| Part I: The study of characteristics of human CD19+CD24highCD38high and CD19+CD24highCD27+ regulatory B cellsObjective: To establish the characteristics of human CD19CD24CD38 and CD19+CD24highCD27+ regulatory B cells(Breg) in peripheral blood.Methods: The proportion, apoptosis, proliferation, B-cell activating factor receptor(BAFF-R), transmembrane activator calcium modulator and cyclophilin ligand interactor(TACI), B cell maturation Ag(BCMA), B-cell activating factor(BAFF) and a proliferation-inducing ligand(APRIL) binding to CD19+CD24highCD38high and CD19+CD24highCD27+ Breg, the expression of T cell Ig domain and mucin domain 3(Tim-3), the expression of CD20, the expression of CD80 and the expression of CD86 of CD19+CD24highCD38high and CD19+CD24highCD27+ Breg in the peripheral blood mononuclear cells of healthy donors were evaluated by flow cytometry. The cytokines IL-10、TGF-β of CD19+CD24highCD38high and CD19+CD24highCD27+ Breg stimulated by CD40 L and Cp G ODN2006 were evaluated by flow cytometry. The results were compared between CD19+CD24highCD38high and CD19+CD24highCD27+ Breg, CD19+CD24highCD38high Breg and non-CD19+CD24highCD38high, CD19+CD24highCD27+ Breg and non-CD19+CD24highCD27+.Results: The proportion of CD19+CD24highCD27+ Breg(14.10% ± 9.47%) was higher than CD19+CD24highCD38high Breg(5.88% ± 3.18%)(P=0.000). There were no statistically significant differences with apoptosis of CD19+CD24highCD38high Breg and CD19+CD24highCD27+ Breg(28.87% ± 14.82% vs. 28.84% ± 18.33%, P=0.994), CD19+CD24highCD38high Breg and non-CD19+CD24highCD38high(28.87% ± 14.82% vs. 23.86% ± 11.17%, P=0.994). The apoptosis of CD19+CD24highCD27+ Breg was higher than non-CD19+CD24highCD27+(28.84% ± 18.33% vs. 14.93% ± 7.83%, P=0.002). Therewere no statistically significant differences with proliferation of CD19+CD24highCD38high Breg and CD19+CD24highCD27+ Breg(21.46% ± 11.88% vs. 17.28% ± 13.18%, P=0.442), CD19+CD24highCD27+ Breg and non-CD19+CD24highCD27+(17.28% ± 13.18% vs. 12.96% ± 11.93%, P=0.570). The proliferation of CD19+CD24highCD38high Breg was higher than non-CD19+CD24highCD38high(21.46% ± 11.88% vs. 6.22% ± 10.07%, P=0.009). CD19+CD24highCD38high Breg and CD19+CD24highCD27+ Breg all expressed BAFF-R, TACI and BCMA, and there were no statistically significant differences with BAFF-R, TACI and BCMA between CD19+CD24highCD38high and CD19+CD24highCD27+ Breg(BAFF-R: 97.17% ± 6.17% vs. 96.46% ± 8.64%, P=0.743; TACI: 65.84% ± 28.60% vs. 71.43% ± 36.51%, P=0.558; BCMA: 91.15% ± 9.62% vs. 90.28% ± 13.61%, P=0.799). The expression of BAFF-R and TACI in CD19+CD24highCD38high Breg were higher than non-CD19+CD24highCD38high(97.17% ± 6.17% vs. 89.56% ± 7.43%、65.84% ± 28.60% vs. 39.07% ± 23.92%, P=0.000、0.002). There was no statistically significant difference with expression of BCMA between CD19+CD24highCD38high Breg and non-CD19+CD24highCD38high(91.15% ± 9.62% vs. 94.13% ± 3.15%, P=0.495). There were no statistically significant differences with expression of BAFF-R, TACI and BCMA between CD19+CD24highCD27+ Breg and non- CD19+CD24highCD27+(98.71% ± 1.10%、56.14% ± 28.75%、94.50% ± 4.08%, P=0.142, 0.130, 0.481). There were no statistically significant differences with BAFF and APRIL binding to receptors between CD19+CD24highCD38high and CD19+CD24highCD27+ Breg(55.24% ± 17.46% vs. 54.42% ± 20.36%, P=0.882; 61.95% ± 31.66% vs. 63.97% ± 35.07%, P=0.835). The level of APRIL binding to CD19+CD24highCD38high Breg was higher than non-CD19+CD24highCD38high(35.29% ± 22.08%, P=0.002), but there was no statistically significant difference with BAFF