Rare Factor VIII-reactive CD4 T cells in non-hemophilic blood characterized by ex vivo expansion and ELISPOTs with added costimulation

Abstract

Abstract Background: Autoimmune responses to self-FVIII can cause a rare but potentially life-threatening autoimmune bleeding disorder referred to as “acquired hemophilia A”. Objectives: (1) To confirm the frequency and specificity of CD4 T cells recognizing factor (F)VIII in the periphery of non-hemophilic individuals; (2) to improve methodology for detecting rare self-reactive CD4 T cells that apparently escaped thymic editing. Methods: CD4 T cells were isolated from healthy non-hemophilic blood donors, incubated with autologous irradiated PBMCs, and stimulated with FVIII 15-mer peptides, anti-CD28 and anti-CD49D. Positive interferon-gamma ELISPOT responses were detected, but they were poorly reproducible due to the low frequency of these autoreactive cells. Therefore, pre-expansion of FVIII-specific T-cell lines, followed by ELISPOT assays with added co-stimulation, was carried out to increase sensitivity and accuracy. Results: Several immunodominant epitopes in FVIII restricted to H LA-DRB1* 01:01, 07:01and 15:01were identified. FVIII-specific T-cell lines generated from individual blood donors showed robust interferon-gamma secretion in response to FVIII, compared to ELISPOT assays with co-stimulation but no FVIII pre-expansion. Conclusions: Our optimized protocol, involving generation of antigen-specific T-cell lines followed by ELISPOTs with added co-stimulation, shows promise for detecting, quantifying and characterizing rare autoreactive T cells in the periphery. Intramural funds, Uniformed Services University