B Cell Reconstitution after Gene Therapy in Patients with Wiