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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2016 Oct 17;113(44):12520–12525. doi: 10.1073/pnas.1607136113

Human Vδ2 T cells are a major source of interleukin-9

Christian Peters a, Robert Häsler b, Daniela Wesch a, Dieter Kabelitz a,1
PMCID: PMC5098669  PMID: 27791087

Significance

We describe in vitro cell culture conditions that induce strong secretion of IL-9 in human peripheral blood γδ T cells. IL-9 plays a role in allergy and increases the antitumor immunity of conventional CD4 and CD8 T cells. Human γδ T cells with a Vδ2 T-cell receptor kill many different tumor cells because they recognize intermediates of a metabolic pathway that is frequently dysregulated in cancer cells. Vδ2 T cells have already been used in cancer immunotherapy, as yet with limited success. Our study demonstrates that TGF-β, together with IL-15, strongly enhances IL-9 production in Vδ2 T cells. We postulate that IL-9–producing Vδ2 T cells might have enhanced therapeutic efficacy upon adoptive transfer into patients who have cancer.

Keywords: γ/δ T cells, human, interleukin-9, transforming growth factor-β

Abstract

Vδ2Vγ9 T cells are the dominant γδ T-cell subset in human peripheral blood. Vδ2 T cells recognize pyrophosphate molecules derived from microbes or tumor cells; hence, they play a role in antimicrobial and antitumor immunity. TGF-β, together with IL-15, induces a regulatory phenotype in Vδ2 T cells, characterized by forkhead box protein P3 (FoxP3) expression and suppressive activity on CD4 T-cell activation. We performed a genome-wide transcriptome analysis and found that the same conditions (TGF-β plus IL-15) strongly enhanced the expression of additional genes in Vδ2 T cells, including IKAROS family zinc finger 4 (IKZF4; Eos), integrin subunit alpha E (ITGAE; CD103/αEβ7), and IL9. This up-regulation was associated with potent IL-9 production as revealed by flow cytometry and multiplex analysis of cell culture supernatants. In contrast to CD4 and CD8 αβ T cells, γδ T cells did not require IL-4 for induction of intracellular IL-9 expression. Upon antigen restimulation of Vδ2 T cells expanded in vitro in the presence of TGF-β and IL-15, IL-9 was the most abundant among 16 analyzed cytokines and chemokines. IL-9 is a pleiotropic cytokine involved in various (patho)physiological conditions, including allergy and tumor defense, where it can promote antitumor immunity. Given the conspicuous sensitivity of many different tumors to Vδ2 T-cell–mediated killing, the conditions defined here for strong induction of IL-9 might be relevant for the development of Vδ2 T-cell–based immunotherapy.

Although less well characterized than other functional T helper-cell subsets, T helper 9 (Th9) cells have been identified on the basis of their selective IL-9 production. IL-9 is a pleiotropic cytokine that promotes T-cell and mast-cell growth and mast-cell accumulation in tissue as well as IgE switching in B cells (1). In line, allergic patients have increased numbers of T cells that produce IL-9 in response to various allergens (2, 3). Moreover, Th9 cells alter intestinal epithelial cell functions (4, 5) and are key players in antiworm immunity (6). Intriguingly, Th9 cells also appear to regulate tumor immunity. Although IL-9 and Th9 cells exerted antitumor activity in some solid tumor models (7, 8), IL-9 actually promoted lymphoma development in other models (9). IL-9 is thus an important cytokine with multiple functions in the regulation of immune responses.

Similar to other functional T-cell subsets, the differentiation of Th9 cells is driven by the cytokine milieu and specific transcription factors. In general, IL-4, together with TGF-β, was found to polarize IL-9–secreting CD4 T cells (10–12), and PU.1 and interferon regulatory factor 4 (IRF4) were identified as crucially important transcription factors (11, 13). The signal strength of T-cell receptor (TCR) ligation and costimulatory signals also regulate IL-9 at the transcriptional level (14).

The γδ T cells expressing Vδ2 paired with Vγ9 (hereafter termed Vδ2 T cells) dominate in the peripheral blood of healthy adult individuals (15). Vδ2 T cells recognize via their TCR, in a CD277/butyrophilin 3A-dependent manner, microbial pyrophosphates and homologous eukaryotic pyrophosphates (isopentenyl pyrophosphate), which are produced by many tumor cells due to their dysregulated mevalonate pathway (16). Hence, Vδ2 T cells play a role in both antiinfective and antitumor immunity (17, 18). Interestingly, Vδ2 T cells exert a surprisingly large functional plasticity (17). In addition to their strong cytolytic activity (19), Vδ2 T cells produce different cytokines, can be converted into forkhead box protein P3 (FoxP3)+ regulatory cells (20, 21), and may acquire professional antigen-presenting capacity (22). Depending on priming conditions, Vδ2 T cells can secrete IFN-γ, IL-4, IL-17, and IL-22, and thus recapitulate cytokine patterns of well-defined functional Th subsets (23, 24).

Here, we identified peripheral blood Vδ2 T cells as a major source of IL-9. In the presence of TGF-β and IL-15 but the absence of IL-4, high levels of IL-9 were secreted already 4 d after initial in vitro activation. Upon antigen restimulation of Vδ2 T cells precultured for 15 d in the presence of TGF-β/IL-15, IL-9 was by far the most abundant among 16 analyzed cytokines and chemokines. Our results might help to enhance selective effector functions of human γδ T cells.

Results

To investigate the impact of differential activation on the gene expression profile and the transcription factor and cytokine/chemokine pattern of Vδ2 T cells, the following four conditions were used for in vitro culture (note: exogenous IL-2 was always present). Purified total γδ T cells were stimulated with the Vδ2-specific phosphoantigen bromohydrin pyrophosphate (BrHPP) in the presence of irradiated peripheral blood mononuclear cells (PBMCs) and in the absence (i) or presence (iii) of TGF-β/IL-15. Alternatively, purified Vδ2 T cells were stimulated with microbeads coupled with anti-CD2/CD3/CD28 antibodies [activation/expander (A/E) beads], again in the absence (ii) or presence (iv) of TGF-β/IL-15 (21).

Gene Expression of Differentially Activated Vδ2 T Cells.

In the presence of TGF-β, the proliferative activity of freshly isolated γδ T cells as measured by 3H-radiolabeled thymidine incorporation was reduced (Fig. S1A). Interestingly, however, the proliferative expansion of Vδ2 T cells that had been cultured for 15 d under conditions (i) to (iv) and restimulated with BrHPP was much higher if TGF-β and IL-15 had been present during initial activation [i.e., conditions (iii) and (iv)] (Fig. S1B). We then analyzed gene expression by means of an Affymetrix whole-genome array in γδ T cells cultured for 8 d under conditions (i) to (iv). The principal component analysis depicted in Fig. 1A indicates that the overall gene expression in Vδ2 T cells cultured with TGF-β/IL-15 clustered differently from those Vδ2 T cells cultured without TGF-β/IL-15. Moreover, BrHPP stimulation [i.e., conditions (i) and (iii)] clearly differed from A/E bead stimulation [i.e., conditions (ii) and (iv)]. The heat map based on hierarchical clustering of 1,056 genes found to be regulated at least 1.5-fold in condition (iv) compared with unstimulated Vδ2 T cells is shown in Fig. 1B. Selected genes with profound modulation are identified in Fig. 1B. The median fold change of these genes induced under conditions (i) to (iv) is listed in Table S1. IL9 was among the most strongly up-regulated genes in TGF-β/IL-15–supplemented conditions (iii) and (iv). All genes listed in Table S1 were strongly modulated (up- or down-regulated) by TGF-β/IL-15 and also were more strongly regulated by A/E bead stimulation compared with BrHPP. A Spearman’s rank coefficient analysis of x-fold gene regulation revealed the highest discrepancy (rs = 0.78) between conditions (i) (i.e., BrHPP-activated, IL-2 only) and (iv) (i.e., A/E bead-activated with TGF-β/IL-15), which differed with regard to both the stimulus and the cytokine milieu (Fig. S2A). This high discrepancy is visualized in the biplot analysis, because many genes up-regulated in condition (iv) were not up-regulated in condition (i) (e.g., IL9), and vice versa [e.g., Eomesodermin homolog (Eomes)] (Fig. S2B).

Fig. S1.

Fig. S1.

