*Purpose: Activation of host T cells that mediate allograft rejection is relied on dendritic cells (DCs) presenting alloantigens to T cells. Our recent study revealed that intact donor MHC-peptide complexes from graft cells were conspicuously transferred to the surface of host DCs (cross-dressing) and cross-dressed DCs sustained effector CD8 T cell function. Here, we studied which population of DCs engages in cross-dressing and whether cross-dressing is sufficient in mediating chronic rejection.

*Methods: In mouse kidney transplantation, MHC class-I molecule H-2K^b was eliminated from either the donor or recipient to restrict OVA antigen presentation to either the cross-dressing or indirect pathway. Recipients without restriction to antigen presentation served as control. Group A, Wild type (WT) control: WT B6 (H-2K^b/b) mice received (Balb/c×B6) F1.OVA (H-2K^d/b) kidneys (n=11); Group B, Cross-dressing : B6 Kb-/- (H-2K^-/-) mice received F1.OVA (H-2K^b/d) kidneys (n=10); Group C, Indirect presentation: B6 WT (H-2K^b/b) mice received F1.OVA Kb-/- (H-2K^d/-) kidneys (n=13). All recipients underwent double nephrectomy and were transferred with 10 million effector OT-I cells that specifically recognize the OVA_257-264 SIINFEKL peptide bound to the H-2K^b on postoperative day 2 (d.2). Graft function and survival were monitored, and histology was analyzed. Flow cytometry was used to measure OT-I cell infiltration in grafts. Cross-dressing events were captured by ImageStream, where H-2K^b– OVA_257-264SIINFEKL complexes were quantified on host conventional DC (cDC), monocyte derived DC (monoDC), and CD11b+CD11c- subsets in kidney grafts.

*Results: Grafts were harvested from Groups A, B and C on d.10 (n=6, 4, and 6) and d.60 (n=5, 6 and 7) with stable graft function. Histology showed comparable changes among three groups on d.10 and d.60. Histological chronic rejection was found on d.60. H-2K^b-SIIFEKL complexes were detected in Groups A, B, and C on cDCs (15.3, 15.7, and 2.2%), monoDCs (72.4, 84.1, and 30.9%), and the CD11b+CD11c- subset (26.8, 59.9, and 3.2%) on d.10. Presence of H-2K^b-SIIFEKL complexes declined on d.60 in all groups on cDCs(3.0, 7.7, and 1.8%), monoDCs (30.7, 56.4, and 14.7%), and the CD11b+CD11c- subset (16.0, 29.3% and 2.0%). Effector OT-I cell counts were comparable among all groups on d.10, but significantly decreased in all groups on d.60. On d.60, graft OT-I cells were significantly less in Groups B and C compared to group A (p<0.05); Group C contained significantly higher number of OT-I cells compared to group B (p<0.005).

*Conclusions: In mouse kidney allograft, monoDCs rather than cDCs represented the primary population that underwent cross-dressing as well as indirectly presented alloantigen. The observation that the CD11b+CD11c- subset underwent significant cross-dressing suggests that cross-dressing began with monoDCs before their maturation. Decline of OT-I cell number suggests indirect presentation plays an increasing significantly role during chronic rejection.

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