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. 2017 Oct 4;7(1):e1379642.
doi: 10.1080/2162402X.2017.1379642. eCollection 2017.

IL-21 promotes the development of a CD73-positive Vγ9Vδ2 T cell regulatory population

Affiliations

IL-21 promotes the development of a CD73-positive Vγ9Vδ2 T cell regulatory population

Clément Barjon et al. Oncoimmunology. .

Abstract

Vγ9Vδ2 T cells contribute to the immune response against many tumor types through their direct cytotoxic activity and capacity to regulate the biological functions of other immune cells, such as dendritic cells and IFN-γ-producing CD8+ T cells. However, their presence in the tumor microenvironment has also been associated with poor prognosis in breast, colon and pancreatic cancers. Additionally, recent studies demonstrated that cytokines can confer some plasticity to Vγ9Vδ2 T cells and promote their differentiation into cells with regulatory functions. Here, we demonstrated that activation of Vγ9Vδ2 T cells isolated from healthy donors and cultured in the presence of IL-21 favors the emergence of a subpopulation of Vγ9Vδ2 T cells that express the ectonucleotidase CD73 and inhibits T cell proliferation in a CD73/adenosine-dependent manner. This subpopulation produces IL-10 and IL-8 and displays lower effector functions and cytotoxic activity than CD73-negative Vγ9Vδ2 T cells. We also showed, in a syngeneic mouse tumor model, the existence of a tumor-infiltrating γδ T cell subpopulation that produces IL-10 and strongly expresses CD73. Moreover, maturation, IL-12 production and induction of antigen-specific T cell proliferation are impaired in DC co-cultured with IL-21-amplified Vγ9Vδ2 T cells. Altogether, these data indicate that IL-21 promotes Vγ9Vδ2 T cell regulatory functions by favoring the development of an immunosuppressive CD73+ subpopulation. Thus, when present in the tumor microenvironment, IL-21 might negatively impact γδ T cell anti-tumor functions.

Keywords: CD73; IL-10; IL-21; adenosine; regulatory functions; tumor microenvironment; γδ T cells.

