Inflammatory Change of FoxP3 ⁺ Regulatory T Cells in Human Acute Graft-Versus-Host Disease: Implication in Immunopathogenesis

FoxP3 ⁺ regulatory T (Treg) cells are a subset of CD4 ⁺ T cells that are requisite for control of immune responses in various settings, thereby preventing excessive inflammation and maintaining homeostasis of the immune system. Paradoxically, in several autoimmune disorders, it has been suggested that Treg cells undergo inflammatory conversion to produce effector cytokines promoting tissue inflammation. Although Treg cells are known to play an important role in the pathogenesis of acute graft-versus-host disease (GvHD) following allogeneic stem cell transplantation (SCT), such inflammatory changes have not been focused on so far especially in the setting of human acute GvHD. Herein, in a prospective cohort of patients with hematologic malignancies who were treated with allogeneic SCT, we investigated the quantitative and qualitative changes of Treg cells in subjects with acute GvHD, including production of inflammatory cytokines from Treg cells. We also studied a cellular mechanism for production of effector cytokines by Treg cells and clinical significance of inflammatory changes of Treg cells.

We first determined the frequency of Treg cells in the peripheral blood of patients with acute GvHD and compared it with that of patients who did not develop acute GvHD. Percentage of CD25 ^hiCD127 ^loFoxP3 ⁺ Treg cells among CD4 ⁺ T cells was significantly lower in patients with acute GvHD. When we analyzed for the composition of Treg cell subsets in more detail, we observed that the proportion of CD45RA ^-FoxP3 ^hi (actively suppressive) Treg cells was decreased and the proportion of CD45RA ^-FoxP3 ^lo (cytokine-producing non-suppressive) Treg cells was increased in patients with acute GvHD.

Next, we assessed the production of inflammatory cytokines from Treg cells following T cell receptor (TCR) stimulation of peripheral blood lymphocytes with anti-CD3/CD28 antibody, using intracellular cytokine staining. As a result, we found that a remarkable proportion of CD4 ⁺CD25 ^hiFoxP3 ⁺ Treg cells of acute GvHD patients produced TNF-α upon TCR stimulation. Interestingly, a significant proportion of TNF-α-producing Treg cells also secreted IL-17A simultaneously. These findings are consistent with the increased CD45RA ^-FoxP3 ^lo fraction among the Treg cell subsets. To explore a potential mechanism underlying inflammatory change of Treg cells, we quantified a variety of soluble factors in systemic circulation before and after onset of acute GvHD. The concentration of IL-6 and IL-21 was significantly higher in the serum drawn after onset of acute GvHD compared to baseline, suggesting that these Th17-polarizing cytokines might be responsible for the inflammatory conversion of Treg cells during acute GvHD.

We further checked the suppressive activity of TNF-α-producing CD4 ⁺CD25 ^hiFoxP3 ⁺ Treg cells of acute GvHD patients. TNF-α ⁺ Treg cells of acute GvHD patients expressed lower level of FoxP3 than the TNF-α ^- counterpart. Similarly, the percentage of CD39 ⁺ cells was lower in TNF-α ⁺ Treg cells than in the TNF-α ^- counterpart. In fact, Treg cell suppressive activity during acute GvHD was significantly reduced compared with that of pre-SCT control when non-Treg CD4 ⁺ T cells or CD8 ⁺ T cells were used as responder cells.

Finally, we examined the clinical significance of TNF-α-producing CD4 ⁺CD25 ^hiFoxP3 ⁺ Treg cells in acute GvHD patients. The frequency of TNF-α ⁺ cells among circulating Treg cells significantly correlated with the severity of acute GvHD, with higher proportion of TNF-α ⁺ Treg cells in patients with the higher grade of acute GvHD. This result indicates that the pathologic conversion of Treg cells to produce TNF-α contributes to immune-mediated tissue injury during acute GvHD.

Taken together, during human acute GvHD, Treg cells undergo functional alteration to produce proinflammatory cytokines including TNF-α and IL-17A and to exhibit the reduced suppressive activity. TNF-α production by Treg cells is closely associated with the clinical severity of acute GvHD.