*Purpose: Our previous studies suggest that a DBA/2 kidney graft is accepted by a MHC-fully mis-matched C57/BL6 recipient. Accepted renal allografts exhibited lymphoid like structures containing numerus Foxp3+ T cells within 2 weeks of transplant, referred to as Treg-rich Organized Lymphoid Structures (TOLS). We have previously shown that depletion of Tregs results in the dissolution of TOLS and the rejection of the kidney allograft by day 6.The aim of this study is to do an in-depth analysis of the TOLS – investigating the time-course change of infiltrated cells in accepted mouse kidney allografts, which immune cells could be involved in the formation of lymphoid like structures, and the role CCR7 plays in the formation of TOLS.

*Methods: DBA/2 donor kidneys were transplanted into C57/BL6 or CCR7 KO recipients that have undergone bilateral nephrectomy. Transplanted animals were sacrificed at week 1, 2, 3, and 6 post-transplant, kidney allografts were examined by H&E, immunohistochemistry staining, and Nanostring RNA analysis. Some renal allografts were digested by collagenase and mononuclear cells were isolated by density centrifugation subsequently, flow cytometry was then used to quantify the percentage of various immune cells.

*Results: H&E staining showed widespread infiltration of immunes cells in the cortex of renal allografts at week 1, and the formation of TOLS can be observed by week 2, localized to small arteries. By week 6, these structures are highly defined, showing the appearance of lymphoid structures. IHC staining showed these structures contain mixtures of CD4+ and CD8+ T cells, Tregs, dendritic cells, B cells, and plasma cells. FACS data suggest 18.8% ± 4.4 of T cells in the TOLS at week 6 were Foxp3+ cells. IHC staining also showed TOLS are PDPN+ (podoplanin), LYVE-1+, and PROX-1+, demonstrating its lymphatic characteristics. TOLS are MECA79-, lacking high endothelial venules that are specific to inflammatory tertiary lymphoid organs. We observed decreased survival of CCR7 KO recipients transplanted with DBA/2 allo-kidneys. TOLS were smaller in kidney allografts from CCR7 KO versus WT recipients (0.40 ± 0.17 mm² vs 1.73± 0.39 mm², respectively, p < 0.001) with a decrease number of B cells when compared to allografts procured from B6 WT recipients (48% vs 75% B220+ cells, respectively, p < 0.001). Nanostring RNA analysis confirmed a B cell signature associated with accepted kidney allografts which was significantly reduced in CCR7 KO recipients.

*Conclusions: TOLS seem to be unique structures in tolerized kidney allografts and their formation is dependent on the CCR7 pathway. They represent novel regulatory tertiary lymphoid organs, different from inflammatory TLOs. Nanostring analysis reveals an increased B cell signature associated with TOLS. How infiltrating immune cells are organized into TOLS and how these structures contribute to tolerance induction need to be further studied.

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