Investigation On CD4+CD25+ Regulatory T Cells In New-onset Patients And Animal Model With SLE

Background and ObjectiveSystemic lupus erythematosus(SLE) is a complicated autoimmune disease characterized by deposition of immune complexes in multiple tissues and organs. The immunological abnormalities in patients with SLE include polyclonal B cell hyperactivity,synthesis of a variety of autoantibodies,increased apoptosis of lymphocyte and deficiency of IL-2 secretion,the etiology and pathogenesis of SLE are still unclear.In 1995,Sakaguchi first demonstrated that there is a subset of CD4+T regulatory T cells in mouse,which constitutively express the IL-2Rαchain (CD25).A number of reports have suggested deficiency of CD4+CD25+T cells be involved in multiple organ and systemic autoimmune diseases.Currently there were some studies investigating CD4+CD25+T regulatory cells in patients with SLE with somewhat different results,one of the possible reasons for discrepancy might be that patients had been treated with a variety of immunosuppressive reagents before they entered the studies.Thus,in present study we investigate numbers and function of CD4+CD25+T cells in patients with new-onset SLE and animal model of lupus to explore the CD4+CD25+ T cells role in SLE.MethodsIn patients,Forty-four new-onset and untreated SLE patients including twenty-four with active(SLEDAI≥10) and twenty with inactive disease (SLEDAI≤5) were enrolled in this study.Twenty-one age- and sex-matched healthy volunteers were also included as controls.Peripheral blood samples were collected and mononuclear cells isolated.The expression of CD25,Foxp3 in CD4+ T cells were analyzed by flow cytometry(FACS).Proliferation assay were performed on isolated CD4+CD25+ and/or CD4+CD25- T cells. Expression of Foxp3 in peripheral blood from SLE patients and normal controls was determined by real-time RT-PCR assay.Correlations between clinical manifestations and CD4+ subpopulations in new-onset SLE patients were analyzed. In animal model,two strain mice were used in this study,(NZBXNZW) F1 and MRL/1pr.The CD4+CD25+T cells in peripheral and center immune organ were detecting using FACS and CD4+CD25+ T cells proliferation and suppressive function were performed in vitro.ResultsCD4+CD25+(3.95-13.04%) and CD4+CD25^high(0.04-1.34%) T cells in peripheral blood in untreated patients with new-onset active lupus were significantly lower than that in the patients with inactive lupus(7.27-24.48%, P＜0.05 and 0.14-3.07%P＜0.01 respectively) and that in normal controls (5.84-14.84%,P＜0.05),Moreover,suppressive capacity of CD4+CD25+T cells in new-onset lupus patients was not impaired as measured by the capacity to inhibit proliferation of CD4+CD25- effector T cells.Interestingly,the decrease in CD4+CD25^high T cells was restored significantly in patients with active lupus after corticosteroid treatment.There was however significantly higher percentage of CD4+Foxp3+ T cells in patients with active(5.30-23.00%) and inactive(7.46-17.38%) new-onset lupus patients,compared to normal control subjects(2.51-12.94%)(P＜0.01).Intriguingly,CD25 expression in CD4+Foxp3+ T cells in patients with active lupus(25.24-62.47%) was significantly lower than that in those patients with inactive lupus(30.35-75.25%, P＜0.05) and normal controls(54.83-86.38%,P＜0.01).Most strikingly,the levels of Foxp3 expression determined by mean fluorescence intensity(MFI) in CD4+CD25^high cells in patients with active SLE were significantly downregulated compared to normal controls(100.52-160.92 vs.122.58-198.1,P =0.012).There was no significant difference in number of CD4+CD25+Foxp3+T cells in patients with either active(1.91-6.75%) or inactive(2.74-7.01%) SLE compared to normal controls(2.11-9.90%)(P=0.524 and P=0.794 respectively), whereas the percentage of CD4+CD25+Foxp3-T cells in active lupus patients (1.19-9.23%)was significantly less than that in inactive lupus(3.73-8.27%, P=0.048) and in normal controls(2.67-11.26%,P=0.039).Interestingly, CD4+CD25-Foxp3+T cells in new-onset lupus(2.97-10.94%) were significantly more frequent than in normal controls(1.01-3.62%)(P＜0.01),and positively correlated with the titers of anti-disDNA antibodies (P=0.029).Similar to CD4+CD25+Foxp3+ T cells,few of these cell expressed CD127.Treatment with glucocorticoids and cyclophosphamide increased CD4+CD25+Foxp3- T cells and reduced CD4+CD25-Foxp3+ T cells in eight of ten active lupus patients,but had no effect on CD4+CD25+Foxp3+T cells.SLE patients had increased numbers of CD4+CD45RO+T cells and decreased numbers of CD4+CD45RA+T cells,compared to normal control (30.08-57.18%vs 22.59-46.29%,P=0.045 for CD4+CD45+T cells and 20.83-47.82%vs 34.90-60.29%,P=0.018 for CD4+CD45RA+ T cells), moreover the percentage of CD4+CD45RO+CD25+Foxp3+T cells in patients with active(2.16-7.51%) and inactive(2.31-7.05%) had no significant difference with that in normal controls(2.28-7.96%)(P=0.961,P=0.742 respectively). However,CD4+CD45RO+CD25-Foxp3+ T cells in active(2.78-5.50%) and inactive(2.85-5.10%) SLE patients were significantly higher than that in normal controls(1.12-3.17%)(P＜0.001,P＜0.001,repectively).The expression of Foxp3 mRNA in patients with new-onset SLE was significantly high compared to normal control.The concentration of IL-6 in plasma from new-onset SLE patients was more higher than that in normal controls and had positive correlation with SLEDAI(P=0.003).Number of CD4+CD25+T cells in spleen(10.65-13.76%) and lymph node (9.85-11.84%)from(NZBXNZW) F1 were significantly lower than those in Balb/c(12.14-15.79%for spleen and 11.40-14.39%for lymph node)(P=0.016,P=0.01,respectively),whereas there were no difference between MRL/lpr and Balb/c.However,the expression of CD69 on CD4+CD25+T cells from MRL/lpr spleen and lymph node was higher than that in(NZBXNZW)F1 and Balb/c.similar to Balb/c,the CD4+CD25+T cells in spleen and lymph node from(NZBXNZW)F1 were CD62L^low and CD44^high,whereas the number of CD4+CD25+ T cells expressed CD62L^high and CD44^low increased.ConclusionsCD4+CD25^high T cells are low in new-onset patients with active SLE. Despite the percentage of CD4+Foxp3+ T cells appears high,the levels of Foxp3 expression in CD4+CD25^high T cells are down regulated in untreated lupus patients.There is a disproportional expression between CD25^high and Foxp3 on CD4+T cells in new-onset patients with active SLE.There is a significant increase in CD4+CD25-Foxp3+T cells,the function of these cells need to be further investigation.There is a discrepancy in the numbers of CD4+CD25+T cells between(NZBxNZW) F1 and MRL/lpr.Thus,it is a necessary to using various animal models of SLE and combine with the results from patients with SLE to elucidate the role of CD4+CD25+T cells in pathogenesis and immune abnormality of SLE...