Effect of intermediate-purity factor VIII (FVIII) concentrate on lymphocyte proliferation and apoptosis: transforming growth factor-β is a significant immunomodulatory component of FVIII
Factor concentrates have been shown to have a variety of immunomodulatory effects in vitro. The presence of plasma-derived factor VIII (pdFVIII) has been shown to diminish lymphocyte proliferative response to mitogens. Recently, we have shown the presence of transforming growth factor-β (TGF-β) as an immunomodulatory component present in plasma-derived FVIII concentrate. However, the addition of neutralizing antibody to TGF-β did not abrogate the inhibitory effect of pdFVIII on monocyte cytokine production, suggesting the presence of other, as yet undetermined, immunomodulatory agent/s in pdFVIII. To further characterize the immunomodulatory effects of pdFVIII, the in vitro effect of pdFVIII concentrate on proliferation and apoptosis of mitogen-stimulated T cells was studied using whole blood and purified T cells. The presence of pdFVIII increased the apoptosis of phytohaemagglutinn (PHA) -stimulated CD4 and CD8 T-cell subsets as determined by Annexin V binding and DNA fragmentation. T-cell subsets showed a pdFVIII dose-dependent inhibition of entry into S-phase and G1 arrest. Addition of neutralizing anti-TGF-β reduced some of these changes. To determine the physiological relevance of these findings, blood samples from five patients receiving FVIII prophylaxis were similarly studied ex vivo and showed significantly increased apoptosis of T-cell subsets as determined by Annexin V staining. TGF-β has been reported to be a potent inhibitor of T-cell proliferation, arresting the cell cycle in G1 phase and causing apoptosis. Together, these findings suggest that TGF-β is a significant immunomodulatory component of pdFVIII concentrates.