Deficit of circulating CD19+ CD24hi CD38hi regulatory B cells in severe aplastic anaemia

Abstract

Immune aplastic anaemia (AA) is caused by cytotoxic T lymphocytes (CTLs) that destroy haematopoietic stem and progenitor cells. Enhanced type 1 T helper (Th1) responses and reduced regulatory T cells (Tregs) are involved in the immune pathophysiology. CD24^hi CD38^hi regulatory B cells (Bregs) suppress CTLs and Th1 responses, and induce Tregs via interleukin 10 (IL-10). We investigated circulating B-cell subpopulations, including CD24^hi CD38^hi Bregs, as well as total B cells, CD4⁺ T cells, CD8⁺ T cells and natural killer cells in 104 untreated patients with severe and very severe AA, aged ≥18 years. All patients were treated with standard immunosuppressive therapy (IST) plus eltrombopag. CD24^hi CD38^hi Bregs were markedly reduced in patients with AA compared to healthy individuals, especially in very severe AA, but residual Bregs remained functional, capable of producing IL-10; total B-cell counts and the other B-cell subpopulations were similar to those of healthy individuals. CD24^hi CD38^hi Bregs did not correlate with responses to IST, and they recovered to levels present in healthy individuals after therapy. Mature naïve B-cell counts were unexpectedly associated with IST response. Markedly reduced CD24^hi CD38^hi Bregs, especially in very severe AA, with recovery after IST suggest Breg deficits may contribute to the pathophysiology of immune AA.

Keywords: aplastic anaemia; flow cytometry; immunosuppressive therapy; lymphocyte subsets; regulatory B cells.

Fig 1

B‐cell subpopulations. (A) Representative dot plots in a healthy individual and a patient with AA. CD3^–CD19⁺CD33^–GPI‐anchor⁺ live B cells were classified based on their expressions of CD24 and CD38: CD24^hiCD38^hi Bregs, CD24^lowCD38^low mature naïve B cells, CD24^hiCD38^low memory B cells, and CD24^lowCD38^hi plasma cells/plasmablasts. (B) Comparisons of CD24^hiCD38^hi Breg frequencies among healthy individuals (n = 29), patients with SAA (n = 37) and VSAA (n = 23). [Colour figure can be viewed at wileyonlinelibrary.com]

Fig 2

B‐cell subpopulation frequencies and response to IST. (A) Initial CD24^hiCD38^hi Bregs did not correlate with IST responses. (B) Among eight lymphocyte subsets studied, only mature naïve B cells correlated with haematological responses. (C) CD24^hiCD38^hi Bregs recovered in 6 months after institution of ATG in both responders (blue dot lines, n = 34) and non‐responders (red solid lines, n = 5). R, responders; NR, non‐responders. [Colour figure can be viewed at wileyonlinelibrary.com]

Fig 3

IL‐10 production from B cells. (A) Representative dot plots of intracellular IL‐10 staining in CD24^hiCD38^hi Bregs and non‐Breg B cells. The control was B cells cultured without brefeldin A. (B) IL‐10⁺ B‐cell frequencies in total B cells were reduced in patients with AA (n = 33) compared to healthy individuals (n = 12). (C) IL‐10⁺ cell frequencies in B‐cell subpopulations in healthy individuals (n = 12) and patients with AA (n = 10). Intracellular IL‐10⁺ B cells were significantly enriched in CD24^hiCD38^hi Bregs in both groups. [Colour figure can be viewed at wileyonlinelibrary.com]