Validation Of A New Tool To Monitor Alloreactive Memory B Cells
CHARBONNIER S. 1, DEVRIESE M. 2, NGUYEN T. 3, BLEIN T. 1, LEON J. 1,4, MEGRET J. 5, USUREAU C. 2, ANDRÉ I. 1, ANGLICHEAU D. 4, FILLATREAU S. 3, TAUPIN J. 2, ZUBER J. 1,4
1 Institut Imagine, INSERM U1163, Paris, France; 2 Service d’Immunologie et histocompatibilité, Hôpital Saint-Louis, Paris, France; 3 Institut Necker Enfants Malades, CNRS UMR8253, INSERM UMR1151, Paris, France; 4 Assistance Publique–Hôpitaux de Paris, Hôpital Necker, Service des Maladies du Rein et du Métabolisme, Transplantation et Immunologie Clinique, Paris, France; 5 Plateforme de Cytométrie en Flux, Structure Fédérative de Recherche Necker, INSERM US24-CNRS UAR3633, Paris, France
High levels of donor-specific anti-HLA alloantibodies represent a major obstacle to organ transplantation. However, serum antibody levels do not capture the risk of anamnestic responses due to the presence of HLA-specific memory B cells. Here, we exploited the case of a highly-sensitized patient, who experienced a dramatic anti-HLA class II, yet not class I, antibody rebound following desensitization, to validate a new cytometry tool for monitoring allospecific memory B cells.
Magnetically purified B cells, collected at the time of antibody rebound, were studied to characterize allospecific memory B cells. First, B cells were stimulated with TLR7/8 agonist and IL-2; second, B cells were co-incubated with fluorescent beads conjugated with single HLA antigens. Next, naïve and switched memory B cells, bound or not to HLA class II-beads, were FACS-sorted and cultured on CD40L-expressing feeder cells, along with a cocktail of cytokines. In both experiments, supernatants were screened for anti-HLA antibodies.
Using polyclonal stimulation of bulk B cells, anti-HLA antibody-secreting cells (ASC) were more frequently differentiated in vitro against HLA class II than HLA class I antigens (64 vs 28%, p < 0.0001). Strikingly, the ability of circulating B cells to give rise to ASC in vitro against HLA II antigens was associated with the rebound of circulating antibodies against HLA II antigens. This finding emphasizes the importance of identifying and characterizing alloreactive memory B cells. To this end, we demonstrated that CD27+ IgD- switched memory B cells, bound to HLA class II-beads, were able to differentiate into anti-HLA ASC on feeder cells, unlike their naïve and unbound B cell control populations.
This study stresses the critical need for monitoring alloreactive memory B cells to tailor desensitization regimen, and offers a new tool to characterize the dynamic and phenotype of HLA-targeted memory B cells.