STK40 regulates T cell development and effector function through COP1-depenendent degradation of c-Jun transcription factor
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ABSTRACT: To examine the effect of Stk40 deletion specifically in T cells during chronic LCMV infection, we have generated a T cell specific knockout mouse line (CD4CreER; Stk40loxp/loxp). We found that mice lacking Stk40 exhibited improved viral clearance and T cell function compared to WT counterparts. To gain a broader understanding of how Stk40 regulates T cell response to chronic LCMV infection, we performed single cell transcriptome analysis on magnetically enriched LCMV-specific dextramer+ CD8 T cells isolated from spleens of Stk40 KO or WT mice at days 15 and 22 after infection with LCMV clone 13.
Project description:Brd4 deficient and WT GP33-tetramer+ cells were sorted from spleens on D8 of LCMV infection. Tamoxifen was administered on days 5-7 of infection to induce to deletion of Brd4. KLRG1hiCD127lo TE, KLRG1loCD127lo EEC, and KLRG1loCD127hi MP cells were sorted for RNAseq analysis.
Project description:Hexokinase 2 (HK2) is one the most highly upregulated enzymes in glycolysis in activated T cells and cancer cells. We genetically abolished HK2 expression in T cells in vivo and infected mice with LCMV to induce T cell proliferation. CD8+ T cells from were sorted from spleens of WT and HK2 null mice 8 days after infection. Additionally, WT CD8+ T cells were compared to WT primary T cell acute lymphoblastic leukemia (T-ALL). T-ALL cells were sorted from spleens of mice 6 weeks after adoptive transfer of bone marrow that overexpresses mutant Notch1 (Notch1-deltaE).
Project description:Cytolytic activity by CD8+ cytotoxic T lymphocytes (CTL) is a powerful tactic in the elimination of intracellular pathogens and tumor cells. The destructive capacity of CTL is progressively dampened during chronic infection - yet the environmental cues and molecular pathways controlling immune “exhaustion” remain unclear. We find CTL immunity is regulated by the central transcriptional response to hypoxia, mediated by the von-Hippel-Lindau/Hypoxia-Inducible-Factor (VHL/HIF) pathway. Deletion of VHL, the primary negative regulator of HIF, leads to lethal CTL-mediated immunopathology during chronic infection, and VHL-deficient CTL display enhanced control of persistent viral infection and neoplastic growth. We find HIF and oxygen influence expression of pivotal CTL transcription, effector and costimulatory-inhibitory molecules, which is relevant to strategies to promote viral and tumor clearance. To understand the role of the VHL/HIF pathway in regulating T cell responses to acute and persistant antigen in vivo, a mixture of ~10^4 WT and Vhl KO virus-specific CD8+ T cells (P14s) was transferred iv into uninfected WT host mice. After infection with either LCMV-Armstrong (acute viral infection) or LCMV-clone13 (persistent viral infection) we double-FACS isolated the responding P14 donor cells from pooled spleens from two sets of host mice to obtain duplicates for microarray for the four conditions, resulting in eight samples (2 WT Arm, 2 VHL KO Arm; 2 WT cl13, 2 VHL KO cl13) at 6 to 7 days post-infection. All conditions were sorted on KRLG1lo P14 cells. Note this was a mixed P14 transfer, so WT and KO cells were responding to infection in the same WT host mice to aid in normalizing effects such as antigen load and cytokine environment.
Project description:To identify mechanisms behind immunosuppression during virus infections, we infected mice with LCMV-Armstrong and LCMV-Clone 13 expression patterns. LCMV-Armstrong induces a T-cell reaction that resolves infection within 8-10 days, while LCMV-Clone13 generates a persisten infection through immunosuppression. We used microarray to uncover splenic gene expression patterns specific to each LCMV infection at 5, 9, and 30 days C57BL6 mice, 6-10 weeks old, were infected with LCMV-Armstrong and LCMV-Clone 13 or left uninfected (naïve). At days 5, 9, and 30 whole spleens were harvested for RNA extraction and hybridization on Affymetric microarray.
Project description:To decipher if NFAT5 controls CD8 T cell exhaustion during chronic infection on a molecular level, we performed scRNA sequencing on sorted P14 WT and NFAT5 KO CD8 from the spleen of a chronic LCMV-infected mouse.
