Human Vδ2 T cells are a major source of interleukin-9 Christian Petersa, Robert Häslerb, Daniela Wescha, and Dieter Kabelitza,1 aInstitute of Immunology, University Hospital Schleswig-Holstein Campus Kiel, D-24105 Kiel, Germany; and bInstitute of Clinical Molecular Biology, University Hospital Schleswig-Holstein Campus Kiel, D-24105 Kiel, Germany Edited by Willi K. Born, National Jewish Health, Denver, CO, and accepted by Editorial Board Member Philippa Marrack September 12, 2016 (received for review May 13, 2016) Vδ2Vγ9 T cells are the dominant γδ T-cell subset in human periph- Vδ2 T cells produce different cytokines, can be converted into + eral blood. Vδ2 T cells recognize pyrophosphate molecules derived forkhead box protein P3 (FoxP3) regulatory cells (20, 21), and from microbes or tumor cells; hence, they play a role in antimicro- may acquire professional antigen-presenting capacity (22). Depend- bial and antitumor immunity. TGF-β, together with IL-15, induces ing on priming conditions, Vδ2 T cells can secrete IFN-γ,IL-4,IL- a regulatory phenotype in Vδ2 T cells, characterized by forkhead 17, and IL-22, and thus recapitulate cytokine patterns of well-defined box protein P3 (FoxP3) expression and suppressive activity on CD4 functional Th subsets (23, 24). T-cell activation. We performed a genome-wide transcriptome Here, we identified peripheral blood Vδ2Tcellsasamajorsource analysis and found that the same conditions (TGF-β plus IL-15) of IL-9. In the presence of TGF-β and IL-15 but the absence of IL-4, strongly enhanced the expression of additional genes in Vδ2T high levels of IL-9 were secreted already 4 d after initial in vitro ac- cells, including IKAROS family zinc finger 4 (IKZF4; Eos), integrin tivation. Upon antigen restimulation of Vδ2 T cells precultured for subunit alpha E (ITGAE; CD103/αEβ7), and IL9. This up-regulation 15 d in the presence of TGF-β/IL-15, IL-9 was by far the most abun- was associated with potent IL-9 production as revealed by flow dant among 16 analyzed cytokines and chemokines. Our results might cytometry and multiplex analysis of cell culture supernatants. In help to enhance selective effector functions of human γδ Tcells. 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 restim- Results ulation of Vδ2 T cells expanded in vitro in the presence of TGF-β and To investigate the impact of differential activation on the gene IL-15, IL-9 was the most abundant among 16 analyzed cytokines and expression profile and the transcription factor and cytokine/che- chemokines. IL-9 is a pleiotropic cytokine involved in various (patho) mokine pattern of Vδ2 T cells, the following four conditions were physiological conditions, including allergy and tumor defense, used for in vitro culture (note: exogenous IL-2 was always pre- where it can promote antitumor immunity. Given the conspicuous sent). Purified total γδ T cells were stimulated with the Vδ2-spe- δ – sensitivity of many different tumors to V 2T-cell mediated killing, cific phosphoantigen bromohydrin pyrophosphate (BrHPP) in the conditions defined here for strong induction of IL-9 might be the presence of irradiated peripheral blood mononuclear cells δ – relevant for the development of V 2T-cell based immunotherapy. (PBMCs) and in the absence (i)orpresence(iii)ofTGF-β/IL-15. Alternatively, purified Vδ2 T cells were stimulated with micro- γ/δ T cells | human | interleukin-9 | transforming growth factor-β beads coupled with anti-CD2/CD3/CD28 antibodies [activation/ expander (A/E) beads], again in the absence (ii)orpresence(iv) lthough less well characterized than other functional T of TGF-β/IL-15 (21). Ahelper-cell subsets, T helper 9 (Th9) cells have been identified on the basis of their selective IL-9 production. IL-9 is a pleiotropic Gene Expression of Differentially Activated Vδ2 T Cells. In the cytokine that promotes T-cell and mast-cell growth and mast-cell presence of TGF-β, the proliferative activity of freshly isolated accumulation in tissue as well as IgE switching in B cells (1). In γδ T cells as measured by 3H-radiolabeled thymidine in- line, allergic patients have increased numbers of T cells that pro- corporation was reduced (Fig. S1A). Interestingly, however, the duce IL-9 in response to various allergens (2, 3). Moreover, Th9 cells alter intestinal epithelial cell functions (4, 5) and are key Significance players in antiworm immunity (6). Intriguingly, Th9 cells also ap- pear to regulate tumor immunity. Although IL-9 and Th9 cells We describe in vitro cell culture conditions that induce strong exerted antitumor activity in some solid tumor models (7, 8), IL-9 secretion of IL-9 in human peripheral blood γδ Tcells.IL-9playsa actually