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Figures

Figure 1.
Figure 1.
Expression of IL-21R in Vγ9Vδ2 T cells and induction of STAT3 phosphorylation by IL-21. (A) Representative histograms showing IL-21R expression in freshly isolated Vγ9Vδ2 T cells (left panel) and after 2 weeks of culture in the presence of IL-21 or not (control) (right panel). Overlay of isotype control (solid grey), control Vγ9Vδ2 T cells (black line) and IL-21-cultured Vγ9Vδ2 T cells (grey line). (B) Western blot analysis of STAT3 phosphorylation induced by IL-21 and total STAT3 in control (Ctrl) and IL-21-cultured Vγ9Vδ2 T cells (IL-21). Data are representative of two independent experiments performed with cells from different donors.
Figure 2.
Figure 2.
Expression of CD39 and CD73 in Vγ9Vδ2 T cells and adenosine production. (A) Representative flow cytometry dot plots showing the percentage CD39+, CD73+ and CD39+CD73+ control (Ctrl), IL-21- (IL-21) and IL-6-cultured (IL-6) Vγ9Vδ2 T cells. This experiment was repeated 4 times and performed each time with cells from different donors. Graphs show the percentage of CD39+ (B), CD73+ (C) and CD39+/CD73+ (D) control (Ctrl) and IL-21-cultured (IL-21) Vγ9Vδ2 T cells. (E) Production of adenosine by control (Ctrl) and IL-21-cultured (IL-21) Vγ9Vδ2 T cells assessed by MALDI-TOF mass spectrometry. *p<0.05 and ***p<0.001 (paired Wilcoxon test). (F) Representative flow cytometry histogram showing CFSE staining of T cells grown in the presence of immobilized anti-CD3 monoclonal antibody and the supernatant from IL-21-amplified Vγ9Vδ2 T cells cultured with or without AMP and APCP (CD73 inhibitor) (left panel) for 4 days. The proliferation index was calculated relative to T cell proliferation in medium alone and is represented as the mean of 6 experiments performed with cells from different donors (right panel). NS non-significant, *p<0.05 (paired Wilcoxon test).
Figure 3.
Figure 3.
Production of IL-10 and IL-8 by Vγ9Vδ2 T cells. Quantification of IL-10 (A) and IL-8 (B) by ELISA in the supernatant of control (Ctrl) and IL-21-cultured (IL-21) Vγ9Vδ2 T cells activated or not with HMBPP for 6h; *p<0.05 and **p<0.01 (paired Wilcoxon test).
Figure 4.
Figure 4.
Effect of IL-21-cultured Vγ9Vδ2 T cells on dendritic cells. Monocyte-derived DC were co-cultured with control (Ctrl) or IL-21-cultured (IL-21) Vγ9Vδ2 T cells for 48 hours. Then, expression of CD83 (A) and CD86 (B) in DC was assessed by flow cytometry analysis. Both the percentage of positive DC (right panels) and the MFI (left panels) are shown. (C) Representative flow cytometry histograms of FITC-conjugated dextran endocytosis by DC with or without LPS (left panel) and by DC co-cultured with HMBPP-activated control (Ctrl, medium panel) and IL-21-cultured (IL-21, right panel) Vγ9Vδ2 T cells for 48h. Each analysis was repeated twice and performed each time with cells from different donors. (D) Percentages of IL-12+ DC following 18h of co-culture with control (Ctrl) and IL-21-amplified (IL-21) Vγ9Vδ2 T cells activated or not with HMBPP. (E) Analysis of T cell proliferation induced by DC loaded or not with CMV and EBV (peptides) after co-culture with control (Ctrl) or IL-21-amplified (IL-21) Vγ9Vδ2 T cells for 18h. Proliferation was assessed at day 6. This experiment was repeated three times and performed each time with cells from different donors. *p<0.05, **p<0.01 and ***p<0.001 (paired Wilcoxon test).
Figure 5.
Figure 5.
Characterization of CD73+ Vγ9Vδ2 T cells. (A) Adenosine production by control (Ctrl), bulk IL-21-cultured (IL-21), CD73 IL-21-cultured (CD73-) and CD73+ (CD73+) IL-21-cultured Vγ9Vδ2 T cells was measured by MALDI-TOF mass spectrometry. Representative results of one of four experiments performed each time with cells from different donors. (B) Relative quantification of IL-10 mRNA in Ctrl, CD73 and CD73+ Vγ9Vδ2 T cells. This experiment was performed twice, each time with cells from different donors. (C) IL-10 production by Ctrl, CD73, CD73+ Vγ9Vδ2 T cells, stimulated or not with HMBPP. This experiment was performed twice, each time with cells from different donors. (D) Graphs show the percentage of IFN-γ+ (left panel) and TNF-α+ (right panel) cells in the CD73 and CD73+ subpopulations of Il-21-cultured Vγ9Vδ2 T cells. (E) Representative flow cytometry histograms showing CD107a membrane expression in CD73 and CD73+ IL-21-cultured Vγ9Vδ2 T cells after HMBPP stimulation or co-cultured with Daudi cells for 6h. (F) The graph show the geometric mean of CD107a membrane expression in non-activated (N/A) CD73 and CD73+ IL-21-cultured Vγ9Vδ2 T cells and after HMBPP stimulation or co-cultured with Daudi cells for 6h. This experiment was performed six times, each time with cells from different donors; *p<0.05, **p<0.01 and ***p<0.001 (paired Wilcoxon test).
Figure 6.
Figure 6.
Analysis of γδ T cell populations in naive or TC1-grafted mice. (A) Percentage of CD73+ γδ T cells from the spleen of naive and from the spleen and tumor of TC1-grafted C57BL6 mice. (B) Relative quantification of IL-21 mRNA in total cells from the spleen of naive mice and from the spleen and tumor of TC1-grafted C57BL6 mice. Results were normalized to CD45 mRNA level. (C) Percentage of IL-10+ γδ T cells and (D) of IL-10+ CD4+ T cells from the spleen of naive and TC1-grafted C57BL6 mice and from TC1 tumors, after cell activation with PMA and ionomycin. (E) CD73 expression (Geo mean) by IFN-γ- and IL-10-producing γδ T cells and CD4+ T cells calculated as the ratio between the expression in tumor-infiltrating cells and in splenocytes of TC1-grafted mice. *p<0.05, **p<0.01 and ***p<0.001 (paired Wilcoxon test).

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