The Role And Mechanism Of TNF-α In The Induction Of Th9 Cells

Objective:Immunotherapy is a promising approach for treating malignancy. Cellular immunity plays a pivotal role in tumor immunotherapy. Antitumor effector T cells include CD4⁺ T helper cells and CD8⁺ cytolytic T cells. Th9 is a latest described Th subset defined by the secretion of IL-9. We and others recently found that Th9/IL-9 mediated potent antitumor effects in vivo, which is superior to other Th subsets. Therefore, there will be significant clinical benefit to explore efficient strategies to induce and expand more Th9 cells. OX40/OX40 L was once considered as the unique inducer of Th9 cells in TNF/TNF receptor family members. Recently, TL1 A and GITR have also been reported to be powerful inducers of Th9 cells, suggesting that other TNF family members, such as TNF-α, may also favor the induction of Th9 cells and promote Th9/IL-9 antitumor properties. Therefore, the purpose of this study is to investigate the roles and mechanisms of TNF-α in promoting Th9 cells differentiation, which may provide new strategies for tumor immunotherapy. Methods:To determine whether TNF-α has any role in Th9 polarization, na?ve CD4⁺ T cells(CD4⁺ CD25^- CD62L^hi) were sorted by flow cytometry. And then cells were activated in the presence or absence of TNF-α under Th9 conditions（anti-CD3/CD28 antibody and TGF-β/IL-4） for induction of Th9 cells. q PCR, ELISA and/or flow cytometry were used to detect the expression of IL-9 m RNA and IL-9 protein in Th9 cells, and also detect cell survival and cell ability of Th9 cells. TNF-α receptors include TNFR1 and TNFR2, so we examined the expression of TNFR1 and TNFR2 in na?ve CD4⁺ T cells by flow cytometry. TNFR1 or TNFR2 blocking antibody was added under Th9 conditions, and then we investigated whether blockade of TNFR1 or TNFR2 could inhibit the induction of Th9 cells by TNF-α. The induction of Th9 cells by TNF/TNF receptor family members, OX40/OX40 L, TL1 A and GITR, are dependent on NF-κB or STAT5 signaling pathways. To determine whether TNF-α activates NF-κB or STAT5 signaling, we used Western Blot to detect phosphorylation of STAT under Th9 conditions. STAT5 inhibitor or NF-κB inhibitor was added to detect whether TNF-α could promote the induction of Th9 cells. Luciferase reporter system assay was performed to further confirm whether NF-κB molecules could bind to and activate the Il9 promoter. Results:TNF-α significantly promoted the differentiation and survival of Th9 cells, and also enhanced Th9 cells activation. Th9 cells induced by TNF-α did not express Th9 related transcription factors Pu.1 and Irf4, and most of Th1-, Th2- and Th17-related transcription factors and cytokines, although Il5 and Il13 was slightly increased. Na?ve CD4⁺ T cells expressed both TNFR1 and TNFR2, blockade of TNFR2 significantly inhibited the induction of Th9 cells by TNF-α, while blockade of TNFR1 could not. TNF-α could activate STAT5, and inhibition of STAT5 inhibited the induction of Th9 cells by TNF-α. Moreover, inhibition of NF-κB suppressed the development of Th9 cells by TNF-α. NF-κB molecules could bind to and activate the Il9 promoter, and then promoted IL-9 expression. Conclusion:TNF-α promotes the differentiation of Th9 cells through TNFR2/STAT5 and TNFR2/NF-κB signaling pathways.