However, the detection of MHC transfer between cells, and particularly the capture of MHC:peptide complexes by dendritic cells (DCs), led us to propose a third, semidirect, pathway of MHC allorecognition. cell responses. In addition, acquired MHC\class I:peptide complexes stimulate T cell responses studies, T cells from OT\1 Rag?/? mice were isolated using a CD8+ T cell isolation kit (Miltenyi Biotech, Surrey, UK). The purity of responder T cells was assessed using PE\conjugated anti\CD8 antibodies (clone 53\6.7). The purity of T cells was consistently between 90% and 95%. CD11c selected DCs were isolated using a CD11c isolation kit (Miltenyi Biotech) following manufacturers instructions. 105 purified CD8+ T cells and 105 CD11c were stimulated in triplicate wells of a 96\well plate. T cell proliferation was measured by [3H] thymidine incorporation after 3 days in culture. Results are shown as mean count per minute of triplicate determinations SD. To measure interferon\ (IFN) production, culture supernatant, taken from the above cultures, were analyzed using an IFN\specific enzyme\linked immunosorbent assay (ELISA) kit, following manufacturer’s instructions (eBioscience). Results are shown as mean pg/mL of triplicate determinations SD. Statistical analysis Data are represented as mean standard error of the mean where appropriate. Graft survival was depicted using KaplanCMeier analysis and groups were compared by log\rank (MantelCCox) Lexacalcitol screening. To determine statistical significance, a Student’s t\test (unpaired, two\tailed) was carried out using the GraphPad Prism software, http://www.graphpad.com/prism/prism.htm. In the figures, p\values <0.05 are indicated by *, p < 0.01 by **, and p < 0.001 by ***, whereas nonsignificant p\values are labeled ns. Values of p < 0.05 were considered significant. Results mOVA\expressing Lexacalcitol skin allografts are rejected in the absence of CD8+ and CD103+ DCs Rejection of skin expressing OVA, a single minor mismatch antigen, has previously been shown in B6 recipient mice 15. Injection of OVA\specific CD8+ T cells, isolated from OT\1 T cell receptor (TCR)Ctransgenic mice, into these transplanted B6 mice indicated the presence of OVA antigen in both sdLNs and spleen following skin transplantation 15. Activation of these T cells may Lexacalcitol be due to acknowledgement of antigen in a variety of ways including antigen offered by donor DCs, direct recognition, or cross\presentation by recipient DCs, or by recipient DCs presenting acquired MHC\peptide complexes from your transplanted tissues. To assess the contribution of cross\presentation in this model, we compared the rejection kinetics of Take action\mOVA skin in B6 mice and Batf3?/? recipient mice (H\2b). Batf3?/? mice lack CD8+ standard DCs (cDCs), the DC subset regarded as the main cross\presenters, as well as the nonlymphoid CD103+ migratory cDC populace. In comparison to B6 mice, Batf3?/? mice reject OVA skin transplants at a slower rate (mean survival time was 25 days on B6 recipients compared to 32 days on Batf3?/? recipients, Physique ?Physique1A)1A) suggesting that either, or both, the CD8+ and the CD103 DC subset contribute to the rejection of skin transplants in the B6 mice. However, Act\mOVA, but not control B6 skin transplants, were rejected by Batf3?/? mice even in the absence of these cross\presenting DC subsets (Physique ?(Figure1A).1A). Next we measured OVA\specific CD8+ T cell response in the Batf3?/? recipient mice receiving Take action\mOVA skin. Injection of CFSE\labeled CD8+ T cells, isolated from OT\1Rag?/? mice, into transplanted mice on days 10, 14, 21, and 30 after transplantation resulted in T cell proliferation, measured 72 h later by CFSE dilution, at all time points (Physique ?(Figure1B).1B). The Lexacalcitol data therefore show that OVA antigen was Lexacalcitol present in the spleen and sdLN for a prolonged period (Physique ?(Figure1B).1B). Interestingly, even when there was very little skin left, day 30, posttransplant, T cell proliferation was still observed. Open in a separate window Physique 1 Take action\ mOVA skin grafts are rejected in the absence of cross\presentation. (A) Batf3?/? mice received either an Take action\mOVA or a B6 skin transplant whereas B6 mice received only Act\mOVA skin. Mice were monitored daily, and Thy1 rejection was deemed as the day when no viable skin remained. ***p < 0.001 (KaplanCMeier). Data are representative of three experiments with five mice.