Single Cell Profiling Identifies Novel B Cell Populations That Drive Chronic Rejection after Lung Transplantation

Abstract

Despite immunosuppressive T cell-targeting therapies, the survival rate after LTx remains as low as 50% after 5 years, mainly due to the development of BOS. The T cell centric paradigm is being increasingly challenged in solid organ transplantation. We recently demonstrated in a new mouse model of lymphocytic bronchiolitis (LB) after orthotopic LTx that B cells played a central role in the development of BOS. Alarming experiences from the past strongly suggest that a systemic B cell depletion must be considered with caution if at all. We hypothesized that a subclassification of B cells based on their individual expression profiles in rejecting versus non-rejecting murine lung grafts can provide insights into which B cell subset should be specifically targeted in priority to prevent lung graft rejection. C57BL/6J mice received C57BL/6J (non-rejecting group) or HLA-A2 knock-in (LB group) lung grafts by an orthotopic LTx surgery. One month later, single cell suspensions of lung grafts were submitted to single cell RNA-sequencing. An unsupervised clustering generated an atlas of 15 cell clusters. While the general distribution of cells amongst the different clusters was homogenous between the two groups, major quantitative shifts were observed. As such, classical monocyte, leukocyte and T cell numbers were increased respectively by 3.93-, 2.78- and 2.27-fold in the LB grafts. B cells, identified based on the expression of Cd79a, Cd79b and Ig genes, were further subdivided into 7 clusters. Clusters 0 and 2 were respectively enriched by 4.12- and 2.92-fold in the grafts that developed LB versus syngeneic lung grafts, while clusters 3 and 4 were overrepresented by 5.21- and 6.78-fold in the absence of LB. Markers for cluster 0 appeared redundant with other clusters, making it difficult to consider for further investigations. Cluster 2 displayed the expression of genes involved either in B cell activation, or differentiation into plasma cells, or regulatory/inhibitory roles, or a combination of the functions above depending on the context. Since the set of markers defining cluster 2 does not clearly relate to any described B cell subtype, we plan to functionally investigate these cells by classical immunology approaches (mixed lymphocyte reaction and ex vivo antibody production).