| Autoimmune hemolytic anemia（AIHA）/Evans syndrome is one kind of immune hemocytopenia in which immune system acting against its own red cell and platelet,resulting in the increasing of RBC or platelet damaged.The life time of RBC or platelet becomes shortened.B lymphocyte plays an important role in AIHA/Evans syndrome.B lymphocyte secretes not only antibodies,but also cytokines.It is also recognized as regulatory B cells（Bregs）which can regulate other immune cells.CD5+B cells are regarded as Bregs and have an important role[1].At present,it is recognized that B lymphocytes are heterogeneous.According to CD5 molecule expression,B lymphocyte can be divided into two subgroups:CD5+and CD5-B lymphocyte[2].Previous studies have found that the number of CD5+B lymphocytes in patients with AIHA/Evans syndrome is increased,which is related to the severity of the disease[3],mainly secreting IL-10[4],and it was speculated that they play an immunomodulatory role in AIHA/Evans syndrome.How does CD5+B cell regulate other cells?Do they regulate other cells by Foxp3 or PD-L1?We intend to detect the expression of CD5 molecules on the surface of B and T lymphocytes in peripheral blood of AIHA/Evans syndrome patients,the concentration of CD5 in serum and their clinical significance will be analyzed.The regulatory molecules,such as Foxp3,PD-L1,PD-L2 and FasL,of CD5+B lymphocytes will be measured.The correlation between the above indexes and clinical indexes was analyzed.Part I CD5 and its clinical significance in AIHA/Evans patientsObjective:To understand the concentration of CD5 molecular and its clinical significance in peripheral blood of patients with AIHA/Evans syndrome.Methods:Flow cytometry（FCM）was used to detect the mean fluorescence intensity（MFI）of CD5 in B and T lymphocytes of patients with AIHA/Evans syndrome（hemolytic patients:18 cases;remission patients:12 cases）,and the level of serum CD5was measured by enzyme-linked immunosorbent assay（ELISA）,13 healthy controls（HCs）were used as negative controls,6 CLL patients as positive controls.The correlation between the above indexes and clinical indexes was analyzed.Result:1、CD5 MFI[（41.67±14.44）%]of CD19+B lymphocytes in the hemolytic group of AIHA/Evans syndrome was higher than that in the remission group[（20.50±9.75）%,P=0.0005]and HCs[（18.43±15.80）%,P=0.018].CD5 MFI of CD19+B lymphocytes was negatively correlated with RBC（R=-0.624,P=0.002）,Hb（R=-0.668,P=0.0005）,and positively correlated with Reticulocyte%（R=0.97,P<0.0001）.2.CD5 MFI of CD4+T lymphocyte in the hemolytic group patients of AIHA/Evans syndrome[（16029±4892）%]was lower than that in the remission patients[（22895±4892）%,P<0.01]and HCs[（22967±5372）%,P<0.01],higher than that in the CLL patients[（8834±6043）%,P<0.05],the difference was statistically significant.3.CD5 MFI of CD8+T lymphocytes in the hemolytic group patients of AIHA/Evans syndrome was lower than that in the remission patients[（14779±4165）%,P<0.05]and HCs[（5096±4271）%,P<0.05],higher than that in the CLL patients[（5096±4271）%,P<0.05].4.The concentration of CD5[（4.08±5.80）ng/ml]in serum in the hemolytic group patients of AIHA/Evans syndrome was higher than that in HCs[（1.44±1.77）ng/ml,（P<0.01）].The concentration of serum CD5[（5.87±7.74）ng/ml]in the hemolytic group patients of AIHA/Evans syndrome was higher than that in the remission patients[（2.30±1.72）ng/ml,P=0.097]and HCs[（1.44±1.77）ng/ml,（P<0.01）].The difference between hemolytic group and control group was statistically significant.The level of serum CD5 was positively correlated with the absolute count of Ret（R=0.415;P=0.023）,complement C4（R=0.430;P=0.036）,CRP（R=0.896;P<0.0001）.Conclusion:CD5 MFI of different lymphocytes is variable in AIHA/Evans syndrome patients.CD5 MFI on B lymphocytes increased,and on T lymphocytes decreased.Serum free CD5 may play an immunomodulatory role in AIHA.Part II Study on regulatory function of CD5+B lymphocytes in AIHA/Evans patientsObjective:To understand the immunic regulation of peripheral blood CD5+B lymphocytes in patients with AIHA/Evans syndromeMethods:The expression of Foxp3,PD-L1,PD-L2 and FasL in CD19+CD5+and CD19+CD5-B lymphocytes were measured by flow cytometry（FCM）in 19 patients with hemolytic patients of AIHA/Evans syndrome,12 patients with remission patients of AIHA/Evans syndrome and HCs.Foxp3,PD-L1,PD-L2 and FasL m RNA in CD19+B lymphocytes was measured by RT-PCR,and the correlation between them and clinical indexes was