OC 47.1 - FVIII-reactive CD4 T cells isolated from non-hemophilic blood respond to multiple epitopes in the FVIII protein

Background: Neutralizing anti-factor VIII (FVIII) antibodies (“inhibitors”) afflict ~30% of severe hemophilia A (HA) patients, while antibodies against self-FVIII can also occur in non-hemophilic individuals. The latter can cause the rare but potentially life-threatening autoimmune disorder “acquired HA”. CD4 T cells provide help for antibody generation, class switching and affinity maturation. Interestingly, a series of non-hemophilic individuals were recently shown by the Maillère group to circulate FVIII-specific CD4 T cells (PMID 29296830) , suggesting thymic escape of FVIII-reactive T cells contributes to the relatively high immunogenicity of FVIII compared to other self-proteins.

Aims: To investigate mechanisms by testing the fine specificity and phenotypes of FVIII-reactive CD4 T cells in non-hemophilic blood donors using ELISPOT assays, HLA-ClassII tetramer staining and flow cytometry.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from blood donors and expanded in vitro via FVIII stimulation. Interferon-gamma ELISPOT assays were then carried out stimulating the lines with pooled synthetic, overlapping FVIII peptides (spanning the FVIII A1, A2, A3, C1 and C2 domains). Positive responses to specific peptide pools were further decoded by ELISPOT assays using IEDB-predicted individual peptides as stimulants. We attempted to identify immunodominant epitopes and isolate FVIII-specific T-cell clones via peptide-loaded HLA-ClassII tetramer staining.

Results: Immunodominant epitopes restricted to HLA-DRB1* 01:01, 07:01 and 15:01 were identified, and a clone restricted to HLA-DRB1*01:01 was isolated. Responses to multiple peptide epitopes clearly confirmed FVIII specificity and ruled out alternative explanations for IFN-gamma secretion upon stimulation with FVIII protein, e.g. molecular mimicry or other off-target antigen stimulation of the responding cells.

Conclusion(s): Acquired HA entails a breakdown of tolerance mechanisms that prevent expansion of FVIII-reactive T cells in the periphery. Identification of HLA-restricted T-cell epitopes recognized by non-hemophilic immune systems will enable us to study autoimmune and tolerance mechanisms and to ask if these are the same epitopes driving hemophilic inhibitor responses.