The Critical Role Of Stat3 On CD4~+T Cells In The Tumor Microenvironment And Delineation Of Treg Lineage In Breast Cancer Microenvironment

Background:Cancer immunotherapy is the boosting area of novel immunology research breakthroughs. It is not only a basic scientific research hot zone for immunologists, but also a frontia of translational medicine for physicians. And the strategy of immunotherapy is more than a complementary method for traditional treatments, but a brandnew clinical thought. Right now the safety and effectiveness are two key problems limit its clinical application.One of the most popular cancer immunotherapy is the T cell-based tumor infiltrating lymphocytes (TIL) adoptive transfer treatment (ACT). But cancerous disease is characterized by successful escaping from immunesurveillance, which would be achieved by recruit of the immune suppressive cells in the microenvironment of tumor site in situ, namely the tolerogenic DCs, tumor associated macrphages (TAM) and the regulatory T lymphocytes (Treg) etc. The existence of these inhibitory immune cells among the TIL cells challenged the safety and effectiveness of cancer immunotherapy, especially the Treg cells. It is a critical problem that confronts us to improve the purity of killer cells, and therefore overcome the disturbance of the cytotoxic T lymphocyte therapeutic effect.Stat3, one member of the Stat family (Signal transducers and activators of transcription), is found to be the key player in the JAK1/Stat3 signaling pathway during tumorigenesis. And Stat3 also functions in the immune system, especially the tumor immune evasion and immune suppression balancing in the tumor microenvironemnt. As a lab tradition, we closely focused on the Treg in tumor microenvironment and tumor antigen recognition form TIL cells. And previously we have published that the TIL cells extracted from ovarian tumor microenvironment contains high percentage of Thl7 cells. It is well established that the IL-6-Stat3-Thl7 axis is a pro-inflammatory pathway involving in the cancerous inflammation, which would promote tumor progression. So the co-existence of Th17 and Treg cells in the tumor microenvironment is an interesting phenomenon for us to dig in.Treg cells could be divided into two sublineages due to their generation, namely natural Treg (nTreg) and induced Treg (iTreg). Then we are eager to ask whether the Treg cells can be further divided into other sublineages due to their functional difference? Do Treg cells in the tumor microenvironment possess higher inhibitory function in order to sustain the escaping cancer from immune surveillance? And we also tried to manipulate the Treg sublineages by ex vivo cytokine induction to inspire the improvement of the ACT treatment. How to dissect the profound effect of Stat3 on CD4+ T cells and how to improve the Stat3-targeting gene therapy were other two main purpose of our project. We wandered whether the knockdown of Stat3 gene not only affects the tumor cells but also the CD4+ T cells in the tumor microenvironment? Therefore we generated the CD4-specific Stat3 gene deletion mice to evaluate the effect of Stat3 gene during the CD4+ T cell mediated anti-tumor immunity. Objects:To investigate the effect of Stat3 deletion on CD4+ T cell development and functions, especially the proliferative and differentiation function in the anti-tumor immunity. And to further evaluate the critical role of Stat3 in the Stat3-/- CD4+ T cell mediated adoptive cell transfer therapy for the B16 mouse melanoma model. Finally to dissect into the signaling pathways which fail the Stat3-/- CD4+ T cells supportive expansion of the CD8+ cytotoxic T lymphocytes. We also succeeded in setup the in vivo/ ex vivo Treg cell function analysis animal model, FoxP3gfp mice. By this animal model we use FCM analysis to find out the possibility of delineation for the Treg lineage of CD4+ T lymphocyte. And then we extended the discoveries in mice to human T cell level to confirm that the Treg cells from tumor microenvironment of breast cancer patients could be also further divided into naive and memory sublineages. Contents:1. Stat3-/- CD4+ T cell mediated specific anti-B16 melanoma adoptive cell transfer treatment model(1) Establish of CD4+ T cell specific Stat3 knockout mouse model and phenotype analysisThe Cre-loxP recombination system is employed to generate tissue-specific Stat3 deficient mice, CD4-Cre/Stat3floxP/floxP (Stat3-/-). We used PCR and Westernblot to confirm the deletion of Stat3 in CD4+ T cells and characterized the Stat3-/- CD4+ T cells cytokine production ability by ELISA. Flow-cytometry analysis revealed the development of CD4+ and CD8+ T cells in this Stat3 knockout mouse.