analyzed.Result:1.The proportion of CD19+CD5+Foxp3/CD19+CD5+B lymphocytes in the hemolytic group patients of AIHA/Evans syndrome[（62.57±21.77）%]was higher than that in the remission patients[（28.97±24.31）%,P<0.01]and HCs[（23.04±16.40）%,P<0.0001].The proportion of CD19+CD5+Foxp3/CD19+B lymphocytes in the hemolytic group patients of AIHA/Evans syndrome[（11.42±15.71）%]was higher than that in the remission patients[（1.81±2.01）%,P<0.05]and HCs[（1.66±1.37）%,P<0.05].The proportion of CD19+CD5+Foxp3+/CD19+CD5+B lymphocytes in patients of AIHA/Evans syndrome was negatively correlated with Hb（R=0.57,P=0.007）,and positively correlated with TBIL（R=0.467,P=0.033）,IBIL（R=0.438,P=0.047）.2.The proportion of CD19+CD5-Foxp3/CD19+CD5-B lymphocytes in the hemolytic group patients of AIHA/Evans syndrome was higher than that in the remission patients[（14.37±13.84）%,P<0.05]and HCs[（13.71±16.76）%,P<0.05].The proportion of CD19+CD5-Foxp3+/CD19+B lymphocytes in the hemolytic group patients[（30.37±23.07）%]was higher than that in the remission patients[（12.63±8.69）%]and HCs[（10.74±12.67）%,P<0.01].The proportion of CD19+CD5-Foxp3+/CD19+CD5-B lymphocytes in patients of AIHA/Evans syndrome was positively correlated with TBIL（R=0.544,P=0.001）、IBIL（R=0.499,P=0.021）;The proportion of CD19+CD5-Foxp3+/CD19+in patients of AIHA/Evans syndrome was negatively correlated with HB（R=0.487,P=0.025）,and positively correlated with TBIL（R=0.632,P=0.002）、IBIL（R=0.584,P=0.005）.3.The proportion of CD19+CD5+Foxp3/CD19+CD5+B lymphocytes[（62.57±21.77）%]was higher than that of CD19+CD5-Foxp3/CD19+CD5-B lymphocytes[（36.04±27.65）%,P<0.05]in the hemolytic group patients of AIHA/Evans syndrome.4.The proportion of CD19+Foxp3+/CD19+lymphocytes in the hemolytic group patients of AIHA/Evans syndrome[（29.77±27.61）%]was higher than that in HCs[（12.39±13.38）%,P<0.05].The proportion of CD19+Foxp3+/CD19+B lymphocytes in the hemolytic group patients of AIHA/Evans syndrome[（41.27±31.40）%]was higher than that in the remission group[（14.44±9.31）%,P=0.09]and HCs[（12.39±13.38）%,P<0.05].The difference between the hemolytic group and HCs group was statistically significant.The proportion of CD19+Foxp3+/CD19+B lymphocytes in patients of AIHA/Evans syndrome was positively correlated with TBIL（R=0.587,P=0.005）,DBIL（R=0.684,P=0.005）,IBIL（R=0.545,P=0.011）,and negatively correlated with HB（R=-0.539,P=0.011）.5.The expression of Foxp3 m RNA in CD19+B lymphocytes in the hemolytic group patients（86.72±91.87）was significantly higher than that in the remission patients（26.10±26.16）and HCs（8.53±14.76）,The difference was statistically significant（P<0.05）.6.The ratio of CD19+CD5+PD-L1+/CD19+CD5+in the hemolytic group patients[（5.93±4.67）%]of AIHA/Evans syndrome was lower than that in remission patients[（16.45±12.91）%,P<0.01]and HCs[（9.67±9.50）%].The proportion of CD19+CD5+PD-L1+/CD19+CD5+in patients of AIHA/Evans syndrome was positively correlated with RBC（R=0.461,P=0.009）,HB（R=0.359,P=0.047）,C3（R=0.485,P=0.008）and C4（R=0.395,P=0.034）,and negatively correlated with RET counting（R=0.371,P=0.040）.The ratio of CD19+CD5-PD-L1+/CD19+in patients of AIHA/Evans syndrome was positively correlated with Ig E（R=0.384,P=0.040）.7.The expression of PD-L1 m RNA in CD19+B lymphocytes in the hemolytic group patients of AIHA/Evans syndrome was not significantly different from that in remission patients and HCs.8.There was no significant difference in PD-L2 of CD5+B lymphocytes among hemolytic group patients,remission patients and controls.The ratio of CD19+CD5-PD-L2+/CD19+in patients of AIHA/Evans syndrome was positively correlated with Ig E（R=0.496,P=0.040）.There was no significant difference in the expression of PD-L2 m RNA in CD19+B lymphocytes of each group.9.The proportion of CD19+CD5+FasL+/CD19+CD5+B lymphocytes[（6.64±6.70）%]was higher than that of CD19+CD5 FasL+/CD19+CD5-B lymphocytes[（1.68±1.54）%,P<0.05]in the hemolytic group patients of AIHA/Evans syndrome.The proportion of CD19+CD5+FasL+/CD19+CD5+B lymphocytes in patients of AIHA/Evans syndrome was positively correlated with C4（R=0.534,P=0.033）,while Ig G（R=0.551,P=0.027）was negatively correlated.There was no significant difference in the expression of FasL m RNA in CD19+B lymphocytes between AIHA/Evans syndrome and HCs.Conclusion:CD5+B lymphocytes from peripheral blood of AIHA/Evans patients may play an immunomodulatory role by Foxp3,but not through PD-L1、PD-L2 and FasL. |
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