Mutiple Myeloma Immune Escape Mediated By Negative Regulation Of Fap?+ Bone Marrow Mesenchymal Stromal Cells On CD4~+ T Cells

Part 1 MM-BMSCs exert immunosuppressive effect on T-cellsObjective:To evaluate the function of MM-BMSCs on T cell proliferation,apoptosis,senescence and differentiation.Methods:Bone marrow was obtained from 20 MM patients which were newly diagnosed and 20 healthy donors,then cultured in human MSCs serum-free medium.After 3 weeks,BMSCs were detached using 0.125%trypsin-0.01%EDTA and analyzed by flow cytometry for expression of CD34,CD45,CD90 and CD105.CD4+ T cells were obtained from healthy donors using CD4+ microbead selection.BMSCs were co-cultured with CD4+ T cells(1:5)in 12 well plates.The experiments were allocated into four groups:HD-BMSCs and MM-BMSCs co-cultured groups,activated CD4+ T cells,and inactivated CD4+ T cells.T cells were obtained from supernatant after 6 days co-culture,CCK-8 was performed to evaluate the effect of BMSC on T-cell proliferation.Flow cytometry was used to measure the effect of BMSC on T-cell apoptosis.The senescence of T cells was detected by P-gal kit,flow cytomerty and RT-PCR were used to observe the expression of activation marker CD28 and human telomerase reverse transcriptase,all above is to evaluate the effect of BMSCs on T-cell senescence.Flow cytometry for CD4,IL-17,CD25,and Foxp3 was carried out to detect the ratio of Treg/Th 17 in T cells and RT-PCR was used to investigate the transcription factor Foxp3 and RORyt,together with levels of IL-10 and IL-17 detected by ELISA to evaluate the effect of BMSC on T-cell differentiation.Results:(1)BMSCs were positive for CD90 and CD 105,negative for CD34 and CD45 analyzed by flow cytometry.(2)Based on the proliferation rate of T cells activated by CD3 functional antibody,both HD-BMSC and MM-BMSC distinctly inhibited the proliferation of CD4+ T cells.However.no significant difference was found between HD-BMSC and MM-BMSC co-culture groups.(3)The apoptotic rates of HD-BMSC and MM-BMSC groups were similar to activated T cell group,suggesting BMSCs did not show obvious inhibitory effect on apoptosis of CD4+ T cells.(4)Blue dyed cells were thought as senescent T cells,the senescent rate was calculated as the percentage of blue dyed cells.Both BMSCs up-regulated the senescent rate of T cells,Compared to HD-BMSCs,MM-BMSCs significantly increased the percentage of SA-P-gal-positive senescent T-cells.(5)The expression of CD28 was decreased in two co-culture groups,MM-BMSCs could trigger the loss of CD28 more obviously.(6)The expression levels of hTERT in two co-culture groups were down-regulated,we found significant inhibitory effect of MM-BMSCs for hTERT compared to HD-BMSCs.(7)Treg/Th17 in CD4+ T cells co-cultured with HD-BMSCs was shifted to Treg,as well as up-regulated IL-10.On the other hand,MM-BMSCs co-cultured T cells were stimulated to Th17 differentiation,accompanied with distinctly increased IL-17.(8)HD-BMSCs mainly induced the expression of Foxp3,while MM-BMSCs facilitated the expression of RORyt.Conclusion:MM-BMSCs exert immunosuppressive effect on T cells:inhibiting T-cell proliferation,promoting T-cell senescence,inducing Th17 differentiation,resulting in immune tolerance and immune escape.Part2 Expression characteristics of FAPa in BMSCsObjective:To investigated the expression characteristics of FAPa in BMSCs in vitro.Methods:After incubation with rabbit anti-human FAPa polyclonal antibody,the location and expression of FAPa were observed by immunofluorescence assay.The expression level of FAPa was observed by Western blot and RT-PCR.Results:FAPa was expressed on BMSC cell membrane as observed by microscope.No significant difference was observe between the expression of FAPa in HD-BMSC and MM-BMSC,which was detected by Western blot and RT-PCR.We speculated that the expression of FAPa was not induced without stimulation of tumor microenvironment,so we added U266 cultured medium in BMSC medium to imitate the multiple myeloma microenvironment,the expression of FAPa was up-regulated in MM-BMSCs treated with TCCM.Conclusion:Expression of FAPa in BMSCs from MM patients is induced in MM microenvironment.Part3 MM-BMSC exert immunosuppressive effect on CD4+ T cells through FAPaObjective:To investigate the immunosuppressive effect of MM-BMSC on CD4+ T cells after treatment with FAP? inhibitor PT-100.Methods:Bone marrow was obtained from 10 MM patients which were newly diagnosed and 10 healthy donors,then cultured in human MSCs serum-free medium.After 3 weeks,BMSCs were detached using 0.125%trypsin-0.01%EDTA.CD4+ T cells were obtained from healthy donors using CD4+ microbead selection.BMSCs were co-cultured with CD4+ T cells(1:5)in 12 well plates.The experiments were allocated into four groups:HD-BMSCs and MM-BMSCs co-cultured groups,activated CD4+ T cells,all groups were treated with FAPa inhibitor PT-100 or not.T cells were obtained from supernatant after 6 days co-culture.CCK-8 was performed to evaluate the effect of BMSC on T-cell proliferation.To investigate the function of PT-100 on co-cultured T cells,T cells was stained by ?