| ObjectiveType 1 diabetes(T1D)is an autoimmune disease,which involves the CD4+ T cell as well as CD8+ T cell-mediated destruction of ? cells.In T1D,B cell-depleting therapy using monoclonal anti-CD20 Ab,rituximab(RTX),effectively delays the decline of islet ? cell function.But the mechanisms underlining the therapeutic efficacy remain to be determined.Among the most common explanations are elimination of antigen presentation cells and reduction of costimulatory signals and pro-inflammatory cytokines produced by B cells.However,in addition to B cells,a small population of T lymphocytes(T cells)was also shown to co-express CD20 in human.This study was designed to examine the existence of CD20+ T cells in peripheral blood of T1D patients and healthy controls(HC)and also to confirm the effect of RTX to this subset.Next we try to examine the proportions of IFN-y-secreting CD4+T cells(Thl cells)and IL-17-secreting CD4+T cells(Th17 cells)and determine the CD20 expression in these two subsets.MethodsA total of 51 T1D patients who came to Jiangsu Province Hospital from Oct 2014 to Feb 2016 and 22 HC were enrolled.The mean ages of these two group were 30.82±17.1 years and 30.2±8.1 years,respectively.The phenotypes of peripheral blood cells from T1D patients and HC were examined by cell surface immunofluorescence staining.Cytokine production cells including Thl cells and Th17 cells and the expression of CD20 were determined by intracellular staining.6 of 55 T1D patients received low dose RTX therapy.Four intravenous infusions of RTX were given on days 1,8,15,22.Visits were made during weekl-4,5-12,13-24 after the fourth infusion.Blood samples were collected and phenotypic analysis of CD20+T cells was made.ResultsFlow cytometry analysis of freshly isolated peripheral blood cells from T1D patients revealed that a mean frequency of 5.91%±2.8%of T cells co-expressed CD20,which was not significantly different from that of HC(7.11%±2.3%,p=0.089).However,CD20 co-expressed on the surface of T cells showed a comparatively lower density with that of B cells.Phenotypic analysis of T cells revealed that CD3+CD20+T cells comprised both CD8+ cytotoxic cells and CD4+ helper cells.Among CD3+CD20+T cells,CD4+cells were increased in T1D patients(38.21%±9.65%)compared with HC(30.32%±5.7%,p=0.089)whereas CD8+ cells were the same(50.11%±9.03%vs.53.95%±9.67%,p=0.108).Interestingly,among CD3+CD20+T cells in T1D patients,CD8+ cells(50.11%±9.03%)were increased compared with CD4+ cells(38.21%±9.65%,p<0.001).Th1 cells were increased in the peripheral blood of T1D patients(15.4%±7.4%)compared with HC(10.1%±3.3%,p=0.001).About 20.1%±.4%of this subset also expressed antigen of CD20 in T1D patients which was higher than that of HC(14.4%±8.0%,p=0.04).Th17 cells were also increased in the peripheral blood of T1D patients(5.1%±2.5%)compared with HC(3.3%±1.3%,p=0.002).Approximately 20.1%±9.4%of Th17 subset co-expressed antigen of CD20 in T1D patients higher than that of HC(12.6%±2.7%,p=0.021).CD3+CD20+T cells were effectively depleted by RTX from peripheral blood of T1D patients.1-4 week after fourth infusion of RTX,mean frequency of CD3+CD20+T cells remained in the peripheral blood was 0.14%±0.19%.ConclusionIn T1D patients,a subset of Tcells co-expressed B cell maker-CD20 dimly.CD3+CD20+CD4+T cells in T1D patients were increased compared with HC.Among CD4+T cells,Thl cells and Th17 cells showed significantly higher expression of CD20 in T1D patients compared with HC.RTX could effectively eliminating CD3+CD20+T cells in the peripheral blood of T1D patients.The pathological effect of this subset in T1D remains to be explored.However,thinking of the potential pro-inflammatory ability,elimination of CD20-expression T cells may also contribute to the therapeutic effect of RTX. |
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