The Immune Regulatory Functions Of Circulating CD5+ B Cells Are Impaired In Adult Patients With Primary Immune Thrombocytopenia
Authors: Hua Fanli , Li Feng , Zou Shanhua , Wang Weiguang , Cheng Yunfeng 
Published: October 2019

Abstract

Background and Objective
Immune thrombocytopenia (ITP) is a typical autoimmune-mediated bleeding disorder in which isolated thrombocytopenia is resulted from increased platelet clearance and/or impaired thrombocytogenesis. In addition to producing antibodies and acting as antigen presenting cells, B cells have been proven to play an important role in regulating immune response mainly via production of interleukin 10 (IL-10). CD5+ B cells are able to produce IL-10, and CD5 is deemed as a hallmark of regulatory B cells. We have previously demonstrated insufficient IL-10 secretion by CD5+ B cells in patients with ITP. The objective of the current study was to investigate the alteration in regulatory functions of peripheral blood CD5+B cells in ITP patients.

Methods
Peripheral blood CD5+ B cells, CD5- B cells and CD4+ T cells were magnetically isolated from 7 adult patients with newly diagnosed ITP and 10 healthy controls. CD4+ T cells were labeled with CFSE (5(6)-Carboxyfluorescein N-hydroxysuccinimidyl ester) before cultured in RPMI1640 in the presence of CD3mAb/CD3mAb/IL-2 for 72 hours with or without CD5+/CD5- B cells at gradient B/T ratios. The proliferation of CD4+ T cells was assessed by the dilution of CFSE expression on a flow cytometry. After 72-hour culture, cells were stained with APC-CD4/FITC-Annexin-V/PI to evaluate the apoptosis of CD4+T cells. The concentrations of IL-4 and IFN-γ in cell culture supernatants were determined by ELISA.

Results
Purified CD5+ B cells from healthy subjects were capable of suppressing the proliferation of CD4+ T cells in a dose-dependent manner when co-cultured for 72 hours. The percentage of CD4+ T cells that underwent proliferation showed a statistically significant reduction (72.8 ± 9.4% vs 61.6 ± 12.8%; p = 0.039) in the presence of CD5+ B cells at 1:3 B/T ratio. With the B/T ratio increased to 1:1, the proportion of proliferating CD4+ T cells decreased to 43.7 ± 16.4% (p < 0.001). The apoptosis of CD4+ T cells was promoted by CD5+ B cells as the percentage of apoptotic CD4+ T cells was increased when co-cultured with CD5+ B cells at 1:1 B/T ratio (3.75 ± 0.82% vs 6.03 ± 2.51%; p = 0.014).
In contrast, CD5- B cells did not suppress the proliferation of CD4+ T cells, quite contrary, the proportion of proliferating CD4+ T cells was slightly increased, though not statistically significant, when co-cultured at 1:1 with CD5- B cells for 72 hours as compared with CD4+ T cell cultured alone (72.8 ± 9.4% vs 78.9 ± 8.5%; p = 0.145). No effect of CD5- B cells on the apoptosis of CD4+T cells was observed.
In ITP patients, CD5+ B cells showed compromised regulatory functions as the proliferation of CD4+ T cells was not altered by CD5+ B cells until the B/T ratio increased to 3:1 (1:1 B/T: 74.6 ± 12.5% vs 72.1 ± 16.2%, p = 0.752; 3:1 B/T: 74.6 ± 12.5% vs 60.7 ± 10.3%, p = 0.042, respectively). The ability of CD5+ B cells to promote the apoptosis of CD4+ T cells was also impaired in ITP patients: no difference in the percentage of apoptotic CD4+ T cells was found when CD5+ B cells (1:1 ratio of B to T) were added to the T cell culture system (2.97 ± 1.07% vs 3.24 ± 1.45%; p = 0.699).
We also evaluated the ability of CD5+ B cells to suppress the secretion of IFN-γ, one of the Th1-type cytokines. In healthy subjects, the IFN-γ secretion by CD4+ T cells was decreased by CD5+ B cells at a minimal B/T ratio of 1:5 (2427.99 ± 632.62 pg/mL vs 1734.41 ± 507.16 pg/mL; p = 0.014) and dramatically dropped to 395.22 ± 136.54 pg/mL when the ratio of B/T increased to 1:1 (p < 0.001). The secretion of IL-4, a representative cytokine of Th2 subset, was not influenced by CD5+ B cells. CD5+ B cells from ITP patients showed no capacity for suppressing the IFN-γ secretion until the ratio of B/T increased as high as to 1:1 (3060.02 ± 493.98 pg/mL vs 2394.57 ± 417.42 pg/mL; p = 0.019) and, even under the condition of 5:1 B/T ratio, CD4+ T cells from ITP patients retained about 30% of the ability of IFN-γ secretion (1067.37 ± 499.70 pg/mL; p < 0.001 ).

Conclusions
CD5+ B cells from normal controls are competent for inhibiting the proliferation of CD4+ T cells and suppressing the secretion of IFN-γ by CD4+ T cells, suggesting potent immune regulatory functions of CD5+ B cells. While in patients with ITP, CD5+ B cells are severely functionally impaired, indicating that the immune regulatory dysfunctions in CD5+ B cells may participate in the pathogenesis of ITP.

Disclosures:
No relevant conflicts of interest to declare.