Alloreactive Memory B Lymphocytes and Anti-HLA Sensitization

Overview

In transplantation, B lymphocytes are major cellular players in the alloreactive humoral response through the production of antibodies targeting allogeneic HLA molecules expressed by the transplant. In subjects sensitized to HLA antigens, the contribution of pre-existing alloreactive memory B lymphocytes (Bmem) to allograft rejection phenomena after transplantation is now recognized. It has been proposed that the identification of these Bmem during the pre-transplant period could contribute to a better assessment of post-transplant immunological risk, allowing optimization of strategies to prevent humoral rejection. However, knowledge regarding the phenotypic and functional heterogeneity of Bmem as well as their clonal diversity is still extremely limited, not allowing discrimination between pathogenic and non-pathogenic alloreactive humoral responses. Such discrimination requires a better understanding of the modalities of differentiation of alloreactive B lymphocyte responses. To this end, this study aims to characterize the clonal, phenotypic and functional properties of alloreactive Bmem in subjects awaiting renal transplantation and sensitized to HLA antigens.

The hypothesis of this study is that analyzing the heterogeneity of alloreactive Bmem in patients carrying HLA antibodies under varying HLA immunization intensity conditions (corresponding to varying intensities of exposure to HLA alloantigens) is likely to provide insight into the differential modalities of overall alloreactive B repertoire formation. Two types of patients sensitized to HLA antigens following immunizing events (at least one transplant or pregnancy) awaiting kidney transplantation will be specifically explored: * patients deemed hyperimmunized based on an incompatible graft rate (IGR) ≥ 85%, carrying a broad HLA antibody repertoire * patients immunized with an IGR between 50 and 85%, carrying a more restricted HLA antibody repertoire. These two groups will be characterized by differential intensities of exposure to HLA alloantigens. Patients with a history of immunizing events other than transfusions will not be investigated, due to the lack of detection of alloreactive Bmem in this situation. "Control" patients will also be included: "naïve" patients carrying HLA antibodies in the absence of a classic immunizing event, in whom alloreactive Bmem are not detectable. The exploration of alloreactive Bmem in these two groups aims to identify potential differences in terms of clonality, phenotype, and functionality, which could clarify several aspects of the alloreactive humoral response: The expected impact of this pilot translational study in kidney transplantation is threefold: * the acquisition of fundamental knowledge in the field of alloantigen-specific human Bmem biology * the acquisition of data prior to the subsequent exploration of the characteristics of the alloreactive B cell response during acute or chronic humoral rejection * and ultimately, the identification of phenotypic, clonotypic, or functional markers that could potentially be used to better discriminate the immunological risk associated with the presence of antibodies and HLA-reactive B cells. The low frequency of alloreactive Bmem, estimated at 20-150 per million B lymphocytes (for a given HLA specificity), as well as the number and epitopic diversity of targeted HLA antigens, complicate the direct assessment of their repertoire and functionalities. High-throughput approaches (RNAseq) in single cells, which have become essential for characterizing the heterogeneity of rare cellular contingents, now make it possible to simultaneously interrogate the repertoire and transcriptome of immune cells. In this project, we will take advantage of these recent methodologies to directly and simultaneously explore the clonality (BCR repertoire), the phenotype of membrane markers and the transcriptome of alloreactive Bmem towards a restricted panel of HLA alleles chosen according to the HLA antibody reactivity profile of the patients analyzed. This methodology allows the acquisition of individual characteristics of several hundred alloreactive B lymphocytes per patient and their comparison with polyclonal Bmem from patients or control subjects. This is a pilot study intended to acquire preliminary data prior to studies on a larger number of patients, before and after transplantation.