The Role Of Transcription Factor T-bet/Eomes And IL-33in Regulating The Anti-tumor Effect Of CD8+Memory T Cells And Effector T Cells

Anti-tumor immune responses are mainly mediated by T cells, the type1cellularimmune response mediated by CD8+CTLs and CD4+Th1cells is the most importantamong those. T-bet and Eomes are known to regulate differentiation and effectorfunctions of Th1and CD8+T cells. Ample clinical evidence shows that longer survivalof cancer patients is associated with increased expression of genes characteristic of type1effector T cells, in particular master transcription regulators T-bet and Eomes. T-betand Eomes have been shown to be important for antitumor effects of CD8+T cells, butthe mechanism is less understood. Immunological memory is one of the most importantcharacteristics of adaptive immune response. Within the overall memory T cellpopulation, at least three distinct subpopulations have been described: effector memoryT cell(T_EM), central memory T cell(T_CM) and T memory stem cells(T_SCM). Besides theirrole in promoting effector functions, T-bet and Eomes are known to be involved in bothfunction and homeostasis of effector and memory T cells. We are interested ininvestigating whether T-bet and Eomes regulate T_SCM, another important subset ofmemory T cells. Although T-bet and Eomes are known to enhance effector functions ofTh1and CD8+T cells, but how is this achieved in the inflammatory environment is lessclear. The effector functions of CD8+T cells are influenced by tissue inflammatorymicroenvironments. IL-33, a member of the IL-1family, acts as a danger signal after itsrelease during cell necrosis. We have found T-bet and Eomes upreguate IL-33receptor（called ST2） in effector CD8T cells. We hypothesize that T-bet and Eomes enhanceeffector CD8T cell function through upregulating IL-33/ST2axis in the inflammatorytissues. To address these questions, therefore we carried out the following studies: Part â… T-bet and Eomes regulate the balance between the effector/centralmemory T cells versus memory stem like T cells and theiranti-tumor effectObjective: To study the role of T-bet and Eomes in regulating the function ofadoptively transferred T cells in tumor-bearing hosts as well as in the generation oftumor-specific memory T cell subsets upon adoptive transfer.Methods: By using adoptive T cell therapy in our animal model, B6-LY5.2/Crmice were challenged with3×10⁵B16F0cells i.d.6days later, mice were irradiated at500rad. On day7, the mice were adoptively transferred with5×10⁵WT, T-bet-/-,Eomes-/-, or T-bet/Eomes DKO pmel-1T cells which have been cultured in Th1condition for3days. Tumor growth was monitored every two days. To examine theprophylactic function of adoptive transfer T cells, B6-LY5.2/Cr mice were irradiated at500rad.24h post irradiation, the mice were adoptively transferred with4×10⁶WT,T-bet-/-, Eomes-/-, or T-bet/Eomes DKO pmel-1T cells which have been cultured inTh1conditions for4days.1month later, the recipient mice were challenged with2×10⁵B16F0cells i.d. Spleen, lymph node and Tumor masses were harvested from mice.Cells were purified for further f^low cytometric analysis on surface marker such as CD44,CD62L as well as IFN_-γ and IL-17production.Result:（1） Tumor growth was inhibited in mice infused with WT, TKO or EKOpmel-1T cells. In constrast, adoptively transferring DKO pmel-1T cells failed to inhibitthe tumor growth;（2）20days post T cell transferred to tumor-bearing mice，thepercentage of adoptively transferred TKO T cells was increased significantly in lymphnodes compared to WT T cells. Either T-bet or Eomes deficiency resulted in modest butstatistically significant reduction in the percentage of the donor T cells among the TILs.T-bet and Eomes double deficiency greatly reduced the percentage of donor T cells intumor tissues. Compared with WT adoptively transferred T cells, the frequency ofIFN_-γ+cells modestly reduced in EKO T cells. T-bet deletion resulted in^lowerfrequency of IFN_-γ+cells. The combined deficiency of T-bet and Eomes led to greatestreduction of the frequency of IFN_-γproducers;（3）30days after infusion, the percentage of CD44^lowmemory cells was greatly increased in DKO pmel-1T cells compared with Tcells of other genotypes. These cells was CD44^lowCD62L^highSca-1+T cells which weresimilar to the phenotype of memory stem T cells;（4） We found the mice infused withWT, TKO or EKO pmel-1T cells were protected from a re-challenge with tumor cells.In contrast, mice receiving DKO T cells succumbed to the challenge with B16cells andgrew tumor;（5）There’s no difference in the proliferation potential in recall responsesamong WT, TKO, EKO and DKO memory T cells, suggested the effector functionsrather than the proliferative potential are controlled by T-bet and Eomes in memory Tcells.Conclusion: In this study, we showed T-bet and Eomes are required for adoptivetumor immunotherapy in mice and we also demonstrated that T-bet and Eomes areimportant for central and effector memory cells, but non-essential for generation andpersistence of stem cell like memory CD8T cells. Our data suggest T-bet and Eomesmay regulate the balance between the effector/central memory T cells versus memorystem like T cells.Part â…¡T-bet-dependent IL-33/ST2axis synergizes IL-12signaling topromote the effector function of CD8+T cellsObjective： To study the role of IL-33/ST2in driving effector function of CD8+Tcells.Methods: Lymphocytes were collected from spleens and lymph nodes obtainedfrom C57BL/6WT, Tbet/（TKO）, Eomes/（EKO）, T-bet/Eomes doubly deficient（DKO） mice. Na ve CD62L+CD44CD8+T cells were purified by FACS or magneticbeads based methods. The na ve CD8T cells are cultured in Tc1, Tc0, Tc2, and Tc17conditions. Cells were stimulated with plate-bound anti-CD3and anti-CD28mAbs incomplete RPMI. Alternatively, naive CD8+T cells were cultured with anti-CD3in thepresence of T cell–depleted cell cycle–arrested splenocytes as antigen-presenting cells.Naive Pmel-1TCR transgenic CD8+T cells were cultured with1μ M gp10025-33inthe presence of antigen presenting cells for4days in various polarizing conditions.F^low cytometric analysis was performed using a FACS f^low cytometer and Real-Time PCR was performed with SYBR green kit to investigate the expression of ST2ineffector CD8+T cells, IFN_-γ production, mRNA levels of IFN_-γ as well as other genes.Result:（1） In this study, we found the ST2mRNA could be highly induced inCD8+T cells cultured in Tc1condition. CD8+T cells cultured in Tc2condition showedminimum ST2expression compared to those cultured in other conditions. TCRsignaling could further increased levels of ST2mRNA in Tc1cells;（2） T-bet isprimarily required for ST2expression in Tc1cells and Eomes seems not involved inregulating ST2mRNA;（3） IL-33synergizes with TCR signaling in the induction ofIFN_-γ production in Tc1cells. Furthermore, we also found IL-33synergizes with TCRsignaling and IL-12in promoting IFN_-γ production, and T-bet is required for IFN_-γproduction driven by IL-12plus IL-33;（4） With further investigation, we found thatIL-12plus IL-33significantly up-regulate IFN_-γat the mRNA level, as well as T-bet andBlimp1and down-regulate TCF-1and LEF-1mRNAs in Tc1Cells;（5） Gadd45bdeficiency reduced levels of detectable active p38in Tc1cells and also dampenedIL-12/IL-33induced IFN_-γ mRNA. In addition, p38inhibitor almost completelyabolished IFN_-γproduction.Conclusion: In our study, we found IL-33receptor ST2is highly expressed ineffector Tc1cells and regulated by T-bet. Our study demonstrated that IL-33canpromote Tc1immune response in Gadd45b/p38-dependent manner.