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Modulation of in vitro expansion of Vδ2 T cells by TGF-β. Vδ2 T cells were activated under condition (i) BrHPP + IL-2, (ii) A/E + IL-2, (iii) BrHPP + IL-2low + IL-15 + TGF-β, or (iv) A/E + IL-2low + IL-15 + TGF-β. (A) Proliferation was determined by 3H-radiolabeled thymidine incorporation 5 d after initial stimulation. Results presented as mean counts per minute of triplicate cultures from nine [positively isolated pan-γδ Τ cells; conditions (ii) and (iv)] or seven [positively isolated Vδ2 T cells; conditions (i) and (iii)] experiments are summarized in a box and whiskers diagram. (B) Differentially activated Vδ2 T cells under conditions (i) to (iv) were cultured for 15 d and then restimulated with BrHPP and cultured for another 7 d (in the presence of 50 IU/mL IL-2). Thereafter, the number of viable γδ T cells was determined as described in SI Materials and Methods. Results of 10 independent experiments are summarized in a box and whiskers diagram, where the median is represented by the horizontal bar and the boxes represent the second and third quartiles. Asterisks indicate significance according to the Student’s t test for paired data (*P ≤ 0.05; **P ≤ 0.01).

Fig. 1.

Fig. 1.

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Transcriptome analysis in differentially expanded Vδ2 T cells. Purified γδ T cells from three healthy donors were differentially stimulated as indicated. Gene expression was determined after 8 d by an Affymetrix HuGene 1.0 st v1 array analysis. (A) Principal component (Comp.) analysis (PCA) of overall gene expression in differentially expanded Vδ2 T cells. (B) Hierarchical clustering [unweighted pair group method with arithmetic mean/correlation] of the 1,056 selected genes, whose median expression was at least 1.5-fold modulated in A/E bead- and TGF-β/IL-15–expanded Vδ2 T cells [condition (iv)] compared with the corresponding freshly isolated cells, and which had a difference of modulation of at least 1.5-fold (compared with all other expansion conditions). Genes of interest are highlighted by displaying their gene symbol. For better comparability between transcripts, data were z-score–normalized before clustering. unstim., unstimulated.

Table S1.

List of x-fold changes of gene expression (with reference to freshly isolated γδ T cells) of γδ T cells activated under conditions (i) to (iv)

Fold change Gene name
Gene symbol (i) (ii) (iii) (iv)
IL9 1.11 1.81 54.83 58.26 IL-9
CCR7 1.26 7.20 9.40 21.16 Chemokine (C-C motif) receptor 7
IKZF4 2.40 3.38 7.50 13.32 IKAROS family zinc finger 4 (Eos)
IL13 1.85 4.49 8.81 13.13 IL-13
ITGAE 1.15 1.41 7.41 12.56 Integrin alpha E (CD103)
IL5 1.53 2.90 3.16 12.38 IL-5
KLF7 −1.18 1.69 5.67 8.10 Krüppel-like factor 7
EOMES 2.58 1.89 −1.17 −1.56 Eomesodermin homolog
TBX21 −1.80 −1.76 −3.25 −3.32 T-box 21 (T-bet)
SMAD3 −1.00 −1.40 −3.12 −10.77 SMAD family member 3
KLRG1 −1.19 −3.10 −4.47 −13.16 Killer cell lectin-like receptor subfamily G-member 1
KLF3 −4.62 −9.12 −28.99 −49.13 Krüppel-like factor 3
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Depicted are the median values of three [(i) and (ii)] or two [(iii) and (iv)] independent experiments.

Fig. S2.

Fig. S2.

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Similarity in the gene regulation patterns between differentially expanded Vδ2 T cells. The x-fold regulation (referring to the respective unstimulated cells) of all 4,567 genes significantly regulated by the different activation conditions (i) to (iv) was compared. (A) Correlation scatter plots and the Spearman’s correlation coefficient (rs) of the log(2)-fold changes (with reference to the corresponding freshly isolated cells) of the gene expression in response to the differential stimulation are depicted. (B) Log(2)-fold changes of the gene expression under conditions (i) to (iv) are depicted in a biplot analysis. The proximity of the genes of interest to the vector of the respective condition illustrates the higher potential of these genes to be modulated under this condition.

Analysis of Gene Expression by Quantitative RT-PCR.

The regulation of 28 genes, including genes of interest from the transcriptome analysis and further lineage-specific transcription factors, cytokines, and surface receptors, was quantified by quantitative RT-PCR in Vδ2 T cells cultured for 8 d (Fig. 2) or 15 d (Fig. S3) under conditions (i) to (iv). Among the genes specifically up-regulated in the presence of TGF-β/IL-15 were FOXP3 (Fig. 2A and Fig. S3A), IKAROS family zinc finger 4 (IKZF4; Eos) (Fig. 2B and Fig. S3B), IL9 (Fig. 2C), TNF superfamily member 13b (TNFSF13B; B cell activating factor of the TNF family, BAFF) (Fig. S3C), integrin subunit alpha E (ITGAE; CD103) (Fig. 2D and Fig. S3D), and IRF4 (Fig. S3D). Interestingly, IL9 was reduced on day 15 compared with day 8 (Fig. 2C and Fig. S3C). Other cytokine genes, including IL13, Epstein–Barr virus induced 3 (EBI3), and interferon, gamma (IFNG) (Fig. 2C and Fig. S3C), were up-regulated under all conditions, although IL13 and EBI3 were more strongly up-regulated in response to A/E bead stimulation. Kruppel-like factor 7 (KLF7) was up-regulated only under condition (iv) (Fig. 2B and Fig. S3B). Among the genes that were generally down-regulated were T-box 21 (TBX21; T-bet), EOMES, SPI1 (PU.1) (Fig. 2A and Fig. S3A), IKZF1 (Ikaros), KLF3 (Fig. 2B and Fig. S3B), TGFB1 (TGF-β1) (Fig. 2C and Fig. S3C), killer cell lectin-like receptor G1 (KLRG1), and SMAD family member 3 (SMAD3) (Fig. 2D and Fig. S3D). Several of these genes [e.g., SPI1, IZKF1, KLRG1, C-C motif chemokine receptor 7 (CCR7)] were more potently regulated by A/E bead stimulation compared with BrHPP stimulation, pointing to an impact of the A/E bead-mediated CD28 costimulation and/or differential TCR signal strength. The down-regulation of EOMES was mainly observed in TGF-β/IL-15–supplemented cultures, whereas A/E bead stimulation and TGF-β/IL-15 had an additive effect on the down-regulation of SMAD3, KLF3, and TBX21.

Fig. 2.

Fig. 2.

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Quantification of gene expression by RT-PCR (day 8). Vδ2 T cells were activated under condition (i) BrHPP + IL-2, (ii) A/E + IL-2, (iii) BrHPP + IL-2low + IL-15 + TGF-β, or (iv) A/E + IL-2low + IL-15 + TGF-β. RT-PCR was performed after 8 d of culture. The differences between the cycle threshold values (ΔCt) of the gene of interest and three different housekeeping genes (G6PDH, HuPo, and RPII) were determined. ΔΔCt values represent the difference between ΔCt values of freshly isolated and differentially stimulated γδ T cells at d 8. The three ΔΔCt values per gene of interest were averaged. Depicted are the mean values ± SEM of various experiments [FOXP3 (n = 7), TBX21 (n = 9), GATA3 (n = 8), SPI1 (n = 6), BCL6 (n = 5), EOMES (n = 5), and RORC tv.2 (n = 5) (A); IKZF2 (n = 9), IKZF4 (n = 9), IKZF1 (n = 7), IKZF3 (n = 7), IKZF5 (n = 7), KLF3 (n = 7), and KLF7 (n = 4) (B); IL4 (n = 7), IL5 (n = 9), IL9 (n = 10), IL10 (n = 8), IL13 (n = 9), EBI3 (n = 5), TNFSF13B (n = 5), IFNG (n = 7), and TGFB (n = 6) (C); and ITGAE (n = 5), KLRG1 (n = 5), CCR7 (n = 3), SMAD3 (n = 3), and IRF4 (n = 3) (D)].

Fig. S3.

Fig. S3.

