To compare the efficacy of these female T cells, we also immunized one female dog in vivo with PBMCs from a DLA-identical male littermate. Male-specific recognition induced by UTY-specific CTLs after in vitro immunization

was comparable to those with T cells after in vivo immunization of a female dog (Figs. 3 and 5) as male-BM was targeted with the highest efficiency, followed by DCs and PBMCs, monocytes and B cells indicating an elevated presentation of male-antigens in BM, as previously assumed by others [11, 12, PXD101 44, 45]. Nevertheless, male-BM represents the most-affected target of female T cells, indicating higher and presumably different UTY-expression/UTY-presentation [46] and confirms the high-potential of UTY in female-to-male-transplantation settings (Figs. 3 and 5). Immunization of the female dog with DLA-identical-male-PBMCs induced UTY-specific CD8+T cells, as indicated by increasing amounts of donor-PBMCs. Other mononuclear-cells, like CD4+, CD14+ and B cells, also increased within the experiment, indicating an intense immune-response (data not shown). IFN-γ-secretion was detectable against the UTY-peptides when loaded on different target cells and hT2-MHC-I-restricted cells. Thereby, immunogenicity of investigated peptides was W248 > K1234 > T368 (T2-cells).

Detailed characterization of the UTY-response of female T cells exhibited a male-specific T cell response acting in an MHC-I-restricted-fashion (Figs. 3 and 5). As these blocking-experiments did not reveal a purely CTL-directed PARP inhibitor T cell reactivity (incomplete blocking), these data could implement an additional CD4+T cell-driven reactivity [43, 47]. With these data, we can evidently exclude xenogeneic-CTL-activity as in vitro data were reproducible in vivo showing male-cell-type-specific comparable results. UTY-mRNA-expression was determined in cell-types of hematopoietic-origin (Fig. 4) and confirmed our in vitro and in vivo data: Only male cells expressed UTY-mRNA (male-BM≫DCs, PBMCs,

monocytes, Neratinib price B-cells), whereas corresponding female cells lack UTY-mRNA proofing male-restricted UTY-expression in hematopoietic-cells [48]. UTY-expression in non-hematopoietic cells was not shown in our study. This is compelling to demonstrate as UTY is ubiquitously expressed and would lead not only to GvL-reaction after adoptive immunotherapy, but also to GvHD after transfusion of CTLs. In order to show that our UTY-derived peptides are good targets for canine-/human-GVL-reactions, canine non-hematopoietic-cells like male-fibro-blasts/keratinocytes as well as gut-, liver- and epithelium-cells should be investigated in further experiments to prove this hypothesis. Three isoform-variants of the hUTY-gene are known (additional isoforms seem to exist) and splice-variants show different expression-profiles and tissue-distributions of resulting peptides [49, 50].