receptors in vivo, we tested anti–VEGF-A treatment on CT26-bearing mice. CT26 cells were implanted subcutaneously but also directly to the liver by subcapsular injection of tumor cells in the left lobe of the liver resulting in the development of liver metastases within 7–10 d, which is more relevant than our subcutaneous tumor model. Consistent with our in vitro results, VEGF-A inhibition decreased the proportion of tumor-infiltrating CD8+ T cells expressing PD-1/Tim-3, PD-1/CTLA-4, and PD-1/Lag-3 (Fig. 4, a and c). Analysis of simultaneous expression of these receptors on CD8+ T cells revealed that anti–VEGF-A treatment decreased the proportion of tumor-infiltrating CD8+ T cells expressing 3–4 receptors when treatment was started when tumors reached ∼10 mm2 (Fig. 4 d). It is of note that anti–VEGF-A treatment has no effect on the expression of 3–4 inhibitory receptors on tumor-infiltrating CD8+ T cells when very large tumors were treated (>90 mm2 at the beginning of the treatment). Mice bearing hepatic metastases (Fig. 4 e, left) were treated with anti–VEGF-A from day 10–20. Analysis of inhibitory receptors revealed a decrease of CD8+ T cells bearing 3–4 inhibitory receptors and mainly of PD-1/Tim-3 expression confirming our results in this more relevant tumor site (Fig. 4, e and f). Interestingly, in two tumor models where intratumoral VEGF-A level is low, VEGF- tumors (MEF-KO) and the MC38 colorectal tumor model (133.3 ± 15.35 pg/ml), the proportion of tumor-infiltrating CD8+ T cells expressing 3–4 inhibitory receptors was limited (<25%; Fig. 4 g). Together, these results showed that targeting VEGF-A can decrease the VEGF-induced expression of inhibitory receptors mediating CD8+ T cell exhaustion, not only in subcutaneously established tumors, but also in hepatic metastases from colorectal cancer.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U970, Paris Cardiovascular Research Center, Université Paris-Descartes, Sorbonne Paris Cité, 75015 Paris, France Service d'immunologie biologique, Service d'oncologie médicale, Service de chirurgie digestive, Service d'hépatogastroentérologie et d'oncologie digestive, Hôpital Européen Georges Pompidou, 75015 Paris, France.

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Related in: MedlinePlus