Responsiveness of PBMCs Isolated from Healthy Individuals towards rFVIII and rFVIIIFc

Introduction: About 30% of patients with severe haemophilia A (HA) develop inhibitory antibodies against coagulation factor VIII (FVIII) during replacement therapy and FVIII is targeted by the immune response in acquired HA. We aimed at analyzing the responsiveness toward recombinant FVIII (rFVIII) and rFVIIIFc in healthy individuals, to see if the Fc-portion influences the cell response.

Methods: PBMCs were isolated from 19 buffy coats of healthy individuals. Each 1x10⁶ PBMCs were stimulated with two concentrations of rFVIII, rFVIIIFc, myelin-oligodendrocyte protein (MOG) as an autoantigen control. Following 16 hour of incubation, cells were stained for flow-cytometric analysis and data from 1x10⁵ CD4⁺ T cells were acquired. Cells were classified into regulatory T cells (Tregs) and non-Tregs based on CD25 and CD127 surface expression and further subdivided into naïve, effector, and memory cells using markers CD45RA and CD197. Antigen-specific activation was measured by surface expression of CD137 for Tregs and CD154 for non-Tregs. The rate of responsiveness to stimuli is presented with two-sided 95% Clopper-Pearson confidence interval (CI95). Intensities of responsiveness were compared using Wilcoxon signed-rank test or Friedman Test. All tests were two-sided and a P < 0.05 was considered significant.

Findings: For analysis, antigen-specific responses were expressed as ratio between antigen-stimulated and non-stimulated CD137⁺ Tregs or CD154⁺ non-Tregs. Tregs reacted significantly more toward 1 IU/100µl rFVIII (84.2%, CI95 60.4–96.6, p = 0.003) and 1 IU/100µl rFVIIIFc (68.4%, CI95 43.4–87.4, p = 0.108) compared with MOG (26,3%, CI95 9.1–51.2, p = 0.039) (P < 0.001). Additionally, Tregs showed a higher intensity of responsiveness against rFVIII and rFVIIIFc than against MOG (P < 0.001). No significant differences were found for the responsiveness of whole CD4⁺ non-Tregs toward the antigen-specific stimuli used in this study. However, the proportion of CD45RA⁺CD197⁺ naïve T cells expressing activation marker CD154 following in vitro incubation was highest for rFVIII-stimulated cells and statistically different to MOG (P < 0.05). Both rFVIII and rFVIIIFc showed a higher proportion of activated naïve T cells compared with the unstimulated control (P < 0.001 and p = 0.002). Thus, in this setting naïve CD4⁺ T cells showed a higher responsiveness toward both rFVIII products.

Conclusion: Our results point out, that the naïve CD4⁺ non-Treg repertoire might have a higher quantity of rFVIII-responsive cells compared with the MOG autoantigen control. Interestingly, no difference in total response and response intensity was detected for the whole CD4⁺ non-Tregs while Tregs reacted more often and stronger toward rFVIII and rFVIIIFc than toward the MOG control, which adds a novel aspect to understanding the immune response against FVIII.

No conflict of interest has been declared by the author(s).