Reduced PTEN Involved in Primary Immune Thrombocytopenia Via Contributing to B Cell Hyper-Responsiveness

Background:

Phosphatase and tensin homolog (PTEN) is thought to mediate B cell activation by negatively regulating the phosphoinositide 3-kinase (PI3K) signaling pathway. This pathway is important for activation, growth, and proliferation. PTEN is involved in maintaining normal B cell function. In immune thrombocytopenia (ITP), enhanced BCR signaling contributes to increased B cell activity, but the role of PTEN in human ITP has remained unclear. Both IL-21/IL-21R signaling and PI3K-PTEN molecules are involved in maintaining normal humoral immunity and deletion of autoreactive B cells. Since B cell overactivity is characteristic ofImmune thrombocytopenia(ITP) we sought to determine whether abnormalities in PTEN might contribute to increase B cell responsiveness in this disease.

Methods:

This study recruited 45 active CITP patients and 33 sex and age matched health volunteers as health controls(HC) Peripheral blood mononuclear cells were isolated from collected anti-coagulated blood. Flow cytometry and real time quantitative PCR were used for detecting the level of PTEN from PBMC cells of HC and CITP patients. Purified B cells from PBMC cells were incubated with human rIL-2 rIL-21r CD40L or anti-IgM alone or in combination for 72h and after that the PTEN level was detected by flow cytometry.The plasma cell generation via IL-21 induction were determined byflow cytometry.

Results:

The expression of PTEN was lower in B cell subpopulations of ITP patients compared with HCs, including IgD⁺CD38^int/high immature B cells, IgD⁺CD38^low/−naïve B cells, and IgD⁻CD38^int/high plasma cells (P<0.001). The levels of PTEN in CD19⁺B cells from patients with active ITP tended to positively correlate with platelet count (r²=0.5472, P=0.0012). In patients with positive platelet-specific autoantibodies, PTEN levels were lower when compared with platelet autoantibody negative patients (717.8±93.4 vs. 990.2 ± 74.4. P=0.0287). After effective treatment, the expression of PTEN was increased (P=0.0011).The patients with active ITP had a significantly higher proportion of CD19⁺IgD⁺CD38^int/hi immature B cells when compared with HCs (14.58±0.89% vs . 7.21± 0.63%,P<0.0001) . Immature B cells from patients with ITP were also found to express a greater density of the activation marker CD95(P=0.0036) and lower IL-21R expression(P=0.0030) compared with HCs. We found that IL-21, CD40L, or anti-IgM resulted in enhanced PTEN expression in normal B cells. However, neither IL-21 alone, CD40L plus anti-IgM, nor the three in combination stimulated PTEN protein upregulation in B cells isolated from patients with ITP. We found that IL-21 increased the proportion of plasma cells that were CD19+CD27+CD38hi or CD138+CD38hi in PBMCs from patients with ITP, although this did not occur in cells from HCs.

Summary/Conclusions:

In summary, PTEN expression is lower in the B cells of patients with ITP, especially in immature B cells. The capacity of IL-21 to induce PTEN was defective in B cells isolated from patients with ITP compared with HCs. B cells from patients with active ITP tend to differentiate into plasma cells through stimulation by IL-21 alone. Our data have established the role that B cell over-activity plays during ITP and decreased expression of PTEN may contribute to B cell hyper-responsiveness and disturbed B cell homeostasis.

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