between CD19+CD24highCD38high Breg and non-CD19+CD24highCD38high(58.95% ± 12.59%, P=0.424). There were no statistically significant differences with BAFF and APRIL binding to their receptors between CD19+CD24highCD27+ Breg and non-CD19+CD24highCD27+(59.05% ± 15.89%,57.76% ± 33.04%, P=0.404, 0.546). Tim-3 of CD19+CD24highCD38high Breg was higher than CD19+CD24highCD27+ Breg(14.27% ± 6.97% vs. 6.91% ± 5.88%, P=0.014) and non-CD19+CD24highCD38high(14.27% ± 6.97% vs. 1.44% ± 1.71%, P=0.000). Tim-3 of CD19+CD24highCD27+ Breg was higher than non-CD19+CD24highCD27+(6.91% ± 5.88% vs. 1.99% ± 2.01%, P=0.002). There were no statistically significant differences with CD20 expression betweenCD19+CD24highCD38high Breg and CD19+CD24highCD27+ Breg(96.89% ± 7.90% vs. 97.12% ± 5.44%, P=0.912), CD19+CD24highCD27+ Breg and non-CD19+CD24highCD27+(97.12% ± 5.44% vs. 96.76% ± 3.38%, P=0.798). CD20 expression of CD19+CD24highCD38high Breg was higher than non- CD19+CD24highCD38high(96.89% ± 7.90% vs. 88.67% ± 9.22%, P=0.004). There were no statistically significant differences with IL-10、TGF-β of CD19+CD24highCD38high and CD19+CD24highCD27+ Breg stimulated by CD40 L and Cp G ODN2006(CD40L: 7.81% ± 2.67% vs. 7.17% ± 2.47%, 7.05% ± 2.66% vs. 8.14% ± 2.44%, P=1.000, 0.404; Cp G ODN2006: 10.13% ± 12.19% vs. 9.88% ± 6.48%, 7.38% ± 2.98% vs. 9.03% ± 3.75%, P=0.421, 0.763). There was no statistically significant difference with CD80 of CD19+CD24highCD38high and CD19+CD24highCD27+ Breg(90.01% ± 8.78% vs. 83.62% ± 16.51%, P=0.113), CD19+CD24highCD27+ Breg and non-CD19+CD24highCD27+(83.62% ± 16.51% vs. 84.42% ± 16.89%, P=0.690). CD80 of CD19+CD24highCD38high Breg was higher than non-CD19+CD24highCD38high(90.01% ± 8.78% vs. 66.78% ± 17.32%, P=0.000). There was no statistically significant difference with CD86 of CD19+CD24highCD38high and CD19+CD24highCD27+ Breg(89.31% ± 11.00% vs. 89.49% ± 10.87%, P=0.515), CD19+CD24highCD27+ Breg and non-CD19+CD24highCD27+(89.49% ± 10.87% vs. 89.63% ± 10.74%, P=0.546). CD86 of CD19+CD24highCD38high Breg was higher than non-CD19+CD24highCD38high(89.31% ± 11.00% vs. 85.37% ± 13.64%, P=0.000).Conclusion: The proportion of CD19CD24CD27 Breg was higher than CD19+CD24highCD38high Breg, the apoptosis of CD19+CD24highCD27+ Breg was higher than non-CD19+CD24highCD27+, the proliferation of CD19+CD24highCD38high Breg was higher than non-CD19+CD24highCD38high. There were no statistically significant differences with BAFF-R, TACI and BCMA between CD19+CD24highCD38high and CD19+CD24highCD27+ Breg. There were no statistically significant differences with BAFF and APRIL binding to their receptors between CD19+CD24highCD38high and CD19+CD24highCD27+ Breg. The expression of BAFF-R and TACI in CD19+CD24highCD38high Breg were higher than non-CD19+CD24highCD38high. APRIL binding to receptors of CD19+CD24highCD38high Breg was higher than non-CD19+CD24highCD38high. Tim-3 of CD19+CD24highCD38high Breg was higher than CD19+CD24highCD27+ Breg, Tim-3 of Bregs were higher than non-Bregs. There were no statistically significant differences with IL-10、TGF-β of CD19+CD24highCD38high andCD19+CD24highCD27+ Breg. CD19+CD24highCD38high and CD19+CD24highCD27+ Breg all expressed CD20. CD80 and CD86 of CD19+CD24highCD38high Breg were higher than non-CD19+CD24highCD38high.Part II: The quantity changes of CD19+CD24highCD38high and CD19+CD24highCD27+ regulatory B cells in chronic graft-versus-host disease and the study of mechanismsObjective: To establish the quantity changes of CD19+CD24highCD38high and CD19+CD24highCD27+ regulatory B cells in chronic