DAPK1 Enhances CD8~+T Cell Anti-virus Function By Death Domain Regulating MTORC1 Activity

Object:mTORC1 senses various signals including growth factor,antigen,nutrition and energy status and regulates the cell growth,differentiation,metabolism and function.It plays key roles in T cell biology:mTORC1 regulates T cell proliferation and differentiation;mediates HIF-1?production and participates in T cell metabolism;regulates the expression effector molecules.In CD8⁺T cells,it has been reported that mTORC1 activation is not regulated by PI3K/Akt,but the mTORC1 activation mechanism and the upstream kinase are not clear.The purpose of the study is to explore whether DAPK1 is involved in the regulation of mTORC1 activation of CD8⁺T cells,and to elucidate its molecular mechanism and its effect on T cell function.Method:1.mTORC1 activation mechanism in CD8⁺T cell1.1 Activation of mTORC1 in CD8⁺T cells:Splenocytes were isolated from wild-type C57BL/6 mouse and stimulated with anti-CD3 for 2 days,followed with stimulation of rhIL-2 for 6 days to generate CTLs.CTLs were treated with PI3K inhibitor IC87114 or ly294002,PKB inhibitor Akti1/2,PDK inhibitor OSU-03012,and rapamycin for 15 minutes.After stimulation with 20ng/ml rhIL-2 for an additional60 min,cells were harvested and cells were lysed.The phosphorylation of ribosomal proteins S6 and 4-EBP1,the substrates of mTORC1,were determined by immunoblotting.1.2 Detection of DAPK1 activity in CD8⁺T cells after TCR or IL-2 stimulation:CTLs were cultured with R1640 medium supplemented with 3%FBS overnight,and anti-CD3/CD28 or rhIL-2 were added.After stimulation with different times,cells were harvested and lysed.Immunoblotting was used to detect the phosphorylation of MLC2,a substrate of DAPK1.1.3.mTORC1 activation in CD8⁺T cells after inhibition of DAPK1 activity:CTLs were cultured with R1640 medium supplemented with 3%FBS overnight,incubated with DMSO or the DAPK1 inhibitor DAPKi for 15 min,and stimulated with 20 ng/ml rhIL-2 for 60 min.Cells were harvested and lysed.Immunoblotting was performed to detect the phosphorylation of the TSC2,S6 and 4-EBP1;and the phosphorylation of S6 was detected by flow cytometry.2.Construction of Cd4-Cre;Dapk1-DD^fl/flC57BL/6 mice2.1.LoxP fragments were inserted into exons 26 and 27,respectively,to construct Dapk1-DD^fl/flC57BL/6 mice.2.2 Dapk1-DD^fl/flC57BL/6 mice were crossed with Cd4-Cre;C57BL/6 mice to generate Cd4-Cre;Dapk1-DD^fl/flC57BL/6 mice.3.The function of DAPK1-DD in T cells3.1.The function of DAPK1-DD in CD8⁺T cells in vitro3.1.1 Detection of mTORC1 activation in WT or Dapk1 DD^-/-CD8⁺T cells:CD8⁺T cells were isolated from the wild-type and Cd4-Cre;Dapk1-DD^fl/fll/fl mouse spleens using CD8⁺magnetic beads.After activation with anti-CD3/CD28 for 3 days,cells were expanded for 3 days with rhIL-2.CTLs were starved overnight in R1640medium supplemented with 3%FBS,stimulated with TCR or IL-2 for different times,and cells were harvested and lysed.Immunoblotting was used to detect the phosphorylation levels of PLC-?,Stat5 TSC2,S6,4-EBP1;Akt-Ser473,and Foxo,the substrates of mTORC2.Co-immunoprecipitation assay was performed to detect the TSC1/TSC2 complexes after TCR stimulation in wild-type and knockout CD8⁺T cells.3.1.2.Apoptosis detection:CD8⁺T cells were isolated from the wild-type and Cd4-Cre;Dapk1-DD^fl/flmouse spleens with magnetic beads.After 3 days of anti-CD3/CD28 activation,Annexin V and propidium iodide?PI?staining were performed.Flow cytometry was used to detect intracellular the fluorescence intensity.3.1.3 Cell proliferation:CD8⁺T cells were isolated with magnetic beads,labelled with CFSE,and stimulated by anti-CD3 and anti-CD28 for 3 days.Cells were harvested and stained with anti-Ki67 