Chronic allograft rejection remains a major obstacle to long-term allograft survival. The immunologic role of tertiary lymphoid organs (TLO) in allograft rejection is unclear. Here, we employed a chronic renal allograft rejection model in mice and intravital 2-photon microscopy to investigate the function of TLO in transplant rejection. CB6F1 (F1) RIP-LTα (preformed TLO) or F1 (no TLO) kidney grafts were transplanted to WT B6 recipients and survival monitored. To investigate immunologic function of TLO, we adoptively transferred B6-RIPLTα CD11c-YFP mice with 10m naïve dsRed OT-I T cells or 10m CTR-labeled NP-specific B cells + 10m CFP+ OT-II cells and immunized with NP-OVA + alum. Intravital 2P imaging of renal TLO was performed at time points 0, 3, 6, 24 or 72 hours after immunization. 4D image analysis was performed and mean speed, displacement, arrest coefficient (AC) and contact times (CT) with DC were calculated for OT-I, OT-II and NP-B cells. F1 RIP-LTα grafts rejected significantly faster (MST= 54) than F1 grafts (MST= 225), demonstrating that TLO contribute to allograft rejection. F1 RIP-LTα grafts contained similar numbers of, but larger TLO than F1 grafts as demonstrated by histology. Mean speed and displacement of OT-I and OT-II cells significantly decreased over time after immunization while AC and mean CT significantly increased. B cell mean speed, displacement and AC increased after immunization. These data are consistent with B cell activation and productive T cell-DC interactions and mirror previously reported data in secondary lymphoid organs. We provide first evidence that TLO provide a local structure for T and B cell activation that propagates anti-graft immune responses in the setting of chronic rejection.
The role of TRM in transplantation is unknown. In this study, we investigated the formation and function of TRM in a mouse kidney transplantation model. (B6xBALB/c) F1.ova kidneys were transplanted to B6 recipients and 1m OT-I Teff cells transferred on day 2. Graft, blood, bone marrow, SLO, and liver were harvested 4 and 8 wks after transplantation. TRM were identified as CD44hiCD62LlowCD69+ CD103+/− cells after excluding in vivo labeled T cells. OT-I and polyclonal TRM were transcriptionally characterized using scRNAseq. TRM residency was tested by parabiosis and re-transplantation. To further establish a causal relationship between the TRM OT-Is and rejection we depleted them at wk 4 post adoptive transfer using anti-Thy1.1 (250 ug IV per day for 7d). Mean serum creatinine (mg/dl) was significantly higher in allo vs syn group at wk 8 (0.7 vs 0.2, p<0.05). Graft histology showed mixed acute and chronic rejection in the allo group. Flow analysis of allograft cells demonstrated TRM cells among OT-I and endogenous T cell populations at 4 & 8 wks. The OT-I population was exclusively TRM phenotype by flow and scRNAseq, rapidly produced IFNγ upon re-stimulation, and was not detected anywhere else. There was no significant difference in mean number of OT-Is between wk 4 and wk 8 (277K vs 234k, p=0.74). OT-I T cells could not be detected in the parabiont kidney graft or tissues or in the secondary host outside the re-transplanted kidney, indicating that the TRM are indeed resident in the graft and do not re-circulate. Depleting the OT-Is preserved kidney function at wk 8 compared to the non-depleted group (Cr= 0.7 vs 0.2, p<0.05). Our findings show that donor-specific TRM form in kidney allografts, are functional, and contribute to rejection.
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