Human CD8⁺ Treg after Allogeneic Stem-Cell Transplantation

Murine CD8⁺ regulatory T cells (Treg) are characterized by expression of the cell surface triad CD44, CD122 and Ly49 and require the transcription factor Helios for stable regulatory function. This regulatory CD8⁺ T cell subset recognizes target cells through the recognition of the Qa-1-molecule (HLA-E in man), resulting in perforin-dependent elimination of target cells. Qa-1 is a MHC class Ib molecule that presents a limited repertoire of peptides that generally serve as an indicator of cellular activation and stress. Until now, CD4⁺ follicular T helper cells (T_FH) and CD8⁺ effector T cells have been identified as direct target cells of CD8⁺ Treg.

The therapeutic success of allogeneic stem-cell transplantation (allo-SCT) as an immunotherapeutic approach to treat hematological diseases is often limited by Graft-versus-Host Disease (GvHD). Major factors for the development of GvHD are allo-reactive CD4⁺ T_FH and CD8⁺ effector T cells. CD4⁺ regulatory T cells (Treg) have been shown to be beneficial in the prevention and/or treatment of GvHD while sparing the Graft-versus-Leukemia effect. The contribution of regulatory CD8⁺ T cell-mediated immune responses in GvHD has not been studied so far. However, targeting CD4⁺ T_FH and CD8⁺ effector T cells, CD8⁺ Treg are feasible candidates as additional important players in immune regulation during GvHD development.

Here we have analyzed the development of CD8⁺ Treg after allogeneic stem-cell transplantation in humans. Using the knowledge derived from murine studies, the killer cell immunoglobulin-like receptor (KIR), the functional homologue of the murine Ly49 receptor, was used as a surface marker on CD8⁺ T cells to determine the human regulatory CD8⁺ T cell subset. In humans, CD8⁺ Treg also express CD44, CD122 and the transcription factor Helios similar to murine CD8⁺ Treg. In vitro proliferation studies with IL-2, IL-12, IL-15 and IL-18 showed dose dependent proliferation kinetics of KIR⁺CD8⁺ Treg vs KIR^-CD8⁺ T cells. Of note, after cytokine stimulation and cell proliferation for up to 12 days, KIR⁺CD8⁺ T cells remained phenotypically stable while KIR^-CD8⁺ T cells did not up-regulate KIR expression. Similar results were obtained in analyses of Helios expression in CD8⁺ Treg during cytokine stimulation. In patients after allogeneic stem-cell transplantation, CD8⁺ Treg development occurs early together with reconstitution of other immune cell subsets. Subsequently, CD8⁺ Treg reconstitution reaches its peak around day +90 after allo-SCT. Compared with samples from healthy donors, CD8⁺ Treg after allo-SCT show similar surface phenotype characteristics, while Helios expression is up-regulated after allo-SCT.

In summary, we have further characterized the KIR⁺CD8⁺ Treg population with distinct phenotypical and functional responses to cytokine stimulation when compared with KIR^-CD8⁺ T cells. This KIR⁺CD8⁺ regulatory T cell subset recovers early in patients after allogeneic stem-cell transplantation with comparable characteristics to CD8⁺ Treg from healthy donors, with the exception of an up-regulated expression of Helios in CD8⁺ Treg after allo-SCT. The rapid generation of CD8⁺ Treg during regeneration of the immune system in these patients implicates an important role for this cell subset during immune reconstitution and may control development of GvHD. Further functional analyses of KIR⁺CD8⁺ T cells in patients after allo-SCT may be helpful in understanding the involvement of CD8⁺ Treg in the pathogenesis of acute and chronic GvHD. Following these results, specific activation/expansion of CD8⁺ Treg may then lead to additional cellular immune-modulatory options for the treatment and prevention of GvHD.