CD8+ T Cells Are Predominantly Protective In a Murine Model Of ITP and Is Required For Effective Steroid Therapy
Authors: Ma Li , Simpson Elisa K. , Li June , Zhu Guangheng , Chen Pingguo , Semple John W. , Freedman John , Ni Heyu 
Published: October 2019

Abstract

Background
Immune thrombocytopenia (ITP) is a common bleeding disorder characterized by autoantibody mediated destruction of autologous platelets. The predominant autoantibody detected in ITP patients target the platelet Glycoprotein (GP)IIbIIIa, however autoantibodies can only be detected in 50-70% of patients. Cytotoxic CD8+ T-cells (CTL) may therefore also be a significant contributing factor for thrombocytopenia, either through direct cytotoxicity against platelets, or decreasing platelet production through interaction with megakaryocytes in the bone marrow. Nevertheless, whether CD8+ T cell mediated cytotoxicity significantly contributes to thrombocytopenia in ITP is controversial. Interestingly, CD8+ regulatory T cells have been demonstrated to play significant roles in other autoimmune diseases, but their function in the context of ITP has not been adequately examined.

Methods and Results
We developed both passive and active murine models to investigate mechanisms of pathogenesis and steroid treatment of ITP. In the passive model, we injected anti-b3 or anti-GPIb antibodies to induce thrombocytopenia; this causes transient antibody-mediated thrombocytopenia. We found that a single intraperitoneal (IP) injection of steroids post-antibody injection was effective at rescuing platelet counts. We also adapted an active model of ITP whereby wild-type (WT) BALB/c mice were transfused with splenocytes from WT platelet immunized β3-/- mice to induce thrombocytopenia. This model encompasses both antibody and cell-mediated ITP and causes sustained thrombocytopenia in the mice. In this model, we found steroid treatment administered either orally or through IP-injection were equally efficacious at ameliorating thrombocytopenia. The successful use of steroids to treat thrombocytopenia in these animal models is representative of the therapeutic effects of steroid treatment seen in human ITP patients.
To study the role of CD8+ T-cells in the response to steroid treatments in ITP, we depleted CD8+ T-cells from splenocytes prior to transfusion into WT mice. Unexpectedly, we found CD8+ T cell depleted splenocyte (lacking in CTL cells), engrafted mice had lower, but not higher, platelet counts. They were also less responsive to dexamethasone treatment compared to non-depleted engrafted mice. Furthermore, transfusion of splenocytes from immunized β3-/- mice in conjunction with antigen-primed CD8+ T-cells (isolated from immunized β3-/- splenocytes) was able to rescue platelet counts in WT mice. Co-transfusion of non-primed CD8+ T cells from β3-/- mice could not rescue platelet counts. These results indicate that platelet-antigen specific CD8+ Tregs play a dominant protective role in attenuating platelet clearance. In further support of our observations, we detected significantly increased CD8+CD25+Foxp3+ Treg percentages in the blood, thymus and spleen of immunized β3-/- mice. Further in vitro splenocyte cultures demonstrated putative regulatory mechanisms of CD8+CD25+ Tregs from immunized β3-/- splenocytes. We found CD8+CD25+ Tregs significantly inhibited CD4+ T and CD19+ B cell proliferation, platelet apoptosis, and platelet associated IgG production in the presence of platelet-antigens, but increased the secretion of the anti-inflammatory cytokine IL-10.

Conclusion
To the best of our knowledge, these are the first reported animal models of steroid treatment of ITP. We demonstrated that steroid therapy was effective and CD8+ T cells are required for this efficacy. We further unveiled a population of CD8+ regulatory T-cells in the CD8+ T cell populations, which were able to rescue platelet counts. This suggests that CD8+CD25+Foxp3+Treg may impart a predominantly protective role, overcoming the cytotoxic function of CD8+ T-cells in ITP. These data provides significant insights into the understanding of immunopathogenesis of ITP, which may be important in designing effective therapy including the potential usage of CD8+ Tregs as a cellular therapeutic method against ITP.

Disclosures:
No relevant conflicts of interest to declare.