Background: Chronic allograft rejection is a major cause of graft loss in solid organ transplantation. Local intra-graft B and T lymphocytes form organized clusters resembling lymphoid follicles, which have been associated with worsened graft outcome. The B cell activating factor BAFF is an important B cell survival factor and may influence the development of tertiary lymphoid organs (TLO). We have studied the expression of TLO-promoting factors and BAFF and their correlation with TLO in a rat model of renal transplantation (RTx). In parallel, we are examining the relationship of intra-graft BAFF levels and TLO formation in renal transplant patients.
Methods: MHC-mismatched rats (Brown Norway ->Lewis) were used in this model of RTx. Rats were subjected to chronic underimmunosuppression (CyA 5mg/KG/every 2 days) and sacrificed on d28 and d56 post-Tx. Expression of TLO-promoting factors (CCL19, CCL21, CXCL13, Lymphotoxin-ß) and BAFF were measured by qPCR, immunohistochemistry or flow cytometry. Intra-graft leukocyte and B cell subsets were assessed by flow cytometry (CD11b/c, CD163, CD3, CD45R, IgM, CD38, CD27) and intra-renal spacial distribution was assessed by immunofluorescence.  Cell proliferation and formation of germinal centres was assessed using Ki67-staining. Specific molecules of T/B cell interaction (CD40/CD40L, ICOS/ICOSL), and B and T cell transcription factors (Bcl-6, Blimp-1) were assessed by qPCR. An interventional anti-BAFF antibody is also being tested in this model. In parallel, renal transplant biopsies from our patient cohort were assessed for BAFF and the occurrence of lymphoid follicles by immunohistochemistry (CD3, CD20).
Results and Discussion: We demonstrated the induction of TLO-promoting factors (CCL19/CCL21, CXCL13, Lymphotoxin-ß, and BAFF) and the development of organized lymphocyte clusters after prolonged underimmunosuppression in a rat model of RTx. We have characterized microanatomical and functional features of these lymphocyte clusters, including T and B cell zones, germinal centres, and expression of costimulatory molecules, B cell differentiation markers and transcription factors related to effector functions of humoral alloreactivity (e.g. Ig class switching, antibody production). The effect of an interventional anti-BAFF antibody on intra-graft lymphocytes is being tested in this rat RTx model. Furthermore, intra-graft BAFF expression and the occurrence of lymphoid aggregates and TLO is correlated in our renal transplant patient collective.
Conclusion: Under prolonged conditions of suboptimal immunosuppression TLO-promoting factors and BAFF are upregulated and organized intra-graft lymphocyte clusters are induced. This may augment local cellular and humoral alloreactivity and thereby contribute to chronic allograft injury. BAFF may act as a pivotal factor for the intra-renal B cell axis and subsequently for TLO formation.