The Role Of IL-24 In Th9 Cell-mediated Antitumor Immunotherapy

Background:Malignant tumor is one of the most urgent problems to be overcome by mankind. Cellular adoptive immunotherapy is a promising method of tumor immunotherapy.Th9 cell is a novel CD4⁺T cell subset characterized by high expression of interleukin（IL）-9. Recently,adoptive immunotherapy with Th9 cells has shown potent antitumor properties in a variety of tumors,including colon cancer,breast cancer,lung adenocarcinoma and melanoma.Therefore,Th9 cells may represent promising effector cells for tumor therapy and exploring the anti-tumor mechanism of Th9 cells contributes to promote the clinical transformation of Th9 cells.At present,studies have shown that IL-9 derived from Th9 cells is identified as a T cell growth factor and is shown to promote T cell proliferation and survival.Th9 cells possess an effector profile with less exhaustion and longer longevity,which may contribute to the antitumor efficacy of Th9 cells.Th9 cell-derived IL-3 prolongs the survival of dendritic cells（DCs）which contribute to the induction of tumor-specific cytotoxic T lymphocytes（CTLs）.Th9 cells secrete granzyme（Gzm）A and Gzm B,which may mediate the tumor cytotoxicity of Th9 cells.However,the key mechanism of Th9 cell-mediated anti-tumor activity remains unclear.We sequenced and analyzed the eukaryotic transcriptome of Th9 cells in order to explore the key antitumor mechanisms of Th9 cells.We found that Th9 cells express high levels of not only IL-9,but also IL-24.IL-24 is a member of IL-10 gene family.IL-24 has two heterodimeric receptors,IL-20R1/IL-20R2 and IL-22R1/IL-20R2.And IL-24 has two molecular forms:an intracellular form and a secreted form.Intracellular IL-24 plays a tumor suppressive role in various types of tumors by inducing cell apoptosis and autophagy, inhibiting angiogenesis and tumor invasion,and sensitizing tumor cells to radiation therapy and chemotherapy.Secreted IL-24 significantly inhibits the proliferation of tumor cells and induced apoptosis of tumor cells.Based on these observations,this study will explore the role of IL-24 in Th9 cell-mediated antitumor activity,in order to explore the new key effector molecules for Th9cell immunotherapy.Our study is mainly divided into the following four parts:Part 1 Expression of IL-24 in Th9 cellsObjective:To explore the high level of cytokines expressed in Th9 cells.Methods:（1）Na（?）ve CD4⁺T cells were sorted from the spleen of C57BL/6 mice and induced to generate Th0 and Th9 cells in vitro,then analyzed the up-regulated cytokine gene expression in Th9 cells by RNA sequencing（RNA-Seq）;（2）The m RNA and protein levels of cytokines expressed at high levels in Th9 cells were detected by Real-time Quantitative polymerase chain reaction（q PCR）,enzyme linked immunosorbent assay（ELISA）and Western Blot;（3）The expression of the cytokines at different time points was detected by q PCR and ELISA.（4）The expression of IL-24 in other cell subtypes was detected by q PCR.Results:（1）Compared with Th0 cells,Th9 cells expressed IL-24 at a higher level;（2）q PCR,ELISA and FCM confirmed that the m RNA and protein levels of IL-24 were higher in Th9 cells than in Th0 cells;（3）Compared with Th0 cells,the expression of IL-24 and IL-9in Th9 cells showed a similar temporal pattern and continued to be highly expressed.（4）IL-24 was expressed in both Th2 and Th9 cells,but was highly expressed in Th9 cells.Conclusion:IL-24 is highly expressed in Th9 cells.Part 2 Effect of IL-24 on Th9 cell differentiation,proliferation and survivalObjective:To explore the role of IL-24 in Th9 cell differentiation and survival.Methods:（1）Na（?）ve CD4⁺T cells were obtained from wildtype（WT）and Il24 knockout(Il24^-/-)mice and cultured under Th9-polarizing conditions,RNA-seq analysis were performed;（2）The levels of IL-9 expression in Th9 cells and Il24^-/Th9 cells were detected by q PCR and ELISA;（3）The proliferation and survival of Th9 cells and Il24^-/Th9 cells in vitro were detected by Flow Cyto Metry（FCM）;（4）The number of cells cultured in vitro was recorded;（5）The IL-24 receptor of Th9 cells was blocked,and the proliferation and survival of different groups of cells were detected by FCM;（6）Th9 cells or Il24^-/Th9 cells were labeled with CFSE and adoptively transferred to C57BL/6 mice.CFSE-stained Th9cells from spleen,lymph nodes（LNs）and lungs were analyzed by FCM at