Lineage-Plasticity and Inflammatory Change of Human FoxP3⁺ Regulatory T Cells in Acute Viral Infection: Implication in Immune-Mediated Tissue Injury

FoxP3⁺ CD4⁺CD25^hi regulatory T (Treg) cells play a major role in maintaining the immune homeostasis by preventing the activation of self-reactive T cells as well as in controlling a series of immune responses in viral infections. Recent studies suggest that lineage-commitment of CD4⁺T cells, including Treg cells, is not a fixed fate, rather a status with a wide range of plasticity. Functional changes and lineage-plasticity of Treg cells during acute viral infection, especially of human, have not been reported so far. Herein, we investigated whether Treg cells show the functional plasticity and whether such changes can affect the regulation of immunopathology in a human acute viral infection. As a model of human acute viral infection, we used a cohort of patients with acute hepatitis A (AHA), since tissue (liver) injury in AHA is mediated exclusively by activated T cells, not by direct cytopathic effect of virus.

To assess the plasticity of Treg cell lineage, first, we examined the production of a variety 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 and multicolor flow cytometry. We found that a significant proportion of FoxP3⁺ CD4⁺CD25^hi Treg cells produced TNF-α following TCR stimulation in patients with AHA, but not in heathy subjects. Analyses at multiple time points during the course of infection showed that TNF-α production from Treg cells decreased in convalescent phase. Likewise, we observed that liver-infiltrating Treg cells also produced TNF-α after TCR stimulation. Moreover, highly-purified CD4⁺CD25^hiCD127^lo/-Treg cells could also produce TNF-α following TCR stimulation, indicating that Treg cells of AHA patients can produce TNF-α in direct response to TCR stimulation.

Next, to exclude the possibility that TNF-α might be secreted from transiently FoxP3-expressing activated non-Treg CD4⁺ T cells, we examined the expression level of CD127 on TNF-α-secreting FoxP3⁺ CD4⁺ T cells. TNF-α⁺ Treg cells expressed CD127 in the level similar to conventional TNF-α^- counterpart, and CD127 expression levels of both Treg populations were much lower than FoxP3^- CD4⁺ T cells. Furthermore, DNA methylation analysis of Treg cell-specific demethylated region (TSDR) after sorting TNF-α⁺ Treg cells revealed completely demethylated pattern in highly conserved CpG island of FOXP3 gene. These findings support that TNF-α is produced from bona fide Treg cells, not from FoxP3-expressing activated non-Treg CD4⁺T cells.

In analysis of immunophenotypes, TNF-α⁺ Treg cells were enriched in CD45RA^-FoxP3^lo population, implying their reduced in vivo suppressive activity. Along with the lower level of FoxP3, TNF-α⁺ Treg cells showed lower level of CD39 expression, a surrogate marker of Treg cell suppressive activity, compared to TNF-α^- Treg cells. Furthermore, TNF-α⁺Treg cells showed a robust evidence of lineage-plasticity toward Th17 lineage, expressing a key transcription factor RORγt. Consistently, they expressed CCR6 and co-produced IL-17A following TCR stimulation, which are the hallmark of Th17 effector function.

To analyze the clinical implication of attenuate suppressive function and plasticity shown by TNF-α⁺ Treg cells, we examined correlation between production of proinflammatory cytokines from Treg cells and severity of liver damage in AHA. As a result, proportion of TNF-α-producing Treg cells closely and linearly correlated with severity of liver damage, suggesting the critical role of TNF-α⁺ Treg cells in the immunopathogenesis of AHA. However, Treg cell suppression assay in the absence or presence of anti-TNF-α antibody showed that Treg cell suppressive function was not affected by TNF-α blockade. This indicates that attenuated function of TNF-α⁺Treg cells is not attributed simply to production of a kind of inflammatory cytokine, rather to more complicated reprogramming mechanism.

Taken together, these data provide a clear evidence of attenuated suppressive activity and Th17-toward lineage plasticity of FoxP3⁺ Treg cells, represented by TNF-α production, in a human acute viral infection. Also, we suggest one possible mechanism that lineage plasticity and inflammatory changes of Treg cells could be implicated in the immunopathogenesis of human diseases.