Elispots and Ex Vivo Expansion of FVIII-Specific T-Cell Clones Confirm Specificity of CD4⁺ T-Cell Responses to Self-FVIII in Healthy Non-Hemophilic Blood Donors

The existence of natural antibodies recognizing endogenous factor VIII (FVIII) and of FVIII-specific CD4⁺ T-cell responses in some healthy, non-hemophilic blood donors has been appreciated for >20 years. The Conti-Fine group measured CD4⁺ T-cell proliferation following in vitro stimulation with FVIII protein or synthetic FVIII peptides. More recently, FVIII-specific CD4⁺ T-cell lines were expanded from PBMCs isolated from large blood volumes donated by healthy individuals, and estimates of specific precursor frequency (~2/million CD4⁺ T cells) were calculated on the basis of interferon (IFN)-gamma ELISPOT assays of FVIII-stimulated cells (Meuniere et al., Blood Advances 1(21): 1842-7). Escape of these self-reactive precursor cells from thymic editing via deletion or anergy and their subsequent persistence in the periphery may contribute to the rare but potentially severe autoimmune reactions to FVIII ("acquired hemophilia A") and to the unusual immunogenicity of therapeutic FVIII administered i.v. to hemophilia A patients.

The present study sought to further characterize CD4⁺ T-cell responses to endogenous FVIII and to map epitopes recognized by these self-reactive cells. We were particularly interested to learn if these cells recognize multiple epitopes in FVIII or if they respond to only several immunodominant epitopes. Accordingly, IFN-gamma ELISPOT assays were carried out by stimulating CD4⁺ T cells with 15-mer FVIII peptides having 12-residue overlaps and spanning the FVIII A1, A2, A3, C1 and C2 domains. For efficient mapping, initial assays utilized large pools of peptides, and positive responses were then "decoded" by ELISPOTs using smaller peptide pools or individual peptides. Blood samples were obtained from healthy controls under approved IRB protocols. The ELISPOT assays utilized CD4⁺ T cells isolated by negative selection, with irradiated autologous PBMCs as antigen presenting cells. Anti-CD49d/CD28 monoclonal antibodies were added for co-stimulation to increase the sensitivity of the assay and cells were cultured with IL-7 to improve cell viability. As a result, this assay required smaller blood volumes, but it should be noted that lower-avidity T-cell responses were likely detected that might be missed in ELISPOT assays without these modifications. Relevance of such low-avidity self-reactive cells is provided by the clinical observation, consistent with basic immunological principles, that risk factors for autoimmune responses to FVIII include old age (pro-inflammatory), trauma, surgery and postpartum status, all of which up-regulate T-cell co-stimulatory factors.

The first subject had HLA-DRB1*01:01 and HLA-DRB1*08:04 alleles. Stimulation with large peptide pools and rFVIII protein indicated recognition of epitopes in at least 3 FVIII domains. Additional ELISPOTs tested the immunogenicity of 15 peptides corresponding to FVIII peptides previously demonstrated to be presented on dendritic cells from 2 individuals with an HLA-DRB1*01:01 allele (van Haren et al., Mol Cell Proteomics. 2011;10(6)), ensuring that our assays included tests of naturally processed FVIII peptides. Two of these peptides, both from the FVIII A1 domain, produced ELISPOT readings above background levels. T cells were then stimulated with these peptides for 19 days, stained with peptide-loaded MHC Class II (HLA-DRB1*01:01) tetramers, sorted and expanded for another 14 days. Tetramer staining then confirmed isolation of CD4⁺ T-cell clones recognizing one of these peptides.

T cells that recognize their cognate antigen with high avidity are significant drivers of allo- and autoimmune responses. Lower-avidity T cells, however, can play significant roles in pro-inflammatory settings. Tetramer staining validated our ELISPOT-based identification of specific epitopes in FVIII. We are now carrying out ELISPOT assays using pooled peptides followed by individual FVIII peptides as stimulants, to estimate the repertoire of FVIII-specific CD4+ T cells in healthy non-hemophilic individuals. Mapping of HLA-restricted T-cell epitopes will also enable future tetramer-based isolation and phenotypic characterization of these rare T cells without expanding them in culture. This will allow us to investigate the interesting question of what peripheral tolerance mechanisms prevent expansion of these self-reactive cells in vivo, except in rare cases of FVIII autoimmunity.

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