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 underlying this study is that analysis of the heterogeneity of alloreactive memory B cells (Bmem) in patients carrying HLA antibodies under different levels of HLA immunization intensity, corresponding to varying degrees of exposure to HLA alloantigens, may provide insights into the mechanisms governing the formation of the overall alloreactive B-cell repertoire.

Two categories of patients sensitized to HLA antigens following immunizing events (at least one transplantation or pregnancy) and awaiting kidney transplantation will be specifically investigated:

Hyperimmunized patients, defined by an incompatible graft rate (IGR) ≥ 85%, carrying a broad HLA antibody repertoire.

Immunized patients with an IGR between 50% and 85%, carrying a more restricted HLA antibody repertoire.

These two groups represent distinct levels of exposure to HLA alloantigens. Patients exposed exclusively to immunizing events other than transplantation or pregnancy will not be included because of the absence of detectable alloreactive Bmem in such settings.

A control group will also be included, consisting of "naïve" patients carrying HLA antibodies in the absence of classical immunizing events and lacking detectable alloreactive Bmem.

Characterization of alloreactive Bmem within these groups aims to identify potential differences in clonality, phenotype, and functionality, thereby clarifying several aspects of the alloreactive humoral response.

The anticipated impact of this pilot translational study in kidney transplantation is threefold:

Acquisition of fundamental knowledge regarding the biology of human alloantigen-specific memory B cells.

Generation of preliminary data supporting subsequent investigations of alloreactive B-cell responses during acute or chronic antibody-mediated rejection.

Identification of phenotypic, clonotypic, or functional markers that may improve discrimination of the immunological risk associated with the presence of HLA antibodies and HLA-reactive B cells.

The low frequency of alloreactive Bmem, estimated at 20-150 cells per million B lymphocytes for a given HLA specificity, together with the number and epitope diversity of targeted HLA antigens, complicates direct assessment of repertoire composition and functional properties. High-throughput single-cell approaches, including RNA sequencing (RNA-seq), have become essential tools for characterizing the heterogeneity of rare cellular populations and now enable simultaneous interrogation of immune-cell repertoires and transcriptomes.

The present project will leverage these recent methodologies to directly and simultaneously investigate clonality (B-cell receptor repertoire), membrane-marker phenotype, and transcriptomic profiles of alloreactive Bmem directed against a restricted panel of HLA alleles selected according to the HLA antibody reactivity profiles of the analyzed patients. This approach enables acquisition of single-cell characteristics from several hundred alloreactive B lymphocytes per patient and comparison with polyclonal memory B-cell populations derived from patients or control subjects.

The study is designed as a pilot investigation intended to generate preliminary data before implementation of larger-scale studies involving greater numbers of patients and analyses conducted before and after transplantation.

Inclusion Criteria:

• Age ≥ 18 years

• Rouen University Hospital patient monitored in the Nephrology and Kidney Transplantation Department, registered on the national kidney transplant waiting list

• Carrier of HLA antibodies, including at least anti-HLA2, identified during the last screening

• Notion of classic immune-promoting events including at least one previous transplant or pregnancy, or absence of a known immune-promoting event (naïve patient group)

• Incompatible graft rate (IGR)

  • IGR ≥ 85% (hyperimmunized patient group with anti-HLA polyreactivity) or
  • IGR between 50% and 85% (immunized patient group with a more restricted repertoire of HLA reactivities) or
  • IGR < 50% (naïve patient group with no known history of immune-promoting events)

• Person who has read and understood the information letter and does not object Not participating in the study

• Affiliation to a social security scheme

Exclusion Criteria:

• Patients who have received rituximab in the previous year (B-cell depleting therapy)
• Patients undergoing an HLA desensitization protocol using plasmapheresis and/or rituximab
• Patients with an active infection
• Persons deprived of their liberty by an administrative or judicial decision or persons placed under judicial protection/guardianship or curatorship

Patients awaiting kidney transplantation and carrying HLA antibodies following immunizing events

The data provided will be the property of the sponsor and will be used solely for its own research activities.