-gal kit,flow cytomerty and RT-PCR were used to observe the expression of activation marker CD28 and human telomerase reverse transcriptase.Flow cytometry for CD4,IL-17,CD25,and Foxp3 was carried out to detect the ratio of Treg/Th17 in T cells and RT-PCR was used to investigate the transcription factor Foxp3 and ROR?yt,as well as levels of IL-10 and IL-17 detected by ELISA to evaluate the role of FAP in MM-BMSC suppressive function.Results:(1)After treatment with 1 pmol/mL and 0.1 pmol/mL PT-100,the proliferation of HD-BMSC and MM-BMSC co-cultured T cells were up-regulated compared to control group,and PT-100 did not show significant effect on T cells without any BMSC.(2)We observed the percentage of senescent CD4+ T cells co-cultured with MM-BMSC was decreased in 1 pmol/mL and 0.1 pmol/mL PT-100.In contrast,PT-100 did not influence the T cell senescence co-cultured with MM-BMSC,suggesting PT-100 inhibited the function of FAP in MM-BMSC especially,reversed senescence inducing effect on T cells.(3)The expression level of CD28 on CD4+ T cells co-cultured with HD-BMSC and MM-BMSC were increased compared with control groups,CD28 expression of HD-BMSC group was up-regulated at the similar level to activated T cells,and that of MM-BMSC treated with PT-100 was increased 4-5 times in comparison with control group.In addition,PT-100 did not influence CD28 expression of T cells directly,suggesting MM-BMSC inhibit CD28 expression through FAPa.(4)PT-100 up-regulated the level of hTERT expressed by T cells co-cultured with HD-BMSC and MM-BMSC without direct influence on T cells.(5)1 pmol/mL PT-100 triggered Treg/Th 17 diverse to Treg,mainly through decreasing the ROR?t expression,although Foxp3 expression was moderately up-regulated in HD-BMSC co-cultured T cells with PT-100.Moreover.we did not observe the effect of PT-100 on T cell cultured without BMSC.(6)PT-100 down-regulated IL-17 secretion in MM-BMSC co-culture group,and did not affect that of other groups.Conclusion:MM-BMSC exert immunosuppressive effect through FAPa.Inhibiting FAPa retarded the negative function of MM-BMSC,thereby proliferation was up-regulated,the expression of CD28 and hTERT were increased resulting in reduced senescence,inducing Treg/Th17 shift to Treg as a result of down-regulated Th17 transcription factor RORyt expression.Part4 Correlated mechanisms of immunosuppressive effect on T cells exerted by MM-BMSC/FAPaObjective:To investigate the relationship between immunosuppressive effect of MM-BMSC/FAPa and PI3K/AKT signaling pathway,explore regulation effect on the level of TGF-?.Methods:Western blot was performed to evaluate the level of AKT and phosphorylated AKT in CD4+ T cell co-cultured with MM-BMSC with or without PT-100.CD4+ T cells were co-cultured with MM-BMSC in 5:1,and the experiment was allocated into MM-BMSC co-culture group and activated CD4+ T cell group,25?mol/mL and 50?mol/mL LY294002 were used to stock the PI3K/AKT signaling pathway,the level of IL-17 and TGF-? were detected by ELISA,RT-PCR was performed to analyze the expression of ROR?t and hTERT,and CD28 expression was obtained by FCM.Western bolt was used to detect the effect of PT-100 on expression of TGF-? in MM-BMSC.CD4+T cells were co-ultured with BMSC(5:1)in 12 well plates,treated with lpmol/mL and 0.1pmol/mL PT-100 or not,the level of TGF-? in the supernatant was measured by ELISA.Results:(1)The level of p-AKT in CD4+ T cells co-cultured with MM-BMSC was up-regulated in comparison with control group,and was down-regulated when treated with 1pmol/mL PT-100.(2)MM-BMSC increased TGF-? secretion of T cells,LY204002 reduced the level of TGF-? by inhibiting PI3K/AKT signaling pathway without direct effect on T clls.(3)Inhibiting PI3K signaling pathway could rescue the CD28 expression of T cells co-cultured with MM-BMSC dramatically,as well as the expression of hTERT.(4)50?mol/mL LY294002 reduced the expression of RORyt,resulting in down-regulated IL-17 secretion.(5)The level of TGF-? in MM-BMSC co-cultured group was up-regulated obviously,and was reduced significantly when treated with 1pmol/mL PT-100.Conclusion:MM-BMSC induced CD4+ T cell senescence and Th17 differentiation by abnormal activation of PI3K/AKT signaling pathway,leading to immunosuppression of T cells.