Female donors produce a strong graft-versus-leukemia (GvL) effect in male patients. Presumably Y-encoded minor histocompatibility antigens (mHA) are recognized by the alloimmune effector lymphocytes. UTY (ubiquitously transcribed tetratricopeptide repeat, Y-linked) is a potential candidate gene for mHA in dogs and men. In the canine model we could show that the minor histocompatibility antigen UTY might be a potential target to further improve the adoptive immunotherapeutic approach. Five HLA-A2 restricted peptides were used for pulsing canine dendritic cells and stimulating canine T cells. T-cells were separated from the blood of female donors using the MACS technique and effector T-cells were generated by incubation of 2x10*6 CD3+ T-cells/ml with autologous mature, peptide-pulsed dendritic cells (DCs) in X-Vivo 15 medium (+ 160 U/ml human IL-2 and 5 ng/ml human IL-7; ratio T:DCs=10:1). T cells were restimulated weekly with peptide-pulsed DCs. After 3 or 4 weeks in culture T-cells were examined for the CD8/CD4 ratio, cytotoxicity assays were assessed after 3–4 re-stimulations in [51Cr]-release assays. The number of peptide-specific T cells was assessed in IFN-g-ELISPOT-assays. UTY-mRNA expression was determined by RT-PCR in several female and male cell types (BM, PBMCs, monocytes, DCs, B-cells). We were able to expand T-cells from 6 female dogs using peptide-pulsed autologous DCs; 3 out of 5 UTY-derived peptides were able to stimulate a specific autologous T-cell response. The number of CD8+ T-cells could be expanded during four weeks of in vitro culture in 3 dogs. In IFN-g-ELISPOT-assays we could demonstrate that the expanded T-cells secrete IFN-g upon recognition of DCs from DLA-identical male littermates. In addition, these T-cells specifically lysed DLA-identical male BM (25-100%), but not autologous or DLA-identical female BM (0–15%) in [51Cr]-release assays (E:T=80:1). Cell types other than BM and DCs (DLA-identical male and female monocytes, PBMCs, B-cells) were not lysed (0–18%). In summary, we could demonstrate that UTY is a naturally processed and presented antigen in the dog. Our data show that HLA-A2 restricted UTY-peptides immunize specific CTLs from female dogs that efficiently recognize DLA-identical male bone marrow and DCs. These findings suggest that UTY may be employed for studies of immunotherapy in the canine model.

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