Developing a Cytotoxic T Cell Assay to Investigate a CD8+ T Cell Pathology in Megakaryopoeisis in Immune Thrombocytopenia

Abstract

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder, characterized by platelet destruction and/or underproduction. The pathophysiology is heterogeneous and can be mediated by autoantibodies and cytotoxic T lymphocytes (CTLs). While platelet destruction in ITP is well documented, there is little support for platelet underproduction due to the inhibition of megakaryocyte growth and considerably less support for CTL-mediated platelet underproduction. Our objective was to develop an assay that could test for CTL-mediated inhibition of megakaryocyte growth (megakaryopoiesis) in ITP, using healthy controls. Peripheral blood from healthy donors was used to prepare hematopoietic stem and progenitor cells (HSPCs). These cells were expanded with StemSpan to culture a large number of megakaryocytes for the CTL assay. Our studies show that CTLs can be stimulated in-vitro using anti-CD3 antibodies and that they can be used after freezing and thawing. We also assessed CTL stimulation via peptide presentation, using viral peptides whom almost 100% of the general population have memory CTL specificity to, in order to activate a lower frequency of CTLs and to model levels of CTL activation in autoimmune disease. Both stimulants were found to stimulate CTLs in healthy donors with donor variability in the IFN-γ ELISpot. The CTL assay was developed by co-culturing thrombopoietin (TPO) stimulated HSPCs with autologous CTLs for 7 days to observe inhibition of megakaryocyte growth. To induce CTL stimulation, CTLs were either incubated with anti-CD3 or HSPCs were incubated with viral peptides before co-culturing with CTLs. Results showed that while viral peptides can be used as an internal control for the CTL assay, it could not serve as a positive control as inhibition was donor dependent. Inhibition of megakaryocyte growth in the presence of anti-CD3 stimulated CTLs was observed in all donors, validating its use as an appropriate positive control to study CD8+ T cell pathophysiology in ITP.