graft-versus-host disease(c GVHD) of allogeneic hematopoietic stem cell transplantation and the study of mechanisms.Methods: The proportion, apoptosis, proliferation, BAFF-R, TACI, BCMA, BAFF and APRIL binding to CD19+CD24highCD38high and CD19+CD24highCD27+ Breg in the peripheral blood mononuclear cells of mild-moderate c GVHD patients, severe c GVHD patients, non-c GVHD patients, immune tolerance patients and healthy donors were evaluated by flow cytometry. The level of plasma BAFF and APRIL of each group were measured by enzyme-linked immunosorbent assay(ELISA). On the basis of blood cell counts calculated the number of CD19+CD24highCD38high and CD19+CD24highCD27+ Breg. The results were compared between each group.Results: The absolute numbers of B cell [(3.83±2.20)×10/L], CD19+CD24highCD38high Breg [(13.31±13.53)×102/ml], CD19+CD24highCD27+ Breg [(27.68±32.88)×102/ml] and the proportion of CD19+CD24highCD38high Breg(3.45%±1.75%), CD19+CD24highCD27+ Breg(2.43%±1.42%) of mild-moderate c GVHD group were lower than non-c GVHD group [(10.85±10.40)×107/L、(87.36±106.40)×102/ml、(57.59±61.85)×102/ml 、 6.26%±3.23% 、 5.06%±3.48%(P<0.05)]. The apoptosis of CD19+CD24highCD38high Breg(48.40% ± 19.15%), the level of plasma BAFF [(2877.90 ± 248.76) ng/L], BAFF binding to CD19+CD24highCD38high Breg(67.84%±19.12%) in mild-moderate c GVHD group were higher than non-c GVHD group [22.23% ± 12.05%、(2495.60 ± 386.78) ng/L 、 60.76%±18.90%(P<0.05)]. The apoptosis of CD19+CD24highCD38high Breg(72.34% ± 19.20%), the level of plasma BAFF [(3171.60 ± 328.50) ng/L], BAFF/CD19+CD24highCD38high Breg ratio(15756.00 ± 25144.61),BAFF/CD19+CD24highCD27+ Breg ratio(8103.10 ± 12623.27), APRIL/CD19+CD24highCD38high Breg ratio(16.35 ± 32.03), APRIL/CD19+CD24highCD27+ Breg ratio(8.53 ± 12.05), TACI of CD19+CD24highCD38high Breg(93.36%±9.76%), BAFF binding to CD19+CD24highCD38high Breg(79.12%±19.62%), BAFF binding to CD19+CD24highCD27+ Breg(79.27%±18.57%) and APRIL binding to CD19+CD24highCD38high Breg(87.64%±23.69%) of severe c GVHD group were higher than non-c GVHD group(P<0.05). The absolute numbers of B cell [(2.58±3.75)×107/L], CD19+CD24highCD38high Breg [(2.97±5.89)×102/ml], CD19+CD24highCD27+ Breg [(7.24±11.27)×102/ml], the proportion of CD19+CD24highCD38high Breg(0.70%±0.40%) and CD19+CD24highCD27+ Breg(2.22%±0.87%), the proliferation of CD19+CD24highCD38high Breg(2.02% ± 1.68%) and CD19+CD24highCD27+ Breg(4.09% ± 2.44%) of severe c GVHD group were lower than non-c GVHD group(P<0.05). The proportion and apoptosis of CD19+CD24highCD38high Breg of severe c GVHD group were lower than mild-moderate c GVHD group(P<0.05). The apoptosis of CD19+CD24highCD38high Breg, the level of plasma BAFF, BAFF/CD19+CD24highCD38high Breg ratio, BAFF/CD19+CD24highCD27+ Breg ratio, APRIL/CD19+CD24highCD38high Breg ratio, APRIL/CD19+CD24highCD27+ Breg ratio, BAFF binding to CD19+CD24highCD38high Breg and CD19+CD24highCD27+ Breg of severe c GVHD group were higher than mild-moderate c GVHD group(P<0.05). In immune tolerance group, the absolute numbers of B cell [(13.30±15.84)×107/L] and CD19+CD24highCD38high Breg [(168.05±220.05)×102/ml], the proportion of CD19+CD24highCD38high Breg(11.40%±7.94%), the apoptosis of CD19+CD24highCD27+ Breg(47.91% ± 20.39%), the proliferation of CD19+CD24highCD38high Breg(33.75% ± 13.47%), the level of plasma BAFF [(3152.80 ± 752.48) ng/L] and APRIL [(87.68 ± 19.32) ng/L], BAFF and APRIL binding to CD19+CD24highCD38high Breg(68.16% ± 17.46%, 81.89% ± 21.98%) were higher than health control group [(4.68 ± 2.96)×107/L 、(27.48 ± 24.99)×102/ml 、5.88%±3.18%、28.84% ± 