Distinct characteristics of unique immunoregulatory canine non-conventional TCRαβ^pos CD4^negCD8α^neg double-negative T...

Conventional CD4^pos 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^pos CD4^negCD8α^neg double-negative (dn) T cells of dogs, a novel FoxP3^pos Treg-like subset was described that, similar to conventional CD4^pos Treg cells, is characterized by high expression of CD25. Noteworthy, human and murine TCRαβ^pos regulatory dn T cells lack FoxP3. Immunosuppressive capacity of canine dn T cells was hypothesized based on expression of inhibitory molecules (interleukin (IL)-10, cytotoxic T-lymphocyte associated protein 4, CTLA4). Here, to verify their regulatory function, the dnCD25^pos (enriched for FoxP3^pos Treg-like cells) and the dnCD25^neg fraction, were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells (PBMC) of Beagle dogs and analyzed in an in vitro suppression assay in comparison to conventional CD4^posCD25^pos Treg cells (positive control) and CD4^posCD25^neg T cells (negative control). Canine dnCD25^pos T cells suppressed the Concanavalin A-driven proliferation of responder PBMC to a similar extent as conventional CD4^posCD25^pos Treg cells. Albeit to a lesser extent than FoxP3-enriched dn and CD4^posCD25^pos populations, even dnCD25^neg T cells reduced the proliferation of responder cells. This is remarkable, as dnCD25^neg T cells have a FoxP3^neg phenotype comparable to non-suppressive CD4^posCD25^neg T cells. Both, CD25^pos and CD25^neg dn T cells, can mediate suppression independent of cell-cell contact and do not require additional signals from CD4^posCD25^neg T cells to secrete inhibitory factors in contrast to CD4^posCD25^pos T cells. Neutralization of IL-10 completely abrogated the suppression by dnCD25^pos and CD4^posCD25^pos Treg cells in a Transwell™ system, while it only partially reduced suppression by dnCD25^neg T cells. Taken together, unique canine non-conventional dnCD25^pos FoxP3^pos Treg-like cells are potent suppressor cells in vitro. Moreover, inhibition of proliferation of responder T cells by the dnCD25^neg 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 in vitro is of high relevance, given the immunotherapeutic potential of manipulating regulatory T cell responses in vivo.