Journal of thrombosis and haemostasis : JTH · 2025
T cells from individuals with and without hemophilia A respond to the same epitopes in factor VIII.
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Vir Pooja, Karim Ahmad Faisal, Gunasekera Devi, Stering Allen I, Lieuw Kenneth, Meeks Shannon L, Pratt Kathleen P
Аннотация
BACKGROUND: Development of neutralizing anti-factor [F]VIII antibodies (inhibitors) follows recognition by CD4+ T cells of epitopes that are presented on the individual's human leukocyte antigen (HLA) class II. Limited blood volumes have presented a major challenge in mapping T-cell epitopes in FVIII, especially as these immune responses typically develop in early childhood.
OBJECTIVES: To determine whether CD4+ T cells from individuals with and without hemophilia A (HA) respond to the same T-cell epitopes.
METHODS: We used interferon-γ enzyme-linked immunospot (ELISPOT) assays with added costimulation to test both unmanipulated CD4+ T cells and in vitro-expanded FVIII-specific CD4+ T-cell lines from donors without HA, to identify responses to FVIII protein and to synthetic 15-mer FVIII peptides.
RESULTS: Tests of both unmanipulated and in vitro-expanded CD4+ T-cell populations from donors without HA identified immunodominant epitopes in FVIII. However, the protocol using expanded T-cell lines produced higher background interferon-γ secretion, which could mask responses to some FVIII epitopes. Importantly, several HLA-DRB1-restricted epitopes identified using T cells from subjects without HA were reproducibly found using T cells from subjects with HA who carried the same HLA-DRB1 alleles.
CONCLUSION: Circulating non-HA CD4+ T cells with self-FVIII specificity, i.e, cells that apparently escaped thymic editing, can respond to the same epitopes recognized by CD4+ T cells from individuals with HA. Therefore, costimulation-enhanced ELISPOT assays can identify clinically relevant T-cell epitopes in FVIII using blood from healthy donors without HA, thereby overcoming blood volume limitations inherent to pediatric patient populations.
OBJECTIVES: To determine whether CD4+ T cells from individuals with and without hemophilia A (HA) respond to the same T-cell epitopes.
METHODS: We used interferon-γ enzyme-linked immunospot (ELISPOT) assays with added costimulation to test both unmanipulated CD4+ T cells and in vitro-expanded FVIII-specific CD4+ T-cell lines from donors without HA, to identify responses to FVIII protein and to synthetic 15-mer FVIII peptides.
RESULTS: Tests of both unmanipulated and in vitro-expanded CD4+ T-cell populations from donors without HA identified immunodominant epitopes in FVIII. However, the protocol using expanded T-cell lines produced higher background interferon-γ secretion, which could mask responses to some FVIII epitopes. Importantly, several HLA-DRB1-restricted epitopes identified using T cells from subjects without HA were reproducibly found using T cells from subjects with HA who carried the same HLA-DRB1 alleles.
CONCLUSION: Circulating non-HA CD4+ T cells with self-FVIII specificity, i.e, cells that apparently escaped thymic editing, can respond to the same epitopes recognized by CD4+ T cells from individuals with HA. Therefore, costimulation-enhanced ELISPOT assays can identify clinically relevant T-cell epitopes in FVIII using blood from healthy donors without HA, thereby overcoming blood volume limitations inherent to pediatric patient populations.