Figure 2.

Recombinant FVIII co-stimulation with lipopolysaccharide plus plasma induces the proliferation of FVIII-specific T cells. Dendritic cells (DC) from HLA-DRB1*11-positive donors were stimulated with recombinant (r)FVIII (1 IU/mL), lipopolysaccharide (LPS) (0.1 mg/mL), and plasma (2.5 mL/mL). Control DC were left untreated. After 24 hours, carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells were co-cultured with DC for 9 days. On day 9, T cells were harvested and stained with HLA-matched FVIII-specific tetramer and anti-CD3 and anti-CD4 antibodies for fluorescence-activated cell sorting (FACS) analyses. (A) Gating strategy for 1 representative donor is shown. In order to analyze only living T cells, gates were set in the forward/side scatter (FSC/SSC) and further on CD3⁺CD4⁺ T cells. T-cell proliferation was assessed by measuring the decrease of CFSE fluorescence intensity in the histogram. Then, CFSE^- cells were defined in the dot plot as CD4⁺tetramer⁺ FVIII-specific T cells. (B) The proliferated FVIII-specific T cells are shown as absolute numbers in events. Data in (B) were obtained from 5 independent experiments (n=6-11). Statistical significance was determined using the Wilcoxon matched-pairs signed rank test. Error bars indicate the means ± standard error of the mean. *P<0.05.