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Quantification of gene expression by RT-PCR (day 15). Vδ2 T cells were activated under condition (i) BrHPP + IL-2, (ii) A/E + IL-2, (iii) BrHPP + IL-2low + IL-15 + TGF-β, or (iv) A/E + IL-2low + IL-15 + TGF-β. RT-PCR was performed after 15 d of culture. The differences between the cycle threshold values (ΔCt) of the gene of interest and three different housekeeping genes (G6PDH, HuPo, and RPII) were determined. ΔΔCt values represent the difference between ΔCt values of freshly isolated and differentially stimulated γδ T cells at day 15. The three ΔΔCt values per gene of interest were averaged. Depicted are the mean values ± SEM of various experiments [FOXP3 (n = 4), TBX21 (n = 4), GATA3 (n = 4), SPI1 (n = 4), BCL6 (n = 4), EOMES (n = 4), and RORC tv.2 (n = 4) (A); IKZF2 (n = 4), IKZF4 (n = 4), IKZF1 (n = 4), IKZF3 (n = 4), IKZF5 (n = 4), KLF3 (n = 3), and KLF7 (n = 3) (B); IL4 (n = 4), IL5 (n = 4), IL9 (n = 5), IL10 (n = 5), IL13 (n = 4), EBI3 (n = 4), TNFSF13B (n = 2), IFNG (n = 4), and TGFB (n = 4) (C); and ITGAE (n = 4), KLRG1 (n = 4), CCR7 (n = 4), SMAD3 (n = 3), and IRF4 (n = 4) (D)]. Significance is indicated according to the Student's t test for paired data (n.s., not significant; *P ≤ 0.05; **P ≤ 0.01).

Cytokine Production of Differentially Activated Vδ2 T Cells.

Next, we analyzed the secretion of a broad panel of cytokines and chemokines in cell culture supernatants of differentially activated Vδ2 T cells by bead-based multiplex analysis. After 4 d of initial stimulation of purified γδ T cells, large amounts (>2,000 pg/mL) of IL-9 and TNF-α were detected, as well as lower concentrations (<750 pg/mL) of other cytokines and chemokines [IL-5, IL-6, IL-13, IFN-γ, C-C motif chemokine 22 (CCL22), and C-X-C motif chemokine 13 (CXCL13)] (Fig. 3A). Importantly, whereas TNF-α was induced by both BrHPP and A/E beads also in the absence of TGF-β/IL-15 [i.e., under conditions (i) and (ii)], the high level of IL-9 secretion (as well as the much lower secretion of IL-6) was selectively induced during initial stimulation in Vδ2 T cells activated with BrHPP and TGF-β/IL-15 [i.e., under condition (iii)] (Fig. 3A). The absence of IL-9 secretion on day 4 in Vδ2 T cells activated with A/E beads and TGF-β/IL-15 was surprising, given that the IL9 gene was similarly up-regulated after 8 d [condition (iv)] (Fig. 2C). Therefore, we also analyzed cytokine production at later time points. Vδ2 T cells activated and cultured for 15 d under conditions (i) to (iv) were washed and restimulated with BrHPP. Cytokines were measured in cell culture supernatants after an additional 4 d. Even larger amounts (4,000–6,000 pg/mL) of IL-9 compared with the initial activation were detected in culture supernatants of Vδ2 T cells that had been initially cultured in the presence of TGF-β/IL-15 [(iii) and (iv)]. Interestingly, comparing both cell cultures expanded in the presence of TGF-β/IL-15, the initially A/E bead-stimulated Vδ2 T cells [condition (iv)] secreted even higher amounts of IL-9 after BrHPP restimulation, compared with the initially BrHPP-stimulated Vδ2 T cells [condition (iii)] (Fig. 3B). In addition to IL-9, BrHPP-restimulated Vδ2 T cells secreted significant amounts (up to 2,500 pg/mL) of IL-5, IL-13, IFN-γ, and TNF-α, whereas the strongest IL-5 and IL-13 secretion was observed in Vδ2 T cells [conditions (ii) and (iv)] that were initially A/E bead-stimulated (Fig. 3B). In line with cytokine measurements in cell culture supernatants, intracellular flow cytometry of IL-9 and IFN-γ expression in Vδ2 T cells after 4 and 8 d of initial activation (without further restimulation) revealed only a few IFN-γ–positive Vδ2 T cells but strong IL-9 expression whenever TGF-β was present (Fig. 4, Left, Medium). To dissect the role of IL-15 versus TGF-β in the induction of IL-9, cultures were supplemented with TGF-β or IL-15 only (Fig. 4, Upper). We found that TGF-β, but not IL-15, was essential for IL-9 induction, but that IL-15 increased the overall proliferation rate. Importantly, we noted significant differences in the intracellular cytokine expression pattern when γδ T cells were activated for 6 h with 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin before the cytokine staining (Fig. 4, Right), which revealed the TGF-β–dependent appearance of IL-9 single-positive, IL-9/IFN-γ double-positive, and IFN-γ single-positive Vδ2 T cells. Under these conditions, IL-9 single-positive Vδ2 T cells were hardly detectable on day 4 but were more abundant on day 8 (Fig. 4, Right). Vδ2 T cells expanded for 15 d under conditions (i) to (iv) and rested afterward for 16 h in the presence of low-dose IL-2 [10 international units (IU)], showed an IL-9 and IFN-γ expression pattern after TPA/ionomycin activation similar to day 4. Even though the IL9 mRNA levels were no longer up-regulated on day 15 (Fig. S3C), IL-9 was produced quickly after TPA/ionomycin activation. Again, IL-9 production was only detected in Vδ2 T cells initially cultured in the presence of TGF-β/IL-15 [conditions (iii) and (iv)]. In addition to IL-9, the production of IFN-γ and TNF-α was enhanced in these cells (Fig. S4A). IL-9 and TNF-α were most abundant in Vδ2 T cells initially stimulated under condition (iv). Slight IL-13 production was detected in the initially A/E bead-stimulated Vδ2 T-cell lines (ii) and (iv) (Fig. S4B). Intracellular expression of the cytotoxic mediators Perforin and Granzyme B was not differentially modulated under the various stimulation conditions (Fig. S4B). When the 15-d Vδ2 T-cell lines (iii) and (iv) were restimulated with BrHPP and cultured for an additional 4 d in the presence of IL-2 only, IL-9 production was still maintained (Fig. S4C).

Fig. 3.

Fig. 3.

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Quantification of cytokines and chemokines in cell culture supernatants. Vδ2 T cells were activated under condition (i) BrHPP + IL-2, (ii) A/E + IL-2, (iii) BrHPP + IL-2low + IL-15 +TGF-β, or (iv) A/E + IL-2low + IL-15 + TGF-β (color-coded as indicated). Concentrations of cytokines and chemokines in cell-free supernatants were quantified by a multiplex bead array. (A) Cytokine secretion in γδ T-cell cultures after 4 d of initial stimulation. (B) Cytokine secretion in 15-d activated Vδ2 T cells, restimulated for 4 d with BrHPP. Shown are the mean values with the SEM of six (IL-1α, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17, IL-22, IL-27, CXCL-13, IFN-γ, and TNF-α, LIF) or four (IL-12 and CCL22) independent experiments. Statistical analysis was performed using the Student’s t test for paired data. Asterisks refer to significant differences in γδ T cells (*P ≤ 0.05; **P ≤ 0.01).

Fig. 4.

Fig. 4.

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Detection of intracellular IL-9 and IFN-γ during primary activation of Vδ2 T cells. Vδ2 T cells were activated under conditions (i) to (iv) and under additional conditions (TGF-β and IL-15, separately) as indicated. Intracellular IL-9 and IFN-γ staining was performed at the indicated time points without (Left, Medium) or with 6 h of TPA/ionomycin activation (Right, TPA/iono). Monensin was added at a concentration of 3 μM 4 h before fixation. Results of one of three independent experiments are shown. The dot plot of the specifically labeled cells is colored red, and the respective isotype control is colored blue.

Fig. S4.

Fig. S4.

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Detection of intracellular mediators in differentially expanded Vδ2 T cells. Fifteen days after primary activation under conditions (i) BrHPP + IL-2, (ii) A/E + IL-2, (iii) BrHPP + IL-2low + IL-15 + TGF-β, or (iv) A/E + IL-2low + IL-15 + TGF-β Vδ2. T cells were cultured for additional 16 h in fresh medium in the presence of 10 IU of IL-2 and then were restimulated for 6 h by TPA/ionomycin (A and B) or were directly restimulated by BrHPP and cultivated in the presence of 50 IU of IL-2 for 4 d (C). (A and C) Intracellular IL-9 and IFN-γ expression from one of four independent experiments. The dot plot from the specific antibody labeling and the corresponding isotype controls are colored red and blue, respectively. (B) Differential mean fluorescence intensity (Diff. mean FI) of multiple experiments [Granzyme B (n = 8), IFN-γ (n = 8), IL-9 (n = 6), IL-13 (n = 7), TNF-α (n = 5), and Perforin (n = 7)]. The median expression is indicated by bars, and asterisks refer to significant difference according to the Student’s t test (*P ≤ 0.05; **P ≤ 0.01).

