The Role Of OX40 In The Regulation Of Double Negative T Cells Conversion And Survival

Objectives: Monitoring T lymphocyte proliferation,especially in vivo,is essential for the evaluation of adaptive immune reactions.Flow cytometry-based proliferation assays have advantages in measuring cell division of different T lymphocyte subsets at the same time by multicolor labelling.In this study,we aimed to establish the use of 5-Ethynyl-2'-deoxyuridine(EdU)incorporation in vivo to monitor T lymphocyte proliferation by flow cytometry with an adoptive transfer model.Methods: Splenocytes from B6 mice were stained with anti-mouse CD3/CD4/CD8/ CD19/CD25 antibodies conjugated with different fluorochromes.Then 4% paraformaldehyde and different concentration of saponin and Triton X-100 and Click reaction were used to explore the effect of fixation,permeabilization reagents and EdU staining on the integrity of cell surface antigens and the fluorescence intensity of APC,PE,PECy7,FITC and Per CP-Cy5.5 during T lymphocytes proliferation detection progress.In order to explore the best EdU administration route and dose in the lymphocytes proliferation in vivo,B6D2F1 mice(H2d/b)were injected with 1×107 MHC-mismatched splenocytes from B6-CD45.1 mice(H2b)through tail vein injection or intraperitoneal injection,and then the mice received injections of EdU intraperitoneally at a dose of 2.5,5,10,20,or 30 mg/kg/day body weight for 3 days.CD45.1+CD3+T cells,CD45.1+CD4+T cells and CD45.1+CD8+T cells were analyzed by flow cytometry for proliferation respectively.B6D2F1 mice were adoptively transferred with 1×107 CD45.1-positive splenocytes labelled with or without CFSE.Then,recipient B6D2F1 mice were injected intraperitoneally with 20 mg/kg EdU or Brd U once a day for three days to evaluate the efficacy of EdU incorporation in the detection of T lymphocyte proliferation in vivo.Results: Fixation followed by permeabilization preserved T cell surface antigens and had no obvious effects on the fluorescence intensity of APC,PE,PE-Cy7,FITC and Per CP-Cy5.5 when the concentration of the permeabilization reagents was optimized.However,the Click reaction resulted in a significant decrease in the fluorescence intensity of PE and PE-Cy7,and surface staining after the Click reaction improved the fluorescence intensity.Thus,an extra step of blocking with PBS with 3% FBS between the Click reaction and cell surface staining is needed.Furthermore,the percentage of EdU-positive cells increased in a dose-dependent manner,and the saturated dose of EdU was 20 mg/kg.Intraperitoneal and intravenous injection had no differences in lymphocyte proliferation detection with EdU in vivo.In addition,T cell proliferation measured by EdU incorporation was comparable to Brd U but was lower than CFSE labelling.Conclusion: 1.It was a feasible method for the measurement of T lymphocyte proliferation with EdU incorporation by flow cytometry in vivo.2.T cell proliferation measured by EdU incorporation was comparable to Brd U.3.EdU incorporation could be used to measure resident immune cell proliferation in vivoBackground & Aims: Converted double negative T cells(DNT)play a key role on immune tolerance in different disease models.However,the origin of DNT was still unclear.Our result showed that DNT could be converted from CD4+ T cell when CD4+ T cell had undergone 4 to 5 rounds of alloantigen-triggered proliferation.Costimulation provided critical signals that enhanced the survival of T cells.The aim of this study was to explore the function and mechanism of OX40 costimulation in DNT conversion and proliferation.Methods: The efficiency of DNT cell conversion was detected when OX40/OX40 L,CD40/CD40 L and CD28/B7 costimulation were blocked by neutralization antibody and gene knockout.Then OX40 expression on DNT was measured.The proliferation and apoptosis of B6 DNT and OX40 KO DNT were compared in vitro and in vivo by a mix lymphocytes reaction or an adoptive transfer model respectively.The proliferation and apoptotic rates in differernt time were detected through EdU incorporation and Annexin V.At the same time,we also evaluated the apoptosis related molecules Bcl-2,Bcl-x L,Survivin and Bcl2 like protein 11(BCL2L11)expression through flow cytometry and real-time PCR.In addition,to identify whether IL-2 regulated the survival of DNT directly by up-regulating OX40 expression,IL-2 or IL-2 Fc protein were used to stimulate B6 DNT and OX40 KO DNT in vitro and in vivo.The proliferation,apoptotic rates and apoptosis related protein were detected after IL-2 stimulation.At last,potential signaling pathway which might be involved in IL-2 regulating OX40 expression on DNT was also addressed.Results: The efficiency of DNT cell conversion decreased when OX40/OX40Lcostimulation were blocked by neutralization antibody and gene knockout.OX40 expression on DNT was higher than activated CD4 T cells,and DNT showed lower level of apoptotic rate.When OX40 was knocked out,the proliferation rates of DNT in vitro and in vivo were lower;in contrast,the apoptotic rates increased.OX40 promoted DNT survival through the induction of the anti-apoptotic molecules,Bcl-2,Bcl-x L,survivin and down-regulation of pro-apoptotic molecule BCL2L11.NF-?B cell signaling took part into this progress.IL-2 stimulation could up-regulate the OX40 expression on DNT.Compared with B6 DNT,B6 DNT + IL-2,and OX40 KO DNT + IL-2 groups,IL-2 stimulation could augment the DNT survival,nevertheless,the proliferation rates of OX40 KO DNT + IL-2 in vitro and in vivo were lower than B6 DNT + IL-2 group,the apoptotic rates were increased.Transcription factor PPAR? agonist could down-regulated the OX40 expression and proliferation rate of DNT,the apoptotic rate was increased with variation of Bcl-2,Bcl-x L and BCL2L11 expression.Conclusions: 1.OX40 costimulation was involved in the process of CD4+ T cells conversion to DNT.2.OX40 ligation promoted DNT survival through the induction of apoptosis related molecules,and NF-?B signaling was involved in OX40-mediated DN T cells survival.3.IL-2 enhanced the survival of DNT in part by up-regulating OX40 expression.