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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 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.
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fig2: 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.

Mentions: To understand whether VEGF-A can act directly on CD8+ T cells, we first analyzed the expression of the two VEGF-A receptors, VEGF-R1 and -R2, on CD8+ T cells in vivo. We observed that these two receptors are expressed only at very low levels in spleens of tumor-free and tumor-bearing mice, but are strongly increased on tumor-infiltrating CD8+ T cells (Fig. 2 a). These VEGFR+ CD8+ T cells also expressed PD-1 (unpublished data). Because tumor-infiltrating T cells bear an activated phenotype unlike splenic CD8+ T cells, these results suggested that VEGFR expression could be associated with T cell activation (Whiteside and Parmiani, 1994). To recapitulate this phenomenon in vitro, we analyzed expression of these receptors after stimulation of purified CD8+ T cells with different doses of anti-CD3 by flow cytometry and confocal microscopy. Expression of VEGF-R1 and -R2 was induced by stimulation with 3 µg/ml of anti-CD3 and became significant at 10 µg/ml after 48 h of culture (Fig. 2, b–d). Because CD8+ T cells can express VEGF receptors after activation, we then analyzed the impact of VEGF-A on these T cells. Anti-CD3 stimulation induced expression of PD-1, but addition of VEGF-A enhanced PD-1 expression on these cells in a dose-dependent manner (Fig. 2, e–f). Furthermore, this effect was blocked when anti–VEGF-A antibody was added to the culture (unpublished data). Thus, VEGF-A directly increases PD-1 expression on activated CD8+ T cells.


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 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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4322048&req=5

fig2: 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.
Mentions: To understand whether VEGF-A can act directly on CD8+ T cells, we first analyzed the expression of the two VEGF-A receptors, VEGF-R1 and -R2, on CD8+ T cells in vivo. We observed that these two receptors are expressed only at very low levels in spleens of tumor-free and tumor-bearing mice, but are strongly increased on tumor-infiltrating CD8+ T cells (Fig. 2 a). These VEGFR+ CD8+ T cells also expressed PD-1 (unpublished data). Because tumor-infiltrating T cells bear an activated phenotype unlike splenic CD8+ T cells, these results suggested that VEGFR expression could be associated with T cell activation (Whiteside and Parmiani, 1994). To recapitulate this phenomenon in vitro, we analyzed expression of these receptors after stimulation of purified CD8+ T cells with different doses of anti-CD3 by flow cytometry and confocal microscopy. Expression of VEGF-R1 and -R2 was induced by stimulation with 3 µg/ml of anti-CD3 and became significant at 10 µg/ml after 48 h of culture (Fig. 2, b–d). Because CD8+ T cells can express VEGF receptors after activation, we then analyzed the impact of VEGF-A on these T cells. Anti-CD3 stimulation induced expression of PD-1, but addition of VEGF-A enhanced PD-1 expression on these cells in a dose-dependent manner (Fig. 2, e–f). Furthermore, this effect was blocked when anti–VEGF-A antibody was added to the culture (unpublished data). Thus, VEGF-A directly increases PD-1 expression on activated CD8+ T cells.

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