Effect of Rituximab on Spleen and Blood B-Cell Subpopulations in Allo-Immunized Children with Sickle Cell Anemia

Introduction:

Transfusion therapy is a mainstay treatment in patients with sickle cell anemia (SCA) but delayed hemolytic transfusion reaction (DHTR) may occur in 4-7 % cases, despite extended red blood cell (RBC) blood group phenotyping and cross matching. This life-threatening acute complication dramatically complexifies patient management and requires that further transfusion be avoided or planned following immunosuppressive therapy. Rituximab (RTX), an anti CD20 monoclonal antibody (Ab) is one of the recommended preventive treatments. The pathophysiology of DHTR is complex and may involve Ab formation, through immune pathways including either T-cell dependent follicular B cells, or T-cell independent marginal zone B cells, depending on the type, structure and function of the allo-RBC antigen. Here, we report the analysis of B cells and subsets in peripheral blood and spleens of children with SCA who required splenectomy for acute splenic sequestration (ASS) or both ASS and DHTR treated with RTX.

Methods:

Consecutive pediatric patients requiring splenectomy at the Robert Debré University Hospital and presenting with either ASS and DHTR treated with RTX or ASS alone between 01/10/2021 and 31/10/2023 were included for analysis, following informed consent. Total blood count and flow cytometry (CF) analysis in blood and fresh spleen samples was performed counting among CD45+ cells, total % of CD19+ B cells and subsets: naïve (CD27^-/IgD⁺), double negative (CD27^-/IgD^-), switched memory (CD27⁺/IgD^-) and unswitched memory (CD27⁺/IgD⁺). Histological evaluation of spleen B cells was also performed on paraffin-embedded spleen samples.

Results and Discussion:

Five children presenting with both ASS and DHTR treated with RTX (R+) and 5 children with ASS and no RTX (R-) were analyzed. Median [min-max] number of RTX infusion (375 mg/ m²) was 2 [2-3]. Median [min-max] age was not significantly different and was 7.2 [2.8-12.2] yrs and 3.6 [2.3;10] yrs, in R+ and R- children, respectively.

Expectedly, a sharp decrease in % of CD19+ B cells in both blood and spleen was evidenced in R+ children compared to R- children (1.5% [0.22-2.9] vs 21.5% [4.5-29.5] and 2.1% [1-10] vs 31.0% [18-48], respectively, p <0.01). This decrease was similar in peripheral blood and spleen in R+ children (p= 0.3), illustrating the effect of RTX not only in blood but also on the splenic CD19+ B cell pool.

In untreated children, we observed a similar % of CD19+ B cell subsets in the blood and the spleen, except for a slightly increased % of switched B cells in the blood compared to the spleen (median of 11% / 6% / 72% / 6% for switched /unswitched /naive/ double neg subsets in the blood and 5% / 1% / 65% / 21% in the spleen).

Regarding B cell subsets in R+ patients, keeping in mind overall low counts of B lymphocytes, we observed no impact of RTX on the % of CD27 memory B cells in either blood or spleen and a relative increase in % of naïve subset in the spleen comparatively to blood, with a median of 49 % / 3% / 3% / 32 % and 42% / 7% / 24% / 30% for switched /unswitched /naive/ double negative subsets, in blood and spleen respectively.

Comparison of absolute counts in B cell subsets in the spleen of R+ and R- patients showed that RTX did not significantly impact unswitched and switched memory B cell numbers while it drastically reduced naïve and double negative B cells (p<0.01).

To further analyze spleen B cells, quantification of B-cell follicles was performed (magnification x 2.5) in all spleen samples and showed a comparable mean (+/- SD) number of B cell follicles of 11 (9) vs 15 (11) in R+ and R- children, respectively. Of note, the number of B follicles was inversely correlated to age at splenectomy (Spearman < 0.01). Further immunochemistry and topographic quantification of spleen B-cell subsets is currently ongoing to further delineate in situ depletion in B cells following RTX and the respective effect of treatment and age in SCA children.

Conclusion:

This study gives insight on the clearing effect of RTX on peripheral and splenic pool of B cells in patients with SCA and DHTR. Although 2 doses of RTX treatment decrease drastically the number of B cells both in the blood and the spleen, subsets of memory B cells may persist in spleens of treated children, a finding which may explain recurrence of hemolytic reactions in patients transfused despite immunosuppression by RTX. In these patients, an alternative treatment with an anti-CD 19+ monoclonal Ab (tafasitamab) could be an option.