promoted lymphoma development in other models (9). role in allergy and increases the antitumor immunity of con- IL-9 is thus an important cytokine with multiple functions in the ventional CD4 and CD8 T cells. Human γδ TcellswithaVδ2 T-cell regulation of immune responses. receptor kill many different tumor cells because they recognize Similar to other functional T-cell subsets, the differentiation of intermediates of a metabolic pathway that is frequently dysre- Th9 cells is driven by the cytokine milieu and specific transcription gulated in cancer cells. Vδ2 T cells have already been used in factors. In general, IL-4, together with TGF-β,wasfoundtopo- cancer immunotherapy, as yet with limited success. Our study larize IL-9–secreting CD4 T cells (10–12), and PU.1 and interferon demonstrates that TGF-β, together with IL-15, strongly enhances regulatory factor 4 (IRF4) were identified as crucially important IL-9 production in Vδ2 T cells. We postulate that IL-9–producing transcription factors (11, 13). The signal strength of T-cell receptor Vδ2 T cells might have enhanced therapeutic efficacy upon (TCR) ligation and costimulatory signals also regulate IL-9 at the adoptive transfer into patients who have cancer. transcriptional level (14). γδ δ γ The T cells expressing V 2pairedwithV9 (hereafter Author contributions: D.W. and D.K. designed research; C.P. and R.H. performed research; termed Vδ2 T cells) dominate in the peripheral blood of healthy C.P., R.H., D.W., and D.K. analyzed data; and C.P. and D.K. wrote the paper. δ adult individuals (15). V 2 T cells recognize via their TCR, in a The authors declare no conflict of interest. CD277/butyrophilin 3A-dependent manner, microbial pyrophos- This article is a PNAS Direct Submission. W.K.B. is a Guest Editor invited by the Editorial phates and homologous eukaryotic pyrophosphates (isopentenyl Board. pyrophosphate), which are produced by many tumor cells due to Data deposition: The data reported in this paper have been submitted to the Gene their dysregulated mevalonate pathway (16). Hence, Vδ2Tcells Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no GSE85482). play a role in both antiinfective and antitumor immunity (17, 18). 1To whom correspondence should be addressed. Email: [email protected]. δ Interestingly, V 2 T cells exert a surprisingly large functional This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. plasticity (17). In addition to their strong cytolytic activity (19), 1073/pnas.1607136113/-/DCSupplemental. 12520–12525 | PNAS | November 1, 2016 | vol. 113 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1607136113 Downloaded by guest on September 24, 2021 proliferative expansion of Vδ2 T cells that had been cultured for in Vδ2 T cells cultured for 8 d (Fig. 2) or 15 d (Fig. S3) under 15 d under conditions (i)to(iv) and restimulated with BrHPP was conditions (i)to(iv). Among the genes specifically up-regulated in much higher if TGF-β and IL-15 had been present during initial the presence of TGF-β/IL-15 were FOXP3 (Fig. 2A and Fig. S3A), activation [i.e., conditions (iii)and(iv)] (Fig. S1B). We then an- IKAROS family zinc finger 4 (IKZF4; Eos) (Fig. 2B and Fig. S3B), alyzed gene expression by means of an Affymetrix whole-genome IL9 (Fig. 2C), TNF superfamily member 13b (TNFSF13B;Bcell array in γδ T cells cultured for 8 d under conditions (i)to(iv). The activating factor of the TNF family, BAFF) (Fig. S3C), integrin principal component analysis depicted in Fig. 1A indicates that the subunit alpha E (ITGAE; CD103) (Fig. 2D and Fig. S3D), and overall gene expression in Vδ2 T cells cultured with TGF-β/IL-15 IRF4 (Fig. S3D). Interestingly, IL9 was reduced on day 15 com- clustered differently from those Vδ2 T cells cultured without pared with day 8 (Fig. 2C and Fig. S3C). Other cytokine genes, in- TGF-β/IL-15. Moreover, BrHPP stimulation [i.e., conditions cluding IL13,Epstein–Barr virus induced 3 (EBI3), and interferon, (i) and (iii)] clearly differed from A/E bead stimulation [i.e., gamma (IFNG)(Fig.2C and Fig. S3C), were up-regulated under all conditions (ii)and(iv)]. The heat map based on hierarchical conditions, although IL13 and EBI3 were more strongly up-regulated clustering of 1,056 genes found to be regulated at least 1.5-fold in in response to A/E bead stimulation. Kruppel-like factor 7 (KLF7) condition (iv) compared with unstimulated Vδ2 T cells is shown in was up-regulated only under condition (iv)(Fig.2B and Fig. S3B). Fig. 1B. Selected genes with profound modulation are identified Among the genes that were generally down-regulated were in Fig. 1B. The median fold change of these genes induced under T-box 21 (TBX21;T-bet),EOMES, SPI1 (PU.1) (Fig. 2A and Fig.