CD20 B Cell Depleting Therapy Is Associated with up-Regulation of CD8⁺CD25^highFoxp3⁺ T Regulatory Cells in a Murine Model of Immune Thrombocytopenia (ITP)

Primary immune thrombocytopenia (ITP) is an autoimmune disease characterized by increased platelet destruction and/or impaired megakaryocyte production, mediated by autoreactive B cells and T cells. B cell depletion therapy by rituximab, a monoclonal human anti-CD20 antibody, has been shown effective in both anti-platelet antibody positive (B cell mediated) and negative (T cell mediated) ITP patients. Those patients responsive to rituximab therapy showed normalized CD4⁺ and CD8⁺ T cell responses (Stasi et al. Blood. 2007), however, the mechanism of T cell regulation by B cell depletion is not clear. One possibility is through normalization of CD4⁺ T helper cells or up-regulation of CD4⁺ regulatory T cells (Tregs) (Stasi et al. Blood. 2008). Another possibility is by suppression of activated conventional CD8⁺ T cells or the up-regulation of CD8⁺ Tregs. We examined the changes of both CD4⁺ and CD8⁺ T cells and Tregs (CD25^highFoxp3⁺) after B cell depletion in vivo in our ITP mouse model. Briefly, BALB/c GPIIIa (CD61) KO mice were either given PBS (ND) or mouse monoclonal anti-CD20 antibody (B-dep, Biogen) at day -1 and day 13 (250ug/mouse, ip). Residual CD19⁺ B cells in peripheral blood were less than 0.1% within 24hours in the latter group. All mice were immunized by transfusions of wildtype (WT) platelets at day 0, 7, 14, and 21 (1×10⁸/mouse, iv). At day 28, we examined the percentages of T cell subsets in the spleens of the immunized mice. B cell-depleted immune CD61 KO mice showed significantly higher percentages of both CD3⁺CD8⁺ T cells and CD8⁺CD25^highFoxp3⁺ T cells (Table 1). There was no significant difference in the CD3⁺CD4⁺ and CD4⁺CD25^highFoxp3⁺ T cell populations. Both ND and B-dep immune CD61 KO splenocytes showed increased cytotoxicity activity against CD61+ PU5-1.8 target cells in vitro compared with naïve CD61 KO splenocytes, indicating the activation of CD8⁺ T cells. To test their in vivo effect on ITP development, splenocytes were engrafted from immune mice into irradiated and AsialoGM-1 treated severe combined immunodeficient (SCID) mice at a dose of 2.5×10⁴/mouse and the mice were monitored for weekly platelet counts. ND and in vitro B cell depleted splenocytes from immune KO mice induced persistent ITP during 3 weeks observation whereas splenocytes from B-dep immune mice did not. To further confirm the role of B cell depletion on CD8⁺ T cell responses, CD8⁺ T cells from either ND or B-dep immune CD61 KO splenocytes were purified and transferred into SCID mice at 3×10⁴/mouse. CD4⁺ T cells from ND immune CD61 KO splenocytes were added at 3×10⁴/mouse to all the SCID mice to support the CD8⁺ T cell survival in vivo. SCID mice received CD8⁺ T cells from B-dep group showed higher platelet count at Day 14. Overall, our results indicate a protective role of CD8⁺CD25^highFoxp3⁺ T cells against the development of cell mediated ITP that is enhanced by B cell depleting therapy in vivo.