(2) B16 mouse melanoma xenograft model for the evaluation of Stat3-/- CD4+ T cell mediated adoptive cell transfer treatmentWe generated the B16 mouse melanoma xenograft model in Stat3-/- mice and wildtype Stat3-/- mice to check the anti-tumor immunity function of this mouse. And we further challenged the B16 mouse melanoma xenograft model with ACT immune therapy to see whether it was the Stat3-/- CD4+ T cells that failed supporting the tumor-antigen specific CD8+ CTL cell expansion to eliminate the B16 tumor.(3) TCR receptor signal pathway downstream crosstalk with Stat3 modulation of apoptosis signal pathwayWe tried to unveil the exact mechanism behind the increased Stat3-/- CD4+ T cell apoptosis during the in vivo anti-tumor ACT model. We succeeded in using the pan-caspase inhibitor Z-VAD-fmk to reverse CD4+ T cell apoptosis upon TCR activation. And then Westernblotting and immunoprecipitation assay were performed to check the interaction of Stat3 with TCR signaling, MAPK signaling and apoptotic Bcl-2 signaling.2. Delineation and functional analysis of Treg cells(1) FCM and suppressive function analysis of nTreg sublineages from the FoxP3gfp mouse animal modelWe applied Flow-cytometry to analyze the mouse nTreg cells, in order to find out whether they can be further divided into naive and memory sublineages according to the surface marker of CD62L and CD44. We also checked the ratio between the naive and memory cells in T effector cells and Treg cells. And we performed suppressive assay for both the naive Treg (CD4+/FoxP3+/CD62L+/CD44-) and memory (CD4+/FoxP3+/ CD62L-/CD44+) Treg cells.(2) The ex vivo cytokine-induced differentiation potential of either nTreg sublineages from the FoxP3gfp mouseNext step we sorted out the naive Treg and memory Treg cells from FoxP3gfp mi by negative MACS and positive FACS. And we checked the purity of each compartments by FCM to confirm they were solid and independent cell groups inside the CD4+ nTreg lineage. We applied different cytokine combinations to induce the sorted naive Treg and memory Treg cells toward Th1/Th2/Th17 paradigm to determine their differentiation potentials.(3) The delineation of Treg cells from PBMC or TILs of breast cancer patients We further took the breast cancer as a disease model to dissect into the tumor microenvironment to find out whether the naive Treg cells played a role in the immune-compromised tumor immunity. The human naive T cell marker, C45RA and CD62L were chosen for the analysis of T cells from breast cancer patients and healthy donors. The FCM analysis was employed to check the TILs from breast cancer tumor microenvironment and the PBMC from the patients as well. Conclusions:1. Stat3 knockout in CD4+ T cells did not affect their development and distribution in mouse immune organs.2. The cytokine-induecd differentiation of Stat3-/- CD4+ T cell into Thl7 were completely defected, while the Thl/Th2/Treg exhibited no significant difference comparing to wildtype Stat3f/f CD4+ T cell. This was the cause of the tremendous decrease of IL-17asecretion from the activated Stat3-/- CD4+ T cells, while other kinds of cytokine secretion were not affected.3. Stat3-/- CD4+ T cells exhibited decreased proliferation and increased apoptosis upon TCR activation. And the pan-caspase inhibitor, Z-VAD-fmk could partially reverse the cell loss and restore the proliferation.4. These Stat3-/- CD4+ T cells could not facilitate the anti-tumor immune functions. And confirmed by repeated B16 ACT immunotherapy model that the deletion of Stat3 caused CD4+ T cells fail to support CD8+ T cell expansion?5. Analysis of TCR signaling and Bcl-2 family members revealed that, TCR activation could phosphorylate Stat3 through ZAP70. And thus downstream modulate Bcl-2 family member expression toward pro-apoptotic rather than anti-apoptotic direction.6. The nTreg cells from FoxP3gfp mouse animal model could be further divided into naive and memory sublineages according to the surface marker of CD62L and CD44. Namely the naive Treg markers are CD4+/FoxP3-GFP+/CD62L+/CD44-, and the memory Treg markers are CD4+/FoxP3-GFP+/CD62L-/CD44+7. The naive and memory sublineages of Treg cells could be sorted out from FoxP3gfp mice splenocytes by negative MACS and positive FACS. And we checked the purity of each compartments by FCM to confirm they were solid and independent cell groups inside the CD4+ nTreg lineage.8. During the in vitro suppressive assay, the naive Treg cells exhibited stronger suppressive function over the memory Treg.9. During the ex vivo cytokine-induced differentiation assay, the sorted naive Treg cell showed more conversion potentials than the memory Treg cells toward Th1/Th2/Th 17 paradigm.10. In the peripheral blood PBMC cells the CD45RA+ naive Treg cells and CD62L+ naive Treg cells both possessed higher percentage among the total CD4+ T cells than healthy donors.11. As say to the TIL cells extracted from breast patient tumor microenvironment, the CD45RA+ naive Treg cells did not increase significantly, while the CD62L+ naive Treg cells almost occupied the majority of TIL cells in some patients.