Requirements for IL-9 Production by CD4 and CD8 T Cells.

To compare the induction of IL-9 in Vδ2 T cells with CD4 and CD8 T cells, we activated purified CD4 and CD8 T cells with immobilized anti-CD3 and soluble anti-CD28 antibodies in the presence of IL-2 and absence of the presence of TGF-β/IL-15 and IL-4 (12). Comparable to Vδ2 T cells, no intracellular IL-9 was detected in the absence of TGF-β (Fig. 5). In contrast to Vδ2 T cells, however, very few CD4 T cells and no CD8 T cells stained positive for IL-9 in the presence of only TGF-β /IL-15. Only in the additional presence of IL-4 was IL-9 expression detectable in CD4 and CD8 T cells (Fig. 5, Bottom). Short-term activation with TPA/ionomycin stimulated expression of IFN-γ, which was strongly enhanced only in CD8 T cells in the presence of TGF-β/IL-15; however, unlike the case in Vδ2 T cells, no IL-9/IFN-γ double-positive cells were detected (Fig. 5, Right). Analysis of the corresponding supernatants from day 8 cell cultures also revealed some IL-9 secretion in TGF-β/IL-15–supplemented CD4 T cells (but less compared with Vδ2 T cells). In the additional presence of IL-4, IL-9 production was induced in CD8 T cells and was enhanced in CD4 T cells, respectively (Fig. S5).

Fig. 5.

Fig. 5.

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Detection of intracellular IL-9 and IFN-γ in differentially activated CD4 and CD8 T cells. CD4 and CD8 T cells were stimulated by immobilized anti-CD3 and soluble anti-CD28 in the presence of IL-2, IL-2low, IL-4, IL-15, and TGF-β as indicated. Intracellular IL-9 and IFN-γ staining was performed 8 d after initial activation without (Left, Medium) or with 6 h of TPA/ionomycin activation (Right, TPA/iono). Monensin was added at a concentration of 3 μM 4 h before fixation. Results of one of four independent experiments are shown. The dot plot of the specifically labeled cells is colored red, and the respective isotype control is colored blue.

Fig. S5.

Fig. S5.

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Detection of IL-9 in supernatants of differentially activated CD4 and CD8 T cells. CD4 and CD8 T cells were stimulated with immobilized anti-CD3 and soluble anti-CD28 antibodies in the presence of IL-2, IL-2low, IL-4, IL-15, and TGF-β as indicated. IL-9 in cell culture supernatants of four independent experiments was quantified by ELISA after 8 d. Supernatants of cultured cells from one donor are represented by one symbol. The mean is indicted by bars.

Profile of Differentially Activated Vδ2 T Cells.

Next, we attempted to correlate the gene expression profile of the differentially activated Vδ2 T cells with surface marker and transcription factor expression. A/E bead stimulation resulted in a decrease of CD27-positive but an increase of CD45RA-positive Vδ2 T cells. The presence of TGF-β/IL-15 [conditions (iii) and (iv)] also led to fewer CD27-positive cells, but strongly induced CD103. Slight KLRG1 expression was only detected in Vδ2 T cells activated by BrHPP in the presence of IL-2 and absence of TGF-β/IL-15 (i.e., condition (i)] (Fig. S6). The kinetics of intracellular expression of lineage-specific transcription factors was also analyzed by flow cytometry (Fig. S7). Induction of GATA-3 was stronger in Vδ2 T cells initially activated with A/E beads [i.e., conditions (ii) and (iv)]. On the other hand, induction of FoxP3 was only detected in Vδ2 T cells activated in the presence of TGF-β/IL-15 [i.e., conditions (iii) and (iv)], and was strongest on day 8 when BrHPP was used for the initial activation [condition (iii)]. The levels of PU.1 were also slightly higher in Vδ2 T cells activated in the presence of TGF-β/IL-15 but decreased during the 15-d culture period. T-bet expression was highest in BrHPP-only stimulated cells [condition (i)], whereas it was lowest whenever TGF-β/IL-15 was present [conditions (iii) and (iv)]. B-cell lymphoma 6 protein (Bcl-6) was not detected at significant levels in any of the differentially activated Vδ2 T cells.

Fig. S6.

Fig. S6.

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Polarization of differentially activated Vδ2 T cells toward different subsets. Vδ2 T cells were activated under conditions (i) to (iv). Surface staining for CD27, CD45RA, CD103, KLRG1, and NKG2D was performed after 15 d. Results of one of three independent experiments are shown. The dot plot of the specifically labeled cells is colored red, and the respective isotype control is colored blue.

Fig. S7.

Fig. S7.

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Kinetics of transcription factor protein expression in differentially activated Vδ2 T cells. Vδ2 T cells were activated under condition (i) BrHPP + IL-2, (ii) A/E + IL-2, (iii) BrHPP + IL-2low + IL-15 + TGF-β, or (iv) A/E + IL-2low + IL-15 + TGF-β (color-coded as indicated). Depicted are the mean values of the differential mean fluorescence intensity of five (PU.1, Helios) or six (GATA-3, T-bet, FoxP3, Bcl-6) independent experiments in a time course. Asterisks indicate a significant difference of a single activation condition or between two groups (separated by bars) (*P ≤ 0.05).

Discussion

Activation by anti-CD3/CD28 antibodies versus cognate antigen recognition differentially regulates gene transcription in CD4 T cells (14) and results in differential TCR signaling kinetics also in γδ T cells (25). This study adds a further facet to the pattern of Vδ2 T-cell gene expression, which has been described as being intermediate between αβ T cells and natural killer cells (26). The results of our present investigation extend these previous studies by demonstrating that gene expression is differentially regulated in human Vδ2 T cells by cognate TCR antigen BrHPP and A/E beads, and TGF-β/IL-15 cytokines provide an additional layer of regulation. This additive effect is exemplified by the lowest Spearman’s rank coefficient between Vδ2 T cells activated by BrHPP, IL-2 [i.e., condition (i)] and A/E, TGF-β/IL-15 [i.e., condition (iv)]. The characteristic gene expression profiles resulting from the differential stimulation of conditions (i) to (iv) correlated with the expression of characteristic surface markers. In this regard, the expression of KLRG1, which coincided with higher expression of the transcriptional repressor KLF3 (27) in BrHPP/IL-2–activated Vδ2 T cells [condition (i)], might indicate differentiation toward a more exhausted phenotype. On the other hand, Vδ2 T cells preactivated in the presence of TGF-β displayed a high proliferative potential upon restimulation, which was accompanied by a loss of KLRG1 and strongly induced CD103 surface expression. This observation is in line with previous findings for murine and human CD8 T cells (28). The TGF-β/IL-15–dependent decrease of CD27 on Vδ2 T cells and the resulting shift toward an effector memory phenotype might implicate enhanced effector potential.

Various cell types have been identified as a cellular source of IL-9. The best characterized is the Th9 subset of CD4 T cells, but IL-9 production has also been found in CD8 T cells (29), innate lymphoid cells (30), and mast cells (31). So far, there is only one report of IL-9 production by γδ T cells in Schistosoma japonicum-infected mice (32). It is commonly accepted that TGF-β and IL-4 are essential requirements for the induction of IL-9 in CD4 T cells in vitro (11, 12). We observed very strong IL-9 induction at the transcriptional and protein levels in human Vδ2 T cells upon in vitro activation in the presence of TGF-β/IL-15, but an absence of exogenous IL-4. Together with our failure to detect IL-4 in any of the Vδ2 T-cell cultures, we conclude that IL-4 is not required for the strong induction of IL-9 in human Vδ2 T cells. Early studies reported that induction of IL-9 in murine CD4 T cells by TGF-β can also occur in the absence of IL-4, but is augmented by additional IL-4 (10). In accordance, we found TGF-β–dependent basal IL-9 production in supernatants of human CD4 T cells, which was further enhanced by IL-4, whereas the intracellular IL-9 expression on day 8 was minimal in the absence of IL-4. Therefore, the initial IL-9 production by CD4 T cells in the absence of exogenous IL-4 might be independent of IL-4 or conditioned by initial endogenous IL-4 secretion. IL-9 production in CD8 T cells always required the presence of both IL-4 and TGF-β. Moreover, intracellular flow cytometry of Vδ2 T cells expanded in the presence of TGF-β/IL-15 revealed that these cells mainly produced IL-9 but also had the potential to produce IFN-γ at the same time when activated by TPA/ionomycin. This observation is striking, because IFN-γ was found to counteract the IL-9 production in naive murine CD4 T cells (10). In line with this observation, but in contrast to our results with Vδ2 T cells, no coexpression of IL-9 and IFN-γ was found in CD4 or CD8 T cells. We conclude that IL-9 production in Vδ2 T cells is differentially regulated compared with αβ T cells, with no requirement for IL-4 for Vδ2 T cells.

