Lytic-activity of cCTLs was assessed after 3–4 stimulations in a [51Cr]-release-assay [39, 40]: Target cells were labelled with 100 μCi [51Cr] for 1.5 h (37 °C) in dog-serum, washed and resuspended in X-Vivo15. [51Cr]-labelled target cells (2000 cells/well) were incubated with effector cells for 4 h (37 °C; E:T = 80:1) in 96-well-microtiter plates. Radioactivity of FK506 culture-supernatant was measured by a γ-counter and percentage of specific-lysis

(cytotoxicity) was calculated:% cytotoxicity = (experimental release-spontaneous release)/(maximum release-spontaneous release) × 100. For the blocking-experiments, we used the monoclonal human canine-cross-reactive MHC-I antibody (clone G46-2.6, end-concentration of 40 μg/ml, BD, Heidelberg, Germany). Canine-IFN-γ-ELISPOT assay (R&D-Systems, Minneapolis, MN, USA) used to quantify peptide-epitope-specific, IFN-γ-releasing effector cells, performed according to the manufacturers’ instructions and examined on day 21 or 28 of T cell stimulation. Precursor frequency of cUTY-specific T cells in dogs’ peripheral blood was evaluated on day 0. Spots were counted by visualization using a dissecting-microscope.

For selleck kinase inhibitor the blocking-experiments, we used the monoclonal human canine-cross-reactive MHC-I antibody (clone G46-2.6, end-concentration of 40 μg/ml, BD). In vivo generation of hUTY-specific-CTLs was tested by immunizing a female dog with PBMCs from a DLA-identical-male dog. On day 0, 50 ml heparinized peripheral-blood was taken from the male-donor

and PBMCs were isolated as described above. 2.5 × 108 cells were resuspended (5 ml warm-RPMI1640) and applied in equal-amounts subcutaneously to the four limbs, followed by a second-immunization on day 14. There, PBMCs (3.2 × 108 in 20 ml RPMI1640) were injected intravenously with 100 ml NaCl. 35 days after the second-injection, blood-derived T lymphocytes were harvested and studied for their UTY-specific reactivity. Distribution of the different cell-populations was monitored at day 0, 14 and 35 via flow-cytometry (donor and Non-specific serine/threonine protein kinase recipient). Mean- and standard-deviation were performed using microsoft® excel xp, and Statistical-calculations were achieved using spss-Version 11.5 (SPSS, Chicago, IL, USA). A statistical significance was accepted for P ≤ 0.05. Canine-female-UTY-specific CTLs were induced in vitro using autologous-DCs derived from monocytes of healthy female dogs (#1, #4, #6). DCs were pulsed with the identified HLA-A2-binding hUTY-derived peptides W248, T368 and K1234. T cells decreased during the first 2 weeks of stimulation, but then the surviving T cells proliferated, resulting in a 1.5-2.9-fold percentage-increase of successfully expanded cCTLs (Fig. 1), whereas the amount of CD4+ T cells decreased (1.6–2.9-fold; data not shown). That means that the absolute T cell number increased after 3–4 weeks of in vitro culture.