Transcription Factor BCL6 Is Required For The Generation But Not Maintenance Of Memory CD8~+ T Cells In Acute Viral Infection

Background:Cytotoxic CD8+T cells are essential for controlling cancer and intracellular pathogens,such as viruses and intracellular bacteria.Upon encountering their cognate antigen presented by antigen-presenting cells,the antigen-specific na?ve CD8+T cells are subjected to activation and clonal expansion,generating large numbers of effectors equipped with cytokines and cytolytic molecules.Following the infected and cancer cells clearance,most of the effectors succumb to apoptosis.Only a small fraction of them survive and then transition into memory cells,which can provide enhanced protection against the same pathogen.Both effector and memory CD8+T cells are heterogeneous.Effectors consist of at least two phenotypically and functionally distinct subsets,i.e.,short-lived and memory precursor effector cells（SLEC and MPEC）.SLEC cells with increased expression of killer cell lectin-like receptor G1（KLRG1）are considered to be short-lived and terminally differentiated;in contrast,MPEC cells with decreased KLRG1 expression have an increased potentiality to differentiate into long-lasting memory cells.Among memory CD8⁺T cells,CD62L⁺central memory T cells（Tcm）are capable of more efficient homeostatic self-renewal,whereas CD62L^–effector memory T cells（Tem）exhibit a greater cytotoxicity and decay over time.B-cell lymphoma 6 protein（BCL6）is a member of zinc finger transcription factor family containing an N-terminal POZ/BTB domain.BCL6 acts as either transcriptional activator or repressor in a sequence-specific manner and plays key roles in various cell types.In the adaptive immune system,BCL6 has been mostly considered as a B cell fate determination gene and has been identified as a“master”differential regulator of follicular helper T cells.Moreover,BCL6 has been reportedly important for the generation and maintenance of memory CD8⁺T cells and forced BCL6 expression can generate increased numbers of memory cells,especially Tcm cells.BCL6 deficient mice used in the previous studies are germline deleted,which may affect the infectious microenvironment caused by abnormal response of CD4⁺T cells and B cells and then influence the generation and maintenance of memory CD8⁺T cells.Moreover,the expression level of BCL6 is only slightly or not at all increased in memory CD8⁺T cells,compared with that in effector cells.Therefore,whether BCL6 plays a role in the maintenance of memory CD8⁺T cells is still elusive.Method:In this study,we separately crossed mice harboring loxP-flanked Bcl6 alleles(Bcl6^fl/fl)with CD4-Cre transgenic mice to get CD4-Cre-Bcl6^fl/fl mice(hereafter designated as Bcl6^-/-)or ERT2-Cre knockin mice to get ERT2-Cre-Bcl6^fl/fl mice(hereafter designated as i Bcl6^-/-).The mice infected by acute lymphocytic choriomeningitis virus（LCMV）was sacrificed and the splenocytes and peripheral blood lymphocytes were detected by Flow Cytometry.Besides,we also crossed CD4-Cre-Bcl6^fl/fl mice with P14 transgenic mice,whose TCR of CD8⁺T cells are specific to LCMV GP_33-41 epitope,and transferred their offspring’s splenocytes into wild type recipients to eliminate the interference of inner environments.The splenocytes and peripheral blood cells of chimeric mice were detected by Flow Cytometry on day 6 or day 8 post LCMV infection.In addition,chromatin immunoprecipitation assay was used to elucidate whether BCL6 could bind directly to the regulator region of Tcf7 gene in CD8⁺effectors.Results:1.We observed several comparable frequencies and numbers of GP33-specific memory cells,KLRG1^lo memory cells,and CD62L⁺Tcm cells in i Bcl6^-/-and control mice while the excision of BCL6 was induced by Tamoxifen after the generation of MPEC cells.Moreover,we did not observe any appreciable differences in the IL-2 production and CD107a/b expression of memory cells.2.The frequency and number of Bcl6^-/-GP33-specific CD8⁺effectors and KLRG1^loo memory precursors in the spleens were both obviously decreased at day 8 after infection.Moreover,the frequencies of BrdU incorporation and Ki-67 expression in Bcl6^-/-P14effectors in the spleens were both reduced at day 6 after infection,but Bcl6^-/-and control P14 effectors exhibited similar rates of apoptosis at day 8 after infection,as measured by Caspase-3/7 activation and Annexin V/7-AAD staining.3.BCL6 was directly associated with regulatory region of Tcf7 gene in antigen-specific CD8⁺effectors.Besides,forced expression of TCF-1 was sufficient to enhance the generation of memory precursors and elevate the production of IL-2 in CD8⁺effector cells.Conclusion:In response to acute viral infection,BCL6 is required for the expansion of CD8⁺effectors and generation of memory precursors by directly regulating the TCF-1 expression during the effector phase.By contrast,in the memory phase,BCL6 is dispensable for the TCF-1 expression and the maintenance of memory CD8⁺T cells.