Characterization of FVIII-specific CD4 T-cell subsets. CD4 T cells were separated by flow cytometry into 4 subsets: naïve T cells (CD45RA⁺ CD45RO⁻ CD127⁺ CD62L⁺ CD197⁺), central memory cells (TCMs; CD45RA⁻ CD45RO⁺ CD127⁺ CD62L⁺ CD197⁺), effector memory cells and effector cells CD45RO⁺ (TEM&TeffRO; CD45RA⁻ CD45RO⁺ CD127⁺ CD62L⁻), and effector memory cells and effector cells CD45RA⁺ (TEMRA&TeffRA; CD45RA⁺ CD45RO⁻ CD127⁺ CD62L⁻; supplemental Figure 1). CD4 T-cell lines were generated from the cells of these subsets by 3 once-per-week rounds of in vitro stimulation with autologous DCs loaded with 0.2 µM of FVIII or 0.25 µM of KLH (for naïve cells only). Specificity of CD4 T-cell lines raised against FVIII or KLH was analyzed by IFN-γ ELISPOT. (A) FVIII-specific T-cell lines obtained from the different CD4 T-cell subsets isolated from 2 representative donors: #393 and #423. (B) Frequencies of FVIII-specific CD4 T cells isolated from the different CD4 T-cell subsets of all donors. Statistical significance was assessed using the Wilcoxon match-pairs signed rank test. *P < .05. Ctrl, control.