antibody,then detected by FCM.3.1.4.Cellular glucose uptake:CD8⁺T cells were isolated from the spleen of wild-type and knockout mice with magnetic beads,then stimulated with anti-CD3/CD28 for 3 days.The cells were washed,resuspended with fresh T-cell medium harvested at time points of 4h,8h,and 12h.Glucose?GO?kit was used to measure the concentration of glucose in the medium and the BCA kit was used to determine the intracellular protein concentrations of T cells,and sugar consumption was calculated.3.1.5.Chemotaxis:Lymphocytes were isolated from spleens and stimulated with anti-CD3 antibody for 2d,followed with rhIL-2 for aother 6d.Cells were harvested on the 4,6,and 8 days and stained anti-CD8 and anti-CD62L.The fluorescence intensity was detected by FCM.At day 8,cells were transferred to 6-well plates.Inhibitors including Akti1/2,rapamycin,and DAPK1i were added for further 12h.and the phosphorylation of KLF2 was detected by western blot.3.2.The function of DAPK1-DD in CD4⁺T cells in vitro3.2.1.DAPK1-DD regulates mTORC1 activation in CD4⁺T cells:CD4⁺T cells were isolated from the wild-type and Cd4-Cre;Dapk1-DD^fl/fl mouse spleens using CD4⁺beads,stimulating with anti-CD3/CD28 for 3 days,then expanded with rhIL-2for another 3 days.Cells were then starved overnight in R1640 medium supplemented with 3%FBS,stimulated with TCR and IL-2 for different times,harvested and lysed.The phosphorylation levels of TSC2,S6 4-EBP1,and AKTSer473were detected by immunoblotting.3.2.2.T cell in vitro Differentiation:Naive T cells CD4⁺CD44^highCD62L^low were purified by flow cytometry from wild-type and Cd4-Cre;Dapk1-DD^fl/fl mouse spleens.Cells were differentiated under different T helper subset differentiation conditions.Flow cytometry was used to detect linage-specific molecules:Th0?IFN-??,Th1?IFN-??,Th2?IL-13?,Th17?IL-17?,and Tregs?Foxp3?.3.2.3 Apoptosis assay:Naive CD4⁺T cells were sorted by flow cytometry and cultured with different T helper subtype differentiation conditions.After 3 days,Annexin V and PI staining were performed,and intracellular fluorescence intensity was detected by flow cytometry.3.2.4.Cell Proliferation Assay:Naive CD4⁺T cells were sorted by flow cytometry from wild-type and Cd4-Cre;Dapk1-DD^fl/fll/fl mouse,labeled with CFSE and differentiated for 3 days.CFSE fluorescence intensity was measured by FCM.3.2.5.DAPK1-DD regulates mTORC1 activationin CD4⁺T cells:Naive CD4⁺T cells were sorted by flow cytometry from wild-type and Cd4-Cre;Dapk1-DD^fl/flmice;and differentiated with different cytokines.After 48 h,cells were harvested,stained with phosphorylated S6 fluorescent antibody,and the intracellular fluorescence intensity was measured by FCM.4.DAPK1-DD and T cell function in steady-state?in vivo experiments?4.1.T cell development analysis:Single cell suspensions were prepared from thymus,spleen,mesenteric lymph node,inguinal lymph node,and axillary lymph node of 6-8weeks wild-type or knockout mice.The cells were stained with anti-CD3,anti-CD4,and anti-CD8,and the percentage of CD3⁺,CD4⁺,and CD8⁺was analyzed by flow cytometry.4.2.T cell activation and effector function:CD8⁺T cells were isolated from the spleens of 6-8 weeks wild-type or knockout mice by magnetic beads,and cultured with PMA,Ionomycin,and Golgi inhibitors for 4 hours.The cells were stained with fluorescent antibodies:anti-CD25,anti-CD44,anti-CD8,anti-IFN-?,and anti-granzyme,and the corresponding fluorescence intensity was measured by flow cytometry.5.Effect of DAPK1-DD on anti-viral infection LCMV infection mouse model:6 to 8 weeks wild-type or knockout mice were intraperitoneally injected with LCMV Amerstrong strain.After 7 days,the mice were sacrificed,and single cell suspension of the spleens