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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 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.
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fig3: 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.

Mentions: PD-1 is the first inhibitory receptor associated with T cell exhaustion. However, other receptors are expressed on exhausted T cells, such as Tim-3, CTLA-4, and Lag-3 (Sakuishi et al., 2010; Woo et al., 2012; Duraiswamy et al., 2013). Density and co-expression of these different molecules have been associated with the magnitude of T cell exhaustion. The more the T cells express these receptors the more they are dysfunctional (Blackburn et al., 2009). We observed that VEGF-A not only enhances PD-1 expression but also increases the percentages of Tim-3– and CTLA-4–expressing CD8+ T cells in a dose-dependent manner (Fig. 3, a–c). The levels of the expression for each molecule (PD-1, Tim-3, CTLA-4, and Lag-3) were also increased in a dose-dependent manner (Fig. 3 d). We then analyzed the simultaneous co-expression of these receptors. Strikingly, compared with the basal expression of 0–1 inhibitory receptor expressed on CD8+ T cells in the absence of exogenous VEGF-A, increasing VEGF-A concentrations induced the simultaneous expression of 3–4 inhibitory receptors on more than 2/3 of T cells (Fig. 3 e). To determine whether VEGF-R1 or VEGF-R2 are involved in VEGF-A–mediated T cell exhaustion, we added neutralizing antibodies to the CD8+ T cell culture in the presence of VEGF-A. Anti–VEGF-R2, but not anti–VEGF-R1 antibody, was able to block the VEGF-A–induced up-regulation of these inhibitory receptors (Fig. 3 f), demonstrating the involvement of VEGF-R2 in this phenomenon. To extend this observation, we analyzed the differential expression of genes coding for inhibitory receptors involved in CD8+ T cell exhaustion. Quantitative RT-PCR analysis showed that other inhibitory receptors, such as CD244/2B4, CD160, and BTLA, were also enhanced by VEGF-A stimulation (Fig. 3 g). These results showed that VEGF-A enhances expression of many different inhibitory receptors involved in CD8+ T cell exhaustion. Furthermore, an increased expression of NFAT was observed after VEGF-A stimulation (Fig. 3 g). NFAT is known to be involved in VEGF-R2 signaling (Liu et al., 2003; Schweighofer et al., 2009), as well as in the control of PD-1 and CTLA-4 expression (Gibson et al., 2007; Oestreich et al., 2008). To determine if VEGF-A–induced T cell exhaustion is dependent on NFAT activation, we used a specific NFAT inhibitor (11R-VIVIT; Le Roy et al., 2012). Interestingly, NFAT inhibition blocked the VEGF-A–induced simultaneous expression of the 4 inhibitory receptors (Fig. 3 h). Among the different signaling pathways activated by VEGFR2 (PI3K–Akt, PLCγ, and Erk), the PLCγ–calcineurin pathway is known to activate NFAT (Schweighofer et al., 2009). Upon VEGF-A treatment, increased PLCγ phosphorylation was observed in CD8+ T cells by Western blot (unpublished data). Furthermore, inhibition of PLCγ and calcineurin by the chemical inhibitor U73122 and cyclosporine A (CsA), respectively, resulted in an inhibition of VEGF-A–induced inhibitory receptor expression on CD8+ T cells (unpublished data). Together, these results show that VEGF-A enhances the expression of inhibitory receptors involved in T cell exhaustion via activation of the VEGFR2–PLCγ–calcineurin–NFAT pathway.


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 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.
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fig3: 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.
Mentions: PD-1 is the first inhibitory receptor associated with T cell exhaustion. However, other receptors are expressed on exhausted T cells, such as Tim-3, CTLA-4, and Lag-3 (Sakuishi et al., 2010; Woo et al., 2012; Duraiswamy et al., 2013). Density and co-expression of these different molecules have been associated with the magnitude of T cell exhaustion. The more the T cells express these receptors the more they are dysfunctional (Blackburn et al., 2009). We observed that VEGF-A not only enhances PD-1 expression but also increases the percentages of Tim-3– and CTLA-4–expressing CD8+ T cells in a dose-dependent manner (Fig. 3, a–c). The levels of the expression for each molecule (PD-1, Tim-3, CTLA-4, and Lag-3) were also increased in a dose-dependent manner (Fig. 3 d). We then analyzed the simultaneous co-expression of these receptors. Strikingly, compared with the basal expression of 0–1 inhibitory receptor expressed on CD8+ T cells in the absence of exogenous VEGF-A, increasing VEGF-A concentrations induced the simultaneous expression of 3–4 inhibitory receptors on more than 2/3 of T cells (Fig. 3 e). To determine whether VEGF-R1 or VEGF-R2 are involved in VEGF-A–mediated T cell exhaustion, we added neutralizing antibodies to the CD8+ T cell culture in the presence of VEGF-A. Anti–VEGF-R2, but not anti–VEGF-R1 antibody, was able to block the VEGF-A–induced up-regulation of these inhibitory receptors (Fig. 3 f), demonstrating the involvement of VEGF-R2 in this phenomenon. To extend this observation, we analyzed the differential expression of genes coding for inhibitory receptors involved in CD8+ T cell exhaustion. Quantitative RT-PCR analysis showed that other inhibitory receptors, such as CD244/2B4, CD160, and BTLA, were also enhanced by VEGF-A stimulation (Fig. 3 g). These results showed that VEGF-A enhances expression of many different inhibitory receptors involved in CD8+ T cell exhaustion. Furthermore, an increased expression of NFAT was observed after VEGF-A stimulation (Fig. 3 g). NFAT is known to be involved in VEGF-R2 signaling (Liu et al., 2003; Schweighofer et al., 2009), as well as in the control of PD-1 and CTLA-4 expression (Gibson et al., 2007; Oestreich et al., 2008). To determine if VEGF-A–induced T cell exhaustion is dependent on NFAT activation, we used a specific NFAT inhibitor (11R-VIVIT; Le Roy et al., 2012). Interestingly, NFAT inhibition blocked the VEGF-A–induced simultaneous expression of the 4 inhibitory receptors (Fig. 3 h). Among the different signaling pathways activated by VEGFR2 (PI3K–Akt, PLCγ, and Erk), the PLCγ–calcineurin pathway is known to activate NFAT (Schweighofer et al., 2009). Upon VEGF-A treatment, increased PLCγ phosphorylation was observed in CD8+ T cells by Western blot (unpublished data). Furthermore, inhibition of PLCγ and calcineurin by the chemical inhibitor U73122 and cyclosporine A (CsA), respectively, resulted in an inhibition of VEGF-A–induced inhibitory receptor expression on CD8+ T cells (unpublished data). Together, these results show that VEGF-A enhances the expression of inhibitory receptors involved in T cell exhaustion via activation of the VEGFR2–PLCγ–calcineurin–NFAT pathway.

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