Stenotrophomonas Maltophilia DnaK Protein Induces T Cell Exhaustion Through JAK2/STAT1

Objective:To clarify the immune interaction between Stenotrophomonas maltophilia(SMA)and T lymphocytes,and to elucidate the pathway and specific molecular mechanism of T Cell Exhaustion induced by SMA.Methods:(1)CD3~+T lymphocytes were isolated and identified by magnetic bead separation and flow cytometry.After Stenotrophomonas maltophilia filter supernatant(SMA-sup)co-culture with T cells,the expression of apoptosis-related proteins and PDL1/PD-1 was detected by WB,and the change of cytokine expression profile was detected by flow cytometry.WB was used to detect the activation of JAK2/STAT1-related proteins in PDL1 up-regulated classical signaling pathway.STAT1 inhibitor Fludarbine was used for recovery experiment,and anti-IFN-γwas used to block the binding of IFN-γto T cell surface receptors for recovery experiment.The expression of apoptosis-related proteins and PDL1/PD-1were detected by WB.RT-q PCR was used to verify relevant immunosuppressive molecules and downstream molecules of the pathway.The changes of cytokine expression profile,apoptosis related proteins and PDL1/PD-1 were detected by WB and flow cytometry after co-culture of SMA-Sup with Jurkat cells and KCL22 cells.(2)Supercentrifugation and transmission electron microscopy were used to separate and identify SMA-OMVs.The cytotoxicity of SMA-OMVS was detected by CCK8 assay and LDH.The changes of cytokines expression profile,apoptosis-related proteins and PDL1/PD-1 were detected by WB and flow cytometry after co-culture of SMA-OMVs and T lymphocytes.(3)The key proteins that SMA may induce T lymphocyte depletion were screened by combining proteomics results;(4)Protein recombination technology to recombine key proteins;WB was used to verify whether key proteins could promote T lymphocyte depletion through JAK2/STAT1 pathway.STAT1 inhibitor Fludarbine was used as a response test.Results:(1)SMA-sup can promote the apoptosis of T lymphocytes,in which the expression of pro-apoptotic proteins Bax,Caspase3,P53 was significantly increased,the expression of anti-apoptotic protein Bcl-2 was significantly decreased,and the expression of PDL1 was also up-regulated.Flow cytometry and RT-QPCR showed the same results.SMA-sup can induce T lymphocytes to secrete IL-2,IL-10 and IFN-γ.After SMA-sup treatment,the expression of JAK2 and STAT1 phosphorylated proteins increased.STAT1 inhibitor Fludarbine;Further anti-IFN-γwas used,and WB results showed no significant changes in the expression of apoptosis-related proteins and PDL1/PD-1D.The expressions of pro-apoptotic proteins Bax,Caspase3 and P53 were significantly decreased,the expressions of anti-apoptotic proteins Bcl-2 were significantly increased,and the expressions of JAK2 and STAT1 phosphorylated proteins were also decreased by WB.RT-q PCR detection of SMA-sup can promote immunosuppressive molecules and JAK2/STAT1 downstream pro-apoptotic and proliferation inhibitory molecules,inhibit the transcription of pro-proliferation molecules,but Fludarbine treatment can block this phenomenon.SMA-sup could not regulate the change of cytokines expression profile in Jurkat cells and KCL22 cells.SMA-sup could inhibit the expression of apoptosis-related proteins in Jurkat cells,in which the expressions of pro-apoptotic proteins Bax,Caspase3 and P53 were significantly decreased,the expressions of anti-apoptotic proteins Bcl-2were significantly increased,and the expressions of JAK2 and STAT1phosphorylated proteins were also inhibited.However,SMA-sup promoted the expression of apoptosis-related proteins in KCL22 cells,including the expression of pro-apoptotic protein Bax,anti-apoptotic protein Bcl-2,and PD-1.(2)SMA-OMVs were successfully separated and characterized by supercentrifugation and transmission electron microscopy.CCK8 test and LDH detection,compared with the control group,The OD460 value and LDH concentration of SMA-OMVs treatment group had no significant change;SMA-OMVs can induce the secretion of IL-10 and TNF-αin T lymphocytes,and OMVs of other Gram-negative bacilli can also stimulate the secretion of IL-10 and TNF-αin a gradient manner.Flow cytometry analysis showed that SMA-OMVs could not promote the apoptosis of T lymphocytes and the expression of PDL1.(3)Combines the result of proteomics,uniprot protein database and related analysis,determine the key protein-SMA–DnaK.(4)SMA-DnaK was successfully expressed and purified by protein recombinant technology.After treating T lymphocytes with SMA-DnaK,apoptosis of T lymphocytes was induced,in which the expression of pro-apoptotic proteins Bax,Caspase3 and P53 were significantly increased,the expression of anti-apoptotic protein Bcl-2 was significantly decreased,and the expression of PDL1 was also up-regulated.Meanwhile,the expression of JAK2 and STAT1 phosphorylated proteins increased.After treatment with STAT1 inhibitor Fludarbine,the expressions of pro-apoptotic proteins Bax,Caspase3 and P53,PDL1 and anti-apoptotic protein Bcl-2 were significantly decreased by WB.Conclusion:Stenotrophomonas maltophilia DnaK activated the JAK2/STAT1pathway in a soluble free state to induce T cell exhaustion.