Distinct characteristics of unique immunoregulatory canine non-conventional TCRαβ CD4CD8α double-negative T cell subpopulations.

Conventional CD4 regulatory T (Treg) cells characterized by expression of the key transcription factor forkhead box P3 (FoxP3) are crucial to control immune responses, thereby maintaining homeostasis and self-tolerance. Within the substantial population of non-conventional T cell receptor (TCR)αβ CD4CD8α double-negative (dn) T cells of dogs, a novel FoxP3 Treg-like subset was described that, similar to conventional CD4 Treg cells, is characterized by high expression of CD25. Noteworthy, human and murine TCRαβ regulatory dn T cells lack FoxP3. Immunosuppressive capacity of canine dn T cells was hypothesized based on expression of inhibitory molecules (interleukin (IL)-10, . Here, to verify their regulatory function, the dnCD25 (enriched for FoxP3 Treg-like cells) and the dnCD25 fraction, were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells (PBMC) of Beagle dogs and analyzed in an suppression assay in comparison to conventional CD4CD25 Treg cells (positive control) and CD4CD25 T cells (negative control). Canine dnCD25 T cells suppressed the Concanavalin A-driven proliferation of responder PBMC to a similar extent as conventional CD4CD25 Treg cells. Albeit to a lesser extent than FoxP3-enriched dn and CD4CD25 populations, even dnCD25 T cells reduced the proliferation of responder cells. This is remarkable, as dnCD25 T cells have a FoxP3 phenotype comparable to non-suppressive CD4CD25 T cells. Both, CD25 and CD25 dn T cells, can mediate suppression independent of cell-cell contact and do not require additional signals from CD4CD25 T cells to secrete inhibitory factors in contrast to CD4CD25 T cells. Neutralization of IL-10 completely abrogated the suppression by dnCD25 and CD4CD25 Treg cells in a Transwell™ system, while it only partially reduced suppression by dnCD25 T cells. Taken together, unique canine non-conventional dnCD25 FoxP3 Treg-like cells are potent suppressor cells . Moreover, inhibition of proliferation of responder T cells by the dnCD25 fraction indicates suppressive function of a subset of dn T cells even in the absence of FoxP3. The identification of unique immunoregulatory non-conventional dn T cell subpopulations of the dog is of high relevance, given the immunotherapeutic potential of manipulating regulatory T cell responses