Transcriptional Factor Nr4a1 Regulates T Cell Tolerance And Exhaustion

T cell tolerance represents as a mechanism that maintains T cell hyporesponsiveness to self tissues to avoid autoimmune diseases.Tolerant T cells is characteristic of their inability to proliferate or to secrete cytokines in response to TCR engagement.T cell tolerance is mediated by both extrinsic and intrinsic mechanisms.Regulatory T cells represent a major extrinsic means.Moreover,T cells have intrinsic mechanisms that results in T cell tolerance,or anergy.A series of publications reported that transcription factors NFAT,Egr2,Egr3 and E3ligase family members Cbl-b,Itch,GRAIL play important roles in intrinsic mechanisms.However,the molecular mechanism underlying T cell tolerance remains unclear.In chronic infection or tumor,sustained antigen stimulation,local immune inhibitory microenvironment and sustained activation of inhibitory receptor cooperatively promote T cell tolerance.Exhausted T cell is characterized of impaired cytokine secretion,poor proliferation and decreased cytotoxic ability,failed control of virus infection and tumor.Immune checkpoint blockade therapy can protect T cell from exhaustion and reinvigorate exhausted T cell,which play significant roles in combating tumor.However there remain some applicable conditions and taboos for the use of immune checkpoint blockade antibodies.It's necessary to find new targets to strengthen the antigen specific T cells to enhance antitumor immunity.Transcriptional factor Nr4a1 is a member of orphan nuclear receptor Nr4a family,which controls the negative selection and apoptosis of unmatured CD4⁺CD8⁺T cells in thymus.Nr4a1 cooperates with family member Nr4a2 and Nr4a3 to control the development of Treg cells in peripheral CD4⁺T cells.Nr4a1 can inhibit the proliferation and effect function of peripheral CD8⁺T cells.This study identified transcription factor Nr4a1 was highly expressed in tolerant CD4⁺T cells through in vitro and in vivo tolerance model.By virtue of of peptide induced tolerance model,colitis model,Eg7 tumor model and LCMV chronic infection model,we investigated the role of Nr4a1 in tolerant and exhausted T cells,and illustrated the underlying mechanism.The methods,results and conclusion of this study are stated as follows:1.Na?ve CD4⁺T cells were stimulated with APC deficient of co-stimulatory molecule to induce the tolerance of CD4⁺T cells.Tolerant T cells were performed with gene expression profiling analysis together with in vitro differentiated Th1,Th2,Th17 cells,which identified Nr4a1 was highly expressed in tolerant CD4⁺T cells.Donor CD4⁺T cells were induced in tolerant or activated state,flow analysis showed that Nr4a1 was substantially increased in tolerant CD4⁺T cells compared to that of activated CD4⁺T cells.2.Na?ve Nr4a1^-/-and WT OT-II cells were sorted and transferred into recipient mice,followed with OT-II peptide injection to induce tolerance of OT-II cells.Flow analysis showed that more Nr4a1^-/-OT-II cells in recipient mice and secreted higher percentage of IL-2and IFN-?,T cell tolerance was low in Nr4a1^-/-OT-II cells,with no Treg cells in both groups.3.Naive CD4⁺T cells from Nr4a1^-/-and WT mice were sorted and transferred into Rag1-/-mice to induce colitis.Mice of Nr4a1^-/-group had severe body loss,shorter colon,huge amount of inflammatory cells infiltrated in colon.Flow analysis showed that more infiltrated CD4⁺T cells were found in lamina propria in Nr4a1^-/-group mice,more IFN-?⁺and IL-17⁺cells in Nr4a1^-/-group mice.Besides there were no Treg cells found in both groups.4.Furthermore,we want to know anti-tumor ability and exhaustion of Nr4a1^-/-CD8⁺T cells.We inoculated E.G7 tumor cells into recipient mice subcutaneously,when tumor was palpable,na?ve WT and Nr4a1^-/-OT-I cells were sorted and transferred into tumor bearing mice,tumor size were measured.We found that tumor growth was apparently inhibited in Nr4a1^-/-OT-I group,tumor infiltrated Nr4a1^-/-OT-I cells showed higher percentage and cell number,reduced percentage of PD-1⁺Tim-3⁺exhausted cells,more IFN-?⁺and TNF-?⁺cell number.These results were found in both immunocompetent mice and immunodeficient Rag1^-/-mice.5.Chimeric bone marrow mice were infected with LCMV-Clone13 virus,after 3 weeks,Nr4a1^-/-CD8⁺T cells had less PD-1⁺Tim-3⁺exhausted cells,more TNF-?⁺cells.Na?ve CD8⁺T were sorted from WT and Nr4a1^-/-P14 mice and transferred into Rag1^-/-mice with equal ratio,after 20 days of LCMV-Clone13 chronic virus infection,Nr4a1^-/-P14 cells had higher percentage,higher expression of Ki-67 and Bcl-2,reduced level of Tim-3 and CTLA-4 but increased level of PD-1 and Lag-3,increased IFN-?secretion and CD107 expression after gp33 peptide stimulation.6.Electrophoretic mobility shift assay demonstrated that overexpression of Nr4a1 in T cells reduced AP-1 binding of downstream effector gene.Overexpression of AP-1 family members C-Jun,Jun-B and Batf in OT-II cells followed with peptide-induced tolerance,similar results were found with that in Nr4a1^-/-OT-II cells.According to above results,we made the following conclusions:1.Nr4a1 was highly expressed in tolerant CD4⁺T cells;2.Nr4a1^-/-CD4⁺T cells increased proliferation and cytokine secretion;3.CD8⁺T cells deficient of Nr4a1 showed enhanced anti-tumor ability and effector function,reduced exhaustion and increased proliferation;4.Nr4a1 reduced AP-1binding of downstream effector gene,overexpression of AP-1 family members C-Jun,Jun-B overcame T cell tolerance due to high expression of Nr4a1.This study thus identifies Nr4a1 as a general key regulator in T cell dysfunction induction,which may be targeted in tumor immunotherapy,chronic infection and autoimmune diseases.