When Vδ2 T cells cultured for 15 d in the presence of TGF-β/IL-15 were restimulated with BrHPP and were cultured for an additional 4 d, even higher levels of IL-9 were detected in cell culture supernatants than after initial stimulation. After restimulation, more IL-9 was produced by initially A/E bead-stimulated [condition (iv)] compared with initially BrHPP-stimulated [condition (iii)] Vδ2 T cells, suggesting that initial CD28 costimulation by A/E beads might induce Vδ2 T cells with a more stable IL-9–producing phenotype. Interestingly, no IL-4 or IL-10 secretion was measured in any of the culture supernatants, whereas Th2 cytokines IL-5 and IL-13 as well as TNF-α and some IFN-γ were detected. The production of IL-5 and IL-13 was not specific to TGF-β/IL-15 conditions, but resulted from the initial CD28 costimulation (by A/E beads), in line with a previous report (33).

A multitude of signaling molecules are involved in the regulation of IL9 gene expression, of which IRF4 and PU.1 have been identified as key transcription factors (11, 13). In accordance, IRF4 and basic leucine zipper ATF-like transcription factor 3 (BATF3) gene expression was enhanced in the presence of TGF-β/IL-15 in our study. Even though we observed the down-regulation of SPI1 (PU.1) expression by RT-PCR on days 8 and 15 after initial activation, this result does not exclude a role for PU.1 in driving IL-9 expression in Vδ2 T cells, because we found a slight increase of PU.1 on protein level in the presence of TGF-β/IL-15. Nonetheless, TGF-β/IL-15 had a more significant negative impact on TBX21 (T-bet) and EOMES gene expression and T-bet protein expression, which were both further down-regulated in the presence of CD28 costimulation (A/E beads). Such a negative effect of TGF-β on T-bet (34) might foster Th9 differentiation, because it also has been shown that T-bet counteracts the development of IL-9–secreting cells (35). Moreover, in response to TGF-β/IL-15, Vδ2 T cells up-regulated Eos and, in line with published data (23, 24), FoxP3.

The (patho)physiological significance of IL-9 producing Vδ2 T cells is currently unknown, but the strongly enhanced CD103 (ITGAE) surface expression might point to a role in the mucosal or epithelial environment. Increased proportions of IL-5– and IL-13–producing γδ T cells are present in the bronchoalveolar lavage of patients with asthma (36). Accordingly, TGF-β, which is produced in response to allergen challenge (37), might drive the differentiation of local Vδ2 T cells into IL-9 producers. Furthermore, we found IL-9 production in Vδ2 T cells accompanied by elevated levels of CXCL13 and TNFSF13B (BAFF). CD4 Th9 cells can coexpress TNFSF13B (38), and CXCL13 can be induced in response to TGF-β (39). Both mediators might be relevant for the interaction with B cells. Because IL-9 promotes IgE production in B cells (40), Vδ2 T cells might thus contribute to the enhanced IgE production (e.g., by coproducing CXCL13, TNFSF13B, and IL-9) in asthma.

A second scenario where IL-9–producing γδ T cells might be highly relevant is tumor defense. Th9 cells themselves can exert enhanced antitumor activity (41), but IL-9 might also act indirectly via mast cells (8) or recruitment of dendritic cells to the tumor site (7). Significant numbers of γδ T cells are present among tumor-infiltrating lymphocytes (TILs) in many cancer types (42), and, in fact, the proportion of γδ T cells among TILs is the best predictive parameter across 25 human tumor entities (43). Many tumors secrete TGF-β, which could induce IL-9 production in tumor-infiltrating γδ T cells. In murine models, IL-9–producing CD8 cytotoxic T lymphocytes are superior effector cells in adoptive cancer immunotherapy, due to their extended lifespan (44, 45). Human Vδ2 T cells have raised great attention as effector cells in immunotherapy because of their broad reactivity toward many different tumors (46). Adoptive transfer of in vitro expanded Vδ2 T cells has already been performed, with promising results in some studies (47). We hypothesize that induction of potent IL-9 production upon in vitro expansion in the presence of TGF-β/IL-15 might also greatly improve the therapeutic efficacy of adoptively transferred Vδ2 T cells.

Materials and Methods

Blood Donors.

Leukocyte concentrates from 30 healthy adult donors (25 male and 5 female) with a mean age of 34.6 y were provided by the Institute of Transfusion Medicine, University Hospital Schleswig-Holstein. Informed consent was obtained from all blood donors. This study was approved by the Ethics Committee of Kiel University Medical Faculty (D402/14).

Cell Culture.

Pan-γδ T cells and Vδ2 T cells were positively isolated and CD4 and CD8 T cells were negatively isolated by magnetic sorting (Miltenyi Biotec). Cell culture was performed in serum-free X-VIVO 15 medium (Lonza) at 37 °C in a humidified atmosphere of 5% CO2 in air. A total of 40 × 103 pan-γδ T cells were stimulated in 96-well, round-bottomed plates in the presence of 50 × 103 irradiated (40 Gy) PBMC feeder cells with 300 nmol/L BrHPP (kindly provided by Innate Pharma). A total of 40 × 103 Vδ2 T cells were stimulated with A/E beads (Miltenyi Biotec), and CD4 and CD8 T cells were stimulated by plate-bound anti-CD3 and soluble anti-CD28, all in the absence of additional feeder cells. Cell cultures were supplemented with 50 IU/mL IL-2 or 10 IU/mL IL-2 (referred to as IL-2low) (Novartis), IL-15 (10 ng/mL), and TGF-β1 (1.7 ng/mL) (R&D Systems/Biotechne) as indicated. Where indicated, CD4 and CD8 T-cell cultures were additionally supplemented with 20 ng/mL IL-4. Fresh cytokines were also added on days 4 and 12, and on day 8, the cells were transferred to a 24-well plate into fresh medium supplemented with the respective cytokines. Additional information is provided in SI Materials and Methods.

Flow Cytometry.

Antibodies and techniques used for cell surface and intracellular staining are listed in SI Materials and Methods.

Measurement of Cytokine Secretion.

Cell culture supernatants were collected at day 8, and up to 16 analytes were measured using a Magnetic Luminex Screening Assay (R&D Systems/Biotechne) (additional information is provided in SI Materials and Methods).

mRNA Expression Analysis.

RNA was isolated using the RNeasy Mini-Kit (Qiagen). Total RNA was hybridized to an Affymetrix Human Gene 1.0 st v1 Array according to the manufacturer’s guidelines. Raw data were normalized using RMA (R; Bioconductor). Expression of selected genes was validated by quantitative RT-PCR. Experimental details and a list of used primers are provided in SI Materials and Methods.

Statistical Analysis.

For statistical analysis, Microsoft Excel 2007 and GraphPad Prism version 5.0 software were used.

Supporting Material.

Table S1 lists the x-fold change in gene expression of 12 genes in Vδ2 T cells activated under conditions (i) to (iv). Table S2 describes the PCR primers. Fig. S1 shows the modulation of Vδ2 T-cell expansion by TGF-β. Fig. S2 compares the gene regulation patterns of differentially expanded Vδ2 T cells. Fig. S3 depicts the gene expression measured by RT-PCR on day 15. Fig. S4 shows different intracellular mediators analyzed in expanded Vδ2 T cells. Fig. S5 shows the concentration of IL-9 detected in the supernatants of activated CD4 and CD8 T cells. Fig. S6 illustrates surface marker expression on differentially activated Vδ2 T cells. Fig. S7 shows the kinetics of transcription factor expression in differentially activated Vδ2 T cells.

Table S2.