were prepared.The cells were stained by fluorescence labelled antibodies:anti-CD4,anti-CD8,anti-CD44,anti-GP33,anti-pS6,anti-IFN?,anti-IL-2 and anti-granzyme.Then fluorescence intensity was measured by flow cytometry.Results1.Regulation of mTORC1 activation by DAPK1 in CD8⁺T cells:IC87114 andAkti1/2 decreased the phosphorylation of Akt Thr308 and Akt Ser473,but did not inhibit the phosphorylation of S6 and 4-EBP1;PDK1 inhibitor OSU-03012 not only inhibited phosphorylation of Akt at Thr308 and Ser473,as well as phosphorylation of S6,4-EBP1 and MLC2;Ly294002 inhibited the phosphorylation of AktThr308,Akt-Ser473,S6,4-EBP1 and MLC2. 2.Activation of mTORC1 and DAPK1 in CD8⁺T cells:TCR or IL-2 stimulation induced,the phosphorylation of MLC2 and mTORC1 activation.DAPK1i inhibitor suppressed the phosphorylation of MLC2,Akt-Ser473,S6 and 4-EBP1. However,but the phosphorylation of Akt-Thr308 was increased upon DAPK1 inhibition. 3.Generation of T cell specific knockout Dapk1-DD mice:LoxP fragments were inserted into the exons of exons 26 and 27 of Dapk1 and Cd4-Cre;Dapk1-DD^fl/fl mice were obtained by crossing Cd4-Cre with Dapk1-DD^fl/flmice.DAPK1-DD was not detected in Dapk1-DD^-/-CD8⁺T cell with a DAPK1 C terminal antibody.4.Effect of DAPK1-DD on signaling pathways in CD8⁺T cell:Compared to wild type,the phosphorylation levels of S6,4-EBP1 and TSC2 were decreased in Dapk1-DD^-/-CD8⁺T cells stimulated by TCR or IL-2;while there was no significant differences in the phosphorylation levels of Aktp-Ser47,Foxo,PLC? or Stat5. 5.Effect of DAPK1-DD on the proliferation,activation,apoptosis and glucose uptake of CD8⁺T cells:Deletion of DAPK1 death domain did not affect the proliferation and apoptosis,but resulted in enchanced homing chemokine expression and reduced glucose uptake.6.Effect of DAPK1-DD on signaling pathways in CD4⁺T cells:Compared to wild type,the phosphorylation levels of S6 and 4-EBP1 were decreased in Dapk1 DD^-/-CD4⁺T cells stimulated by TCR or IL-2;while there was no significant differences in the phosphorylation levels of Aktp-Ser473.The phosphorylation of S6 in different T helper subtypes were reduced. 7.The effect of DAPK1-DD on proliferation,apoptosis and differentiation of CD4⁺ T cells:Compared with the wild type,there were no statistically differences in differentiation,CFSE dilution,and apoptosis in Dapk1-DD^-/-CD4⁺T cells. 8.Effect of DAPK1-DD on T cell development and CD8⁺T cells activation in homeostasis:Under steady state,the numbers and proportions of CD3⁺,CD4⁺, and CD8⁺T cells from the thymus,spleen,mesenteric lymph nodes,axillary lymph nodes,and inguinal lymph nodeswere comparable between control and xx deficient mice;the expressions of effector molecules i.e CD25,CD44,IFN-?,and granzymes in CD8⁺T cells were comparable between control and DAPK1-DD deficient mice. 9.Effect of DAPK1-DD on anti-viral infection in vivo:After infection of LCMV, compared with controls,the numbers of total splenic CD4⁺,CD8⁺,and virus-specific CD8⁺T cells in the knock out mice were reduced.The phosphorylation of S6 and the expressions of effector molecules i.e.IFN-?, granzymes,and IL-2 were reduced in the DAPK1-DD deficient mice.Conclusion1.The activation of mTORC1 in CD8⁺T cells is independent of PI3K/Akt.2.The activation of mTORC1 in CD8⁺T cells is regulated by PDK1/DAPK1/TSC2.3.DAPK1 binds to TSC2 through DD and phosphorylates TSC2-Ser939,then enhances mTORC1 activation.4.DAPk1-DD-deficient CD8⁺T cells have reduced glucose uptake.5.The expressions of homing chemokines are enhanced in DAPK1-DD-deficient CD8⁺T cells.6.DAPKk1-DD does not affect the proliferation and apoptosis in CD8⁺T cells and CD4⁺T cells.7.Deletion of DD resulted in impaired anti-viral activities of CD8⁺T cells.