18.33%、21.46% ± 11.88%、(2218.80 ± 229.32) ng/L、(69.83 ± 10.27) ng/L、55.24% ± 17.46%、61.95% ± 31.66%(P<0.05)]. In immune tolerance group, the absolute numbers of CD19+CD24highCD27+ Breg [(42.11 ± 33.25)×102/ml], the proportion of CD19+CD24highCD27+ Breg(4.14% ± 2.21%), the apoptosis of CD19+CD24highCD38high Breg(15.18% ± 14.48%) and the proliferation of CD19+CD24highCD27+ Breg(7.01% ± 5.62%) were lower than health control group[(90.48 ± 57.31)×102/ml、14.10% ± 9.47%、28.87% ± 14.82%、17.26% ± 13.18%(P<0.05)]. In non-c GVHD group, the absolute numbers of B cells, the apoptosis of CD19+CD24highCD27+ Breg, the level of plasma APRIL, BAFF and APRIL binding to CD19+CD24highCD38high Breg and APRIL binding to CD19+CD24highCD27+ Breg were higher than health control group(P<0.05). In non-c GVHD group, the absolute numbers and proportion of CD19+CD24highCD27+ Breg and the apoptosis of CD19+CD24highCD38high Breg were lower than health control group(P<0.05). In immune tolerance group, the absolute numbers and proportion of CD19+CD24highCD38high Breg and the level of plasma BAFF were higher than non-c GVHD group(P<0.05). TACI of CD19+CD24highCD27+ Breg in immune tolerance group(58.50% ± 28.08%) was lower than non-c GVHD group(78.18% ± 28.75%)(P<0.05). In each group, there were no statistically significant differences with BAFF-R and BCMA of CD19+CD24highCD38high Breg and CD19+CD24highCD27+ Breg(P<0.05).Conclusion: After allogeneic hematopoietic stem cell transplantation, CD19+CD24highCD38high Breg and CD19+CD24highCD27+ Breg from c GVHD patients were less frequent than Bregs from patients without c GVHD, and Bregs from severe c GVHD patients were less frequent than mild-moderate c GVHD patients. In immune tolerance group, the proportion of CD19+CD24highCD38high Breg were higher than health control group, but the proportion of CD19+CD24highCD27+ Breg were lower than health control group. The changes of quantity of Breg associated with apoptosis and proliferation.Part III: The function changes of CD19+CD24highCD38high and CD19+CD24highCD27+ regulatory B cells in chronic graft-versus-host disease and the study of mechanismsObjective: To establish the function changes of CD19CD24CD38 and CD19+CD24highCD27+ regulatory B cells in chronic graft-versus-host disease(c GVHD) of allogeneic hematopoietic stem cell transplantation and the study of mechanisms.Methods: The cytokines IL-10 、 TGF-β of CD19+CD24highCD38high and CD19+CD24highCD27+ Breg in the peripheral blood mononuclear cells of mild-moderatecGVHD patients, severe cGVHD patients, non-cGVHD patients, immune tolerance patients and healthy donors stimulated by CD40 L and Cp G ODN2006 were evaluated by flow cytometry. The STAT3 phosporylation(p-STAT3), level of My D88, CD40, TLR9, CD80, CD86 and Tim-3 of CD19+CD24highCD38high and CD19+CD24highCD27+ Breg were evaluated by flow cytometry. The results were compared between each group.Results: In mild-moderate and severe cGVHD patients, the proportion of IL-10 of CD19+CD24highCD38high Breg(2.32% ± 0.42%、0.56% ± 0.54%) stimulated by CD40 L were lower than without c GVHD patients(5.88% ± 3.11%)(P<0.05), there was no statistically significant difference with TGF-β in each group. In severe c GVHD patients, the proportion of IL-10 of CD19+CD24highCD27+Breg(2.02% ± 1.90%) stimulated by CD40 L were lower than without c GVHD patients(4.94% ± 3.81%)(P<0.05), there was no statistically significant difference with TGF-β in each group. After stimulated by Cp G ODN2006, there were no statistically significant differences with IL-10 and TGF-β of