different time points.Results:（1）The knockout of IL-24 in Th9 cells had no effects on the expression of Th9-related transcription factors,such as Spi1,Irf4,Stata5,Irf1 or Foxo1.However,knockout of IL-24 in Th9 cells induced differential expression of genes:Ccnd3,Il10,Erdr1,Slfn and Lsp1;（2）Il24^-/-Th9 cells had lower expression of IL-9 compared to regular Th9 cells;（3）knockout of IL-24 enhanced Th9 cell proliferation,but also increased Th9 cell apoptosis in vitro;（4）Knockout of IL-24 in Th9 cells decreased the number of Th9 cells in vitro;（5）Blocking IL-24 receptor inhibited Th9 cell proliferation and promotes Th9 cell apoptosis in vitro;（6）Knockout of IL-24 in Th9 cells decreased the survival of Th9 cells in vivo.Conclusion:IL-24 contributes to the survival of Th9 cells.Part 3 Effect of IL-24 on antitumor immunotherapy of Th9 cellsObjective:To explore the effect of IL-24 on antitumor efficacy of Th9 cells in vitro and in vivo.Methods:（1）WT or Il24^-/-Th9 cells were co-cultured with multiple myeloma（MM）cells and separated by Transwell inserts,and tumor cell proliferation and apoptosis were detected by FCM;（2）The IL-24 receptor of MM cells was blocked,and Th9 cells were isolated and co-cultured with MM cells through Transwell.The proliferation and survival of different groups of MM cells were detected by FCM.（3）C57BL/6 mice were injected intravenously with B16-OVA melanoma cells and followed by the adoptive transfer of Th9cells cultured from OT-II or OT-II-Il24^-/-mice.Tumor lung metastasis was examined;（4）C57BL/6 mice were injected subcutaneously with B16-OVA cells and followed by the adoptive transfer of Th9 cells cultured from OT-II or OT-II-Il24^-/-mice.The development of the tumor was monitored over time.（5）CFSE-labeled OT-II or OT-II-IL24^-/-Th9 cells were adopted into C57BL/6 mice via the tail vein.FCM and q PCR were used to detect the potential anti-tumor factors in vivo.Results:（1）The deficiency of IL-24 abrogated Th9-induced inhibition of tumor cell proliferation and survival;（2）After blocking IL-24 receptor of MM cells,the anti-tumor effect of Th9 cells was decreased in vitro;（3）The knockout of IL-24 largely abrogated Th9-induced inhibition of tumor lung metastasis;（4）The deficiency of IL-24 in Th9 cells largely abrogated Th9-induced inhibition on tumor growth;（5）OT-II-Il24^-/-Th9 cells did not change the proportion of CD8⁺T cells and the expression of Gzm B in mice.Conclusion:IL-24 promotes the antitumor effect of Th9 cells.Part 4 Effect of Foxo1 on expression of IL-24 in Th9 cellsObjective:To explore the mechanism of IL-24 expression in Th9 cells.Methods:（1）The data of RNA-seq described in Part I（1）were analyzed again to find the differentially expressed genes of transcription factors;（2）q PCR further verified the expression levels of these transcription factors in Th9 and Il24^-/-Th9 cells;（3）Foxo1inhibitor（Foxo1i）was added during Th9 cell differentiation.The expression levels of IL-9and IL-24 and the proliferation and survival of Th9 cells in vitro were detected by q PCR.（4）C57BL/6 mice were injected i.v.with B16-OVA melanoma cells and followed by the adoptive transfer of Th9 or Foxo1i-Th9 cells from OT-II mice.Tumor lung metastasis was examined;（5）C57BL/6 mice were injected s.c.with B16-OVA cells and followed by the adoptive transfer of Th9 or Foxo1i-Th9 cells cultured from OT-II mice.The development of the tumor was monitored over time;（6）Adoptive immunotherapy with Foxo1i-Th9 cells was performed on B16-OVA tumor-bearing mice,while recombinant IL-24 was administered and tumor growth was monitored.Results:（1）We found 14 up-regulated and 4 down-regulated transcription factors in Th9cells compared to Th0 cells;（2）QPCR analysis further confirmed that the expression of Erg,Npas2,Ahr,Foxo1,Elk3,Stat5b and Runx1 was increased in Th9 and Il24^-/-Th9 cells;（3）Foxo1i treatment decreased the expression of both Il9 and Il24 in Th9 cells.Furthermore,Foxo1i treatment increased Th9 cell proliferation,but had minor effects on Th9 cell apoptosis;（4）Foxo1i-Th9 cells increased the lung tumor burden in mice as compared with regular Th9 cells;（5）Foxo1i-Th9 cells exhibited less inhibition on B16-OVA tumor growth as compared to regular Th9 cells;（6）The addition of IL-24 partially restored the antitumor efficacy of Foxo1i-Th9 cells.Conclusion:Foxo1 is the transcription factor which contributes to the expression of IL-24 in Th9 cells.