Project description:Chronic viral infections incapacitate adaptive immune responses by 'exhausting' virus-specific T cells, inducing their deletion and reducing productive T cell memory. Viral infection rapidly induces death receptor Fas (CD95) expression by dendritic cells (DCs) making them susceptible to elimination by the immune response. Lymphocytic Choriomeningitis Virus (LCMV) Clone 13, which normally establishes a chronic infection, is rapidly cleared in C57Black/J mice with conditional deletion of Fas in DCs. The immune response to LCMV is characterized by an extended survival of virus-specific effector T cells. Moreover, transfer of Fas-negative DCs from non-infected mice to already-infected animals results in either complete clearance of the virus or a significant reduction of viral titers. Thus, DC-specific Fas expression plays a role in regulation of anti-viral responses and suggests a strategy for stimulation of T cells in chronically infected animals and humans in order to achieve the clearance of persistent viruses. We compared gene expression between splenic DCs from B6.FasKI and B6.CD11c-Cre.FasKI mice. DCs were isolated on day 5 after LCMV infection with 3 mice in each group, for a total of 6 samples. Spleens were collagenase-DNAse digested and sorted by flow to isolate DCs.
Project description:Wnt signal transduction during an immune response is involved in the establishment of functional CD8 T cell memory P14 ConductinLacZ CD8 T cells (CD45.2) were transferred in CD45.1+.2+ recipient mice. The day after, recipients were infected with 200pfu LCMV WE. At day 8 after infection, cells from spleen and lymph nodes were harvested, magnetically enriched for CD8 T cells, loaded with the beta-galactosidase substrate (FDG) and sorted as 7AAD-negative CD45.- negative, beta-galactosidase positive versus negative cells. P14 CD8 T cells were sorted at day 8 after LCMV infection according to LacZ activity for RNA extraction and hybridization on Affymetrix microarrays.
Project description:CD4 and CD8 T cells display functional defects during chronic infection such as loss of certain cytokines. Recent studies have suggested that CD4 T cells may actually gain other functions, however. Here, we analyzed gene expression profiles from LCMV-specific CD4 and CD8 T cells throughout the response to either acute LCMV or chronic LCMV infection. This alllowed us to identify CD4-specific changes during chronic infection compared to acute infection but also revealed shared core regulators between CD4 and CD8 T cells. LCMV-specific CD4 and CD8 T cells were isolated 6, 8, 15 and 30 days post infection with LCMV Armstrong or LCMV clone 13. Naïve CD4 and CD8 T cells were also isolated from naïve mice as comparisons. Four replicates of each sample were hybridized. The only exception is LCMV-specific CD4 T cells isolated 6 days post infection with LCMV-Arm where only three replicates were hybridized.
Project description:Understanding the response of memory CD8 T cells to persistent antigen re-stimulation and the role of CD4 T cell help is critical to the design of successful vaccines for chronic diseases. However, studies comparing the protective abilities and qualities of memory and naïve cells have been mostly performed in acute infections, and little is known about their roles during chronic infections. Herein, we show that memory cells dominate over naïve cells and are protective when present in large enough numbers to quickly reduce infection. In contrast, when infection is not rapidly reduced, memory cells are quickly lost, unlike naïve cells. This loss of memory cells is due to (i) an early block in cell proliferation, (ii) selective regulation by the inhibitory receptor 2B4, and (iii) increased reliance on CD4 T cell help. These findings have important implications towards the design of T cell vaccines against chronic infections and tumors. 16 samples are analyzed: 3 replicates of secondary effector CD8 P14 T cells at day 8 post-acute lymphocytic choriomeningitis virus (LCMV) infection; 4 replicates of secondary effector CD8 P14 T cells at day 8 post-chronic LCMV infection; 4 replicates of primary effector CD8 P14 T cells at day 8 post-acute LCMV infection; and 5 replicates of primary effector CD8 P14 T cells at day 8 post-chronic LCMV infection.
Project description:We report the transcriptional profile of phosphatidylserine (PS) positive and PS negative CD44+CD62L- effector CD8+ T cells from LCMV-Armstrong infected mice sorted from spleens on day5 amd day10 after infection. PS was stained in vivo by injection of MFG-E8-eGFP. PS+ cells acquire PS through binding of extracellular vesicles (EVs). Binding of EVs by effector CD8+ T cells is strongly enhanced during LCMV infection and peaks between day5 and day10 post infection. Effector T cell gene signature is increased in EV+ CD8+ T cells compared to their EV- counterparts.