*Purpose: Chronic allograft rejection remains a major obstacle to long-term allograft survival. Tertiary lymphoid organs (TLOs) are ectopic lymphoid structures that arise in non-lymphoid tissues in the setting of chronic inflammation. They have been documented extensively in human renal allografts and have been associated with chronic rejection. Their immunologic role in allograft rejection is unclear. Therefore, fundamental understanding of TLO function is necessary. Here, we employed a chronic renal allograft rejection model in mice and intravital time-lapse 2-photon microscopy to investigate the function of TLO in transplant rejection.

*Methods: CB6F1 RIP-LTα (preformed TLO) or CB6F1 (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 10e6 naïve dsRed OT-I T cells or 10e6 CTR-labeled NP-specific B cells + 10e6 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. Three-dimensional 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.

*Results: CB6F1 RIP-LTα grafts rejected significantly faster (MST= 54) than CB6F1 grafts (MST= 225), demonstrating that TLO contribute to allograft rejection. Grafts from both groups harvested at the time of rejection demonstrated interstitial fibrosis, lymphocytic infiltrates and TLO, positive for B, T and HEV-marker PNAd. CB6F1 RIP-LTα grafts contained similar numbers of, but larger TLO than CB6F1 grafts. 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.

*Conclusions: Here, we show that TLO form in and contribute to allograft rejection. We provide first evidence that TLO provide a local structure for T and B cell activation that might propagate anti-graft immune responses in the setting of chronic rejection. Further studies will elucidate the formation and maintenance of TLO and the consequences of local T and B cell activation. Data will be applicable to other conditions with TLO formation such as autoimmune disease and cancer.

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