CD4⁺ T-cell proliferation in response to FVIII glycosylated peptides. (A) A graphic representation of the synthetic 15 amino-acid peptides corresponding to N-glycosylation site 2118 was shown. A non-glycosylated peptide, 15 amino acids in length, centered around site N2118 (2118NGP1) was modified with the addition of either a single GlcNAc residue (2118GlcP1) or a high mannose glycan Man6GlcNAc2 (2118MP1). (B) CD4⁺ T-cell proliferation levels in response to 1 U of FIX as a non-specific control, no stimulant as the negative control, and increasing doses of rFVIII (0.1 and 1 U). (C) CD4⁺ T-cell proliferation rates measured after stimulation with 2118NGP1, 2118GlcP1, and 2118MP1 synthetic peptides, respectively. (D) Summary of the comparison of CD4⁺ T-cells proliferative rate after stimulation with different synthetic peptides from multiple experimental runs. The background was subtracted for each run. The data are presented as means with standard deviation from 3 separate experiments (**P < .01; ***P < .001; ****P < .0001). (E) CD4⁺ T-cell proliferation rates in response to FVIII and MP1 in the presence or absence of mannan, respectively (*P < .05).