CD19+CD24highCD38high Breg in each group, the proportion of IL-10 of CD19+CD24highCD27+Breg in mild-moderate and severe c GVHD patients(4.02% ± 1.91%、1.69% ± 1.25%) were lower than without c GVHD patients(8.80% ± 5.98%)(P<0.05), there was no statistically significant difference with TGF-β in each group. In mild-moderate and severe c GVHD patients, the level of p STAT3 of CD19+CD24highCD38high Breg(6.48% ± 10.20%、3.06% ± 4.80%) were lower than without c GVHD patients(14.79% ± 7.05%)(P<0.05). There was no statistically significant difference with My D88 of CD19+CD24highCD38high Breg in each group. In severe c GVHD patients, the level of p STAT3 and My D88 of CD19+CD24highCD27+Breg(3.31% ± 2.09%, 41.31% ± 29.52%) were lower than without c GVHD patients(27.98 ± 12.65, 69.41% ± 24.09%)(P<0.05). Tim-3 of CD19+CD24highCD38high and CD19+CD24highCD27+ Breg in severe c GVHD group(2.07% ± 0.58%、2.59% ± 2.44%) were lower than non-c GVHD group(11.35% ± 9.72%,16.34% ± 22.52%)(P<0.05). In immune tolerance group, Tim-3 of CD19+CD24highCD27+ Breg(23.49% ± 23.19%) was higher than health control group(6.91% ± 5.88%)(P<0.05). There were no statistically significant differences with level of CD40, TLR9, CD80 and CD86 of CD19+CD24highCD38high and CD19+CD24highCD27+ Breg in each group(P<0.05).Conclusion: After allogeneic hematopoietic stem cell transplantation,CD19+CD24highCD38high Breg and CD19+CD24highCD27+ Breg from c GVHD patients were impairment in IL-10 production than Bregs from patients without c GVHD, but there was no statistically significant difference with TGF-β production. Impairment in IL-10 production of CD19+CD24highCD38high Breg associated with reduced p STAT3, impairment in IL-10 production of CD19+CD24highCD27+ Breg associated with reduced p STAT3 and My D88. Tim-3 of CD19+CD24highCD38high and CD19+CD24highCD27+ Breg in c GVHD group were lower than non-c GVHD group. In immune tolerance group, Tim-3 of CD19+CD24highCD27+ Breg was higher than health control group.Part IV: Screening of biological marker gene associated with B cell in c GVHD patientsObjective: Screening of biological marker gene associated with B cell in c GVHD patientsMethods: Microarrays were used to detect expressed gene profiles of peripheral blood mononuclear cells total RNA from subjects in the mild-moderate c GVHD, severe c GVHD, non-c GVHD, immune tolerance and healthy donors groups. Found out the genes associated with B cell in c GVHD patients reached statistical significance after a false discovery rate(FDR) correction was applied to the data. Real-time quantitative RT-PCR was used to confirm the B cell genes.Results: 284 genes were found by microarrays between cGVHD patients and patients without c GVHD, 5 genes(CDKN2A, SOX4, ZBTB32, CD70, IL21R) associated with B cell. 800 genes were found by microarrays between immune tolerance patients and patients without c GVHD, 3 genes(NFAM1, CD27, BST1) associated with B cell. Real-time quantitative RT-PCR confirmed significantly lower expression of CDKN2 A in c GVHD relative to without c GVHD patients(P=0.001). Significantly higher expression of CD27 and lower expression of BST1 in immune tolerance relative to without c GVHD patients(P=0.038, P=0.022).Conclusion: Significantly lower expression of CDKN2 A in cGVHD relative to without c GVHD patients. Significantly higher expression of CD27 and lower expression of BST1 in immune tolerance relative to without c GVHD patients. The changes of thesegenes may be potential biomarkers for c GVHD and immune tolerance. |
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