List of primers used for RT-PCR and real-time PCR

Gene symbol Transcript variant Database accession no. Protein product Primer direction Primer
BCL6 Tv.1–3 NM_001130845.1 Bcl-6 Fw. AgTCCCCAACCAAgCTgA
NM_001134738.1 Rev. AgAgCCCgTCATggACCT
NM_001706.4
CCR7 NM_001838.3 CCR7 Fw. ggggAAACCAATgAAAAgC
Rev. ACCTCATCTTgACACAggCATA
EBI3 NM_005755.2 EBI3 Fw. CCTgCAgTggAAggAAAgg
Rev. AgggTCCAggAgCAATCC
Eomes (Not intron spanning) NM_005442.2 Eomes Fw. gCTCACTCTTCCCgTACCAg
Rev. CATggAgCCgTAggggTA
FOXP3 Tv.1–2 NM_014009.3 FoxP3 Fw. TCACCTACgCCACgCTCAT
NM_001114377.1 Rev. TCATTgAgTgTCCgCTgCTT
G6PDH Tv.1–2 NM_000402.4 G6PDH Fw. ACAgAgTgAgCCCTTCTTCAA
NM_001042351.2 Rev. ATAggAgTTgCgggCAAAg
GATA3 Tv.1–2 NM_001002295.1 GATA-3 Fw. CTCATTAAgCCCAAgCgAAg
NM_002051.2 Rev. TCTgACAgTTCgCACAggAC
IFNG NM_000619.2 IFN-γ Fw. TCAgCTCTgCATCgTTTTgg
Rev. gTTCCATTATCCgCTACATCTgAA
IKZF1 Tv.1 only NM_006060.5 Ikaros Fw. ACgCACAAATCCACATAACCT
Rev. CCACATTTgTgAggTTTACCAA
IKZF2 Tv.1–2 NM_016260.2 Helios Fw. ggAACgCTgCCACAACTATC
NM_001079526.1 Rev. TCCTTACAATCTTCCATAggAggTA
IKZF3 Tv.1–15 NM_001284515.1 Aiolos Fw. AgAggCCgAgTAgCCACAg
NM_001284514.1 Rev. CAgTTCCgCATTTgTTTgTATATC
NM_001257414.1
NM_001257413.1
NM_001257412.1
NM_001257411.1
NM_001257410.1
NM_001257409.1
NM_001257408.1
NM_183232.2
NM_183231.2
NM_183230.2
NM_183229.2
NM_183228.2
NM_012481.4
IKZF4 NM_022465.3 Eos Fw. ATACACCACCCgCACTCC
Rev. CAAgAAATCCggAACACACC
IKZF5 NM_022466.5 Pegasus Fw. gACTgTgACggTgACgAAgA
Rev. gAAAAgCCATACAgAggTCTCC
IL10 NM_000572.2 IL-10 Fw. gATgCCTTCAgCAgAgTgAA
Rev. gCAACCCAggTAACCCTTAAA
IL13 NM_002188.2 IL-13 Fw. AgCCCTCAgggAgCTCAT
Rev. TgATgCTCCATACCATgCTg
IL4 NM_172348.1 IL-4 Fw. gAACAgCCTCACAgAgCAgA
Rev. AggCAgCgAgTgTCCTTCT
IL5 NM_000879.2 IL-5 Fw. CACTgAAgAAATCTTTCAgggAAT
Rev. CCgTCTTTCTTCTCCACACTTT
IL9 NM_000590.1 IL-9 Fw. TggACATCAACTTCCTCATCA
Rev. TgCCCAAACAgAgACAACTg
ITGAE NM_002208.4 CD103 Fw. AggAACTTCTATgAAAAgTgTTTTgAg
Rev. CTgTCCCgAAggTCAAACTC
IRF4 Tv.1–2 NM_002460.3 IRF4 Fw. gCCAAgATTCCAggTgACTC
NM_001195286.1 Rev. CTggCTAgCAgAggTTCTACg
KLF3 NM_016531.5 KLF3 Fw. CCCCTTAATgAACTCAgTgTCC
Rev. gggATTCCACAggTAAAggTC
KLF7 NM_003709.2 KLF7 Fw. CAgCTTTACCATCCCTggAg
Rev. CCAAgTCCTCACCAAAggTC
SPI1 Tv.1–2 NM_003120.2 PU.1 Fw. CCACTggAggTgTCTgACg
NM_001080547.1 Rev. CTggTACAggCggATCTTCT
RPII NM_000937.4 RPII Fw. CAAgTTCAACCAAgCCATTg
Rev. CCAgCATAgTggAAggTATTCA
RPLP0 Tv.1–2 NM_001002.3 Hupo Fw. TCTACAACCCTgAAgTgCTTgAT
NM_053275.3 Rev. CAATCTgCAgACAgACACTgg
RORC Tv.2 only NM_001001523.1 ROR-γT Fw. AgAAggACAgggAgCCAAg
Rev. CAAgggATCACTTCAATTTgTg
SMAD3 NM_005902.3 SMAD3 Fw. gTCTgCAAgATCCCACCAg
Rev. AgCCCTggTTgACCgACT
TBX21 NM_013351.1 T-bet Fw. gACTCCCCCAACACAggAg
Rev. gggACTggAgCACAATCATC
TNFSF13B Tv.1 only NM_006573.4 Baff Fw. gACTgAAAATCTTTgAACCACCA
Rev. TTgCAAgCAgTCTTgAgTgAC
TGFB1 NM_000660.5 TGF-β1 Fw. gCACgTggAgCTgTACCA
Rev. AAgATAACCACTCTggCgAgTC
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Fw., forward; Rev., reverse; Tv., transcript variant.

SI Materials and Methods

Cell Culture.

PBMCs were separated by Ficoll–Hypaque density gradient centrifugation. PBMCs used in the present study comprised an average of 4.1% ± 1.1% γδ T cells, 0.3% ± 0.2% Vδ1 T cells, and 3.8% ± 1.3% Vδ2 T cells. Pan-γδ T cells were positively isolated from PBMCs by magnetic sorting (anti-TCRγ/δ MicroBead Kit; Miltenyi Biotec). Vδ2 T cells were positively isolated using the phycoerythrin (PE)-labeled anti-Vδ2 clone B6 in combination with anti-PE beads (Miltenyi Biotec). Optimized separation conditions [i.e., use of two consecutive magnetic activated cell sorting (MACS) columns] resulted in a purity of >98%. To avoid (pre)activation, purified T cells were cultured for 22 h at 37 °C. For the specific activation of Vδ2 T cells within total γδ T cells, Vδ2-specific BrHPP was used (48). A/E beads (Miltenyi Biotec) used for activation of isolated Vδ2 were coated with 10 μg/mL anti-CD3, 10 μg/mL anti-CD28, and 1 μg/mL anti-CD2 mAb. For stimulation, one bead per target cell was used (21). The purity of the BrHPP- and A/E bead-expanded Vδ2 T cells after 15 d of expansion was >98%. For stimulation of CD4 and CD8 T cells, plates were coated with anti-CD3 (clone OKT3; Janssen-Cilag) at 2 μg/mL, and soluble anti-CD28 (clone CD28.2; BD Biosciences) was added directly to the cell cultures at 1 μg/mL.

Determination of Cellular Expansion.

Cell proliferation was measured by uptake of 3H-radiolabeled thymidine. After restimulation of 20 × 103 differentially expanded Vδ2 T cells, the absolute cell number of viable Vδ2 T cells was measured after an additional 7 d of in vitro culture by a flow cytometric method termed standard cell dilution assay (SCDA) (49). In brief, cells from 96-well, round-bottomed plates were washed and stained with anti-TCRγδ–PE/Cy7 (clone 11F2; BD Biosciences) and anti-Vδ2–PE (clone B6; BD Biosciences). After one washing step, cells were resuspended in sample buffer containing a defined number of allophycocyanin-labeled fixed standard cells and 0.2 μg/mL propidium iodide. Based on the known number of standard cells, the absolute number of viable Vδ2 T cells in a given microculture was determined as previously described (21, 49).

Flow Cytometry.

In addition to mAb used for surface staining in SCDAs, the following mAbs were used for intracellular staining: anti-Granzyme B (clone GB11), anti–IFN-γ (clone 4S.B3), anti–IL-9 (clone MH9A3), anti–IL-13 (clone JES10-5A2), anti–TNF-α (clone 359-81-11), anti-Perforin (clone dG9), IgG1 (clone MOPC-21), and IgG2b (clone 27-35) isotype controls (all from BD Biosciences). For intracellular cytokine detection, cells were stimulated for 6 h with 20 ng/mL TPA (Sigma–Aldrich) and 1 μg/mL ionomycin (EMD Millipore/Calbiochem) as indicated; the cells were always treated with 3 μM monensin (EMD Millipore/Calbiochem) 4 h before fixation. Thereafter, cells were fixed and permeabilized using the Cytofix/Cytoperm/Permwash-Kit (BD Biosciences). For intracellular staining of transcription factors, we used the following mAbs: anti–Bcl-6 (clone K112-91), anti-FoxP3 (clone 259D), anti–GATA-3 (clone L50-823), and IgG1 (clone MOPC-21) isotype controls (all from BD Biosciences) and anti-Helios (clone 22F6), anti–T-bet (clone 4B10), anti-PU.1 (clone 7C6B05), and IgG (clone HTK888) isotype controls (all from Biolegend). For detection of transcription factors, cells were fixed and permeabilized using the transcription factor staining buffer set from Affymetrix/eBioscience. All samples were analyzed on a FACS-Fortessa flow cytometer (BD Biosciences) using BD FACSDiva Software v. 8. For further analysis, FlowJo software v. 10 was used.

Measurement of Cytokine Secretion.

A Magnetic Luminex Screening Assay (R&D Systems/Biotechne) was used to measure simultaneously up to 16 analytes (IL-1α, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-17, IL-22, IL-27, CXCL-13, CCL22, IFN-γ, TNF-α, and LIF) on a Luminex LX100 system. Data acquired using the Luminex xPonent 2.3/3.1 firmware (software) represent the median of the fluorescence intensity of the respective analytes. For each standard curve, a curve fit was applied according to the manufacturer’s manual. The sample concentrations were interpolated from the resulting regression equation. Each value was measured in experimental duplicates. For the detection of only IL-9 in supernatants, an IL-9 DuoSet ELISA (R&D Systems/Biotechne) was used.

Quantitative RT-PCR.

RNA was isolated using the RNeasy Mini-Kit (Qiagen). The RT of 250 ng of mRNA was performed at 37 °C for 1 h using 3 μg of random hexamer primers (Invitrogen/Thermo Fisher Scientific,), 25 nmol of dNTP (Bioline), 200 IU of Moloney murine leukemia virus (M-MLV) Reverse Transcriptase (Promega), and M-MLV Reverse Transcriptase Reaction Buffer (Promega) in a total volume of 20 μL in the presence of 20 IU of RNasin Plus RNase Inhibitor (Promega). The expression levels of the genes of interest in Vδ2 T cells activated under conditions (i) to (iv) were quantified by RT-PCR. To this end, 0.8 μL of cDNA and 10 pmol of a forward primer and a reverse primer were added to a reaction mix containing ImmoMix (Bioline), ROX Reference Dye (Invitrogen), SYBR Green (Invitrogen/Thermo Fisher Scientific), and 2.5 mM MgCl2 in a final volume of 20 μL. The quantitative RT-PCR assay was performed in three steps, with 15 s at 95 °C, 15 s at 60 °C, and 10 s at 70 °C. The gene expression was analyzed on an ICycler Thermal Cycler (582BR; Biorad) with an ICycler Optical Module (584BR; Biorad). The quality of the PCR products was controlled by melt curve analysis. A list of specific primers purchased from TIB Molbiol used for RT-PCR is provided in Table S2.

Transcriptome Analysis.

Total RNA isolated by an RNeasy Mini-Kit was processed as previously described (50) and hybridized to an Affymetrix Human Gene 1.0 st v1 Array according to the manufacturer’s guidelines. Raw data were normalized using RMA (R; Bioconductor). Transcripts that showed a median expression lower than the median expression of antigenomic background controls on the Affymetrix Human Gene 1.0 st v1 Array were categorized as not expressed, and thus excluded from further analysis. Transcripts were considered as differentially expressed when they were categorized as expressed and the fold change (based on the ratios of the medians) was >1.5 or <−1.5. A cluster analysis was performed using TIBCO Spotfire 6.5 by hierarchical clustering with correlation as a distance measure. The principal component analysis was performed using the same software, although only the two strongest components were displayed. All data were z-score–normalized before cluster analysis and principal component analysis.

Acknowledgments

We thank Ina Martens for expert technical assistance. This work was supported by the Else-Kröner-Fresenius Foundation (D.K.), a research grant from the Medical Faculty (to C.P.), and the Cluster of Excellence ExC 306 Inflammation-at-Interfaces project (D.K. and R.H.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. W.K.B. is a Guest Editor invited by the Editorial Board.

Data deposition: The data reported in this paper have been submitted to the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no GSE85482).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1607136113/-/DCSupplemental.

References

  • 1.Kaplan MH, Hufford MM, Olson MR. The development and in vivo function of T helper 9 cells. Nat Rev Immunol. 2015;15(5):295–307. doi: 10.1038/nri3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Devos S, et al. Allergen-induced interleukin-9 production in vitro: Correlation with atopy in human adults and comparison with interleukin-5 and interleukin-13. Clin Exp Allergy. 2006;36(2):174–182. doi: 10.1111/j.1365-2222.2006.02422.x. [DOI] [PubMed] [Google Scholar]
  • 3.Xie J, et al. Elevated antigen-driven IL-9 responses are prominent in peanut allergic humans. PLoS One. 2012;7(10):e45377. doi: 10.1371/journal.pone.0045377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Nalleweg N, et al. IL-9 and its receptor are predominantly involved in the pathogenesis of UC. Gut. 2015;64(5):743–755. doi: 10.1136/gutjnl-2013-305947. [DOI] [PubMed] [Google Scholar]
  • 5.Gerlach K, et al. TH9 cells that express the transcription factor PU.1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells. Nat Immunol. 2014;15(7):676–686. doi: 10.1038/ni.2920. [DOI] [PubMed] [Google Scholar]
  • 6.Licona-Limón P, et al. Th9 cells drive host immunity against gastrointestinal worm infection. Immunity. 2013;39(4):744–757. doi: 10.1016/j.immuni.2013.07.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Lu Y, et al. Th9 cells promote antitumor immune responses in vivo. J Clin Invest. 2012;122(11):4160–4171. doi: 10.1172/JCI65459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Purwar R, et al. Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells. Nat Med. 2012;18(8):1248–1253. doi: 10.1038/nm.2856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Chen N, Lu K, Li P, Lv X, Wang X. Overexpression of IL-9 induced by STAT6 activation promotes the pathogenesis of chronic lymphocytic leukemia. Int J Clin Exp Pathol. 2014;7(5):2319–2323. [PMC free article] [PubMed] [Google Scholar]
  • 10.Schmitt E, et al. IL-9 production of naive CD4+ T cells depends on IL-2, is synergistically enhanced by a combination of TGF-beta and IL-4, and is inhibited by IFN-gamma. J Immunol. 1994;153(9):3989–3996. [PubMed] [Google Scholar]
  • 11.Staudt V, et al. Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells. Immunity. 2010;33(2):192–202. doi: 10.1016/j.immuni.2010.07.014. [DOI] [PubMed] [Google Scholar]
  • 12.Dardalhon V, et al. IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+ IL-10+ Foxp3(-) effector T cells. Nat Immunol. 2008;9(12):1347–1355. doi: 10.1038/ni.1677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Chang HC, et al. The transcription factor PU.1 is required for the development of IL-9-producing T cells and allergic inflammation. Nat Immunol. 2010;11(6):527–534. doi: 10.1038/ni.1867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Tan C, et al. Phenotypes of Th lineages generated by the commonly used activation with anti-CD3/CD28 antibodies differ from those generated by the physiological activation with the specific antigen. Cell Mol Immunol. 2014;11(3):305–313. doi: 10.1038/cmi.2014.8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Hayday AC. [gamma][delta] cells: A right time and a right place for a conserved third way of protection. Annu Rev Immunol. 2000;18:975–1026. doi: 10.1146/annurev.immunol.18.1.975. [DOI] [PubMed] [Google Scholar]
  • 16.Gu S, Nawrocka W, Adams EJ. Sensing of pyrophosphate metabolites by Vγ9Vδ2 T cells. Front Immunol. 2015;5:688. doi: 10.3389/fimmu.2014.00688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Vantourout P, Hayday A. Six-of-the-best: Unique contributions of γδ T cells to immunology. Nat Rev Immunol. 2013;13(2):88–100. doi: 10.1038/nri3384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Bonneville M, O’Brien RL, Born WK. Gammadelta T cell effector functions: A blend of innate programming and acquired plasticity. Nat Rev Immunol. 2010;10(7):467–478. doi: 10.1038/nri2781. [DOI] [PubMed] [Google Scholar]
  • 19.Kabelitz D, Wesch D, Pitters E, Zöller M. Characterization of tumor reactivity of human Vγ9Vδ2 γδ T cells in vitro and in SCID mice in vivo. J Immunol. 2004;173(11):6767–6776. doi: 10.4049/jimmunol.173.11.6767. [DOI] [PubMed] [Google Scholar]
  • 20.Casetti R, et al. Cutting edge: TGF-beta1 and IL-15 Induce FOXP3+ gammadelta regulatory T cells in the presence of antigen stimulation. J Immunol. 2009;183(6):3574–3577. doi: 10.4049/jimmunol.0901334. [DOI] [PubMed] [Google Scholar]
  • 21.Peters C, Oberg HH, Kabelitz D, Wesch D. Phenotype and regulation of immunosuppressive Vδ2-expressing γδ T cells. Cell Mol Life Sci. 2014;71(10):1943–1960. doi: 10.1007/s00018-013-1467-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Brandes M, Willimann K, Moser B. Professional antigen-presentation function by human gammadelta T Cells. Science. 2005;309(5732):264–268. doi: 10.1126/science.1110267. [DOI] [PubMed] [Google Scholar]
  • 23.Ness-Schwickerath KJ, Jin C, Morita CT. Cytokine requirements for the differentiation and expansion of IL-17A- and IL-22-producing human Vgamma2Vdelta2 T cells. J Immunol. 2010;184(12):7268–7280. doi: 10.4049/jimmunol.1000600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Wesch D, Glatzel A, Kabelitz D. Differentiation of resting human peripheral blood gamma delta T cells toward Th1- or Th2-phenotype. Cell Immunol. 2001;212(2):110–117. doi: 10.1006/cimm.2001.1850. [DOI] [PubMed] [Google Scholar]
  • 25.Lafont V, Liautard J, Sable-Teychene M, Sainte-Marie Y, Favero J. Isopentenyl pyrophosphate, a mycobacterial non-peptidic antigen, triggers delayed and highly sustained signaling in human gamma delta T lymphocytes without inducing down-modulation of T cell antigen receptor. J Biol Chem. 2001;276(19):15961–15967. doi: 10.1074/jbc.M008684200. [DOI] [PubMed] [Google Scholar]
  • 26.Pont F, et al. The gene expression profile of phosphoantigen-specific human γδ T lymphocytes is a blend of αβ T-cell and NK-cell signatures. Eur J Immunol. 2012;42(1):228–240. doi: 10.1002/eji.201141870. [DOI] [PubMed] [Google Scholar]
  • 27.Turner J, Nicholas H, Bishop D, Matthews JM, Crossley M. The LIM protein FHL3 binds basic Krüppel-like factor/Krüppel-like factor 3 and its co-repressor C-terminal-binding protein 2. J Biol Chem. 2003;278(15):12786–12795. doi: 10.1074/jbc.M300587200. [DOI] [PubMed] [Google Scholar]
  • 28.Schwartzkopff S, et al. TGF-β downregulates KLRG1 expression in mouse and human CD8(+) T cells. Eur J Immunol. 2015;45(8):2212–2217. doi: 10.1002/eji.201545634. [DOI] [PubMed] [Google Scholar]
  • 29.Visekruna A, et al. Tc9 cells, a new subset of CD8(+) T cells, support Th2-mediated airway inflammation. Eur J Immunol. 2013;43(3):606–618. doi: 10.1002/eji.201242825. [DOI] [PubMed] [Google Scholar]
  • 30.Mohapatra A, et al. Group 2 innate lymphoid cells utilize the IRF4-IL-9 module to coordinate epithelial cell maintenance of lung homeostasis. Mucosal Immunol. 2016;9(1):275–286. doi: 10.1038/mi.2015.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Chen CY, et al. Induction of interleukin-9-producing mucosal mast cells promotes susceptibility to IgE-mediated experimental food allergy. Immunity. 2015;43(4):788–802. doi: 10.1016/j.immuni.2015.08.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Yu X, et al. Characteristics of γδ T cells in Schistosoma japonicum-infected mouse mesenteric lymph nodes. Parasitol Res. 2014;113(9):3393–3401. doi: 10.1007/s00436-014-4004-8. [DOI] [PubMed] [Google Scholar]
  • 33.Inami M, et al. CD28 costimulation controls histone hyperacetylation of the interleukin 5 gene locus in developing th2 cells. J Biol Chem. 2004;279(22):23123–23133. doi: 10.1074/jbc.M401248200. [DOI] [PubMed] [Google Scholar]
  • 34.Neurath MF, et al. The transcription factor T-bet regulates mucosal T cell activation in experimental colitis and Crohn’s disease. J Exp Med. 2002;195(9):1129–1143. doi: 10.1084/jem.20011956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Goswami R, et al. STAT6-dependent regulation of Th9 development. J Immunol. 2012;188(3):968–975. doi: 10.4049/jimmunol.1102840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Krug N, et al. Cytokine profile of bronchoalveolar lavage-derived CD4(+), CD8(+), and gammadelta T cells in people with asthma after segmental allergen challenge. Am J Respir Cell Mol Biol. 2001;25(1):125–131. doi: 10.1165/ajrcmb.25.1.4194. [DOI] [PubMed] [Google Scholar]
  • 37.Torrego A, Hew M, Oates T, Sukkar M, Fan Chung K. Expression and activation of TGF-beta isoforms in acute allergen-induced remodelling in asthma. Thorax. 2007;62(4):307–313. doi: 10.1136/thx.2006.063487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Jabeen R, et al. Th9 cell development requires a BATF-regulated transcriptional network. J Clin Invest. 2013;123(11):4641–4653. doi: 10.1172/JCI69489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Kobayashi S, et al. TGF-β induces the differentiation of human CXCL13-producing CD4(+) T cells. Eur J Immunol. 2016;46(2):360–371. doi: 10.1002/eji.201546043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Dugas B, et al. Interleukin-9 potentiates the interleukin-4-induced immunoglobulin (IgG, IgM and IgE) production by normal human B lymphocytes. Eur J Immunol. 1993;23(7):1687–1692. doi: 10.1002/eji.1830230743. [DOI] [PubMed] [Google Scholar]
  • 41.Miao BP, et al. Inhibition of squamous cancer growth in a mouse model by Staphylococcal enterotoxin B-triggered Th9 cell expansion. Cell Mol Immunol. 2015 doi: 10.1038/cmi.2015.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Lo Presti E, Dieli F, Meraviglia S. Tumor-infiltrating γδ T lymphocytes: Pathogenic role, clinical significance, and differential programing in the tumor microenvironment. Front Immunol. 2014;5:607. doi: 10.3389/fimmu.2014.00607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Gentles AJ, et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat Med. 2015;21(8):938–945. doi: 10.1038/nm.3909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Lu Y, et al. Tumor-specific IL-9-producing CD8+ Tc9 cells are superior effector than type-I cytotoxic Tc1 cells for adoptive immunotherapy of cancers. Proc Natl Acad Sci USA. 2014;111(6):2265–2270. doi: 10.1073/pnas.1317431111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Lu Y, Wang Q, Yi Q. Anticancer Tc9 cells: Long-lived tumor-killing T cells for adoptive therapy. Oncoimmunology. 2014;3:e28542. doi: 10.4161/onci.28542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Kabelitz D, Kalyan S, Oberg HH, Wesch D. Human Vδ2 versus non-Vδ2 γδ T cells in antitumor immunity. Oncoimmunology. 2013;2(3):e23304. doi: 10.4161/onci.23304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Buccheri S, Guggino G, Caccamo N, Li Donni P, Dieli F. Efficacy and safety of γδT cell-based tumor immunotherapy: A meta-analysis. J Biol Regul Homeost Agents. 2014;28(1):81–90. [PubMed] [Google Scholar]
  • 48.Espinosa E, et al. Chemical synthesis and biological activity of bromohydrin pyrophosphate, a potent stimulator of human gamma delta T cells. J Biol Chem. 2001;276(21):18337–18344. doi: 10.1074/jbc.M100495200. [DOI] [PubMed] [Google Scholar]
  • 49.Pechhold K, Pohl T, Kabelitz D. Rapid quantification of lymphocyte subsets in heterogeneous cell populations by flow cytometry. Cytometry. 1994;16(2):152–159. doi: 10.1002/cyto.990160209. [DOI] [PubMed] [Google Scholar]
  • 50.Häsler R, et al. A functional methylome map of ulcerative colitis. Genome Res. 2012;22(11):2130–2137. doi: 10.1101/gr.138347.112. [DOI] [PMC free article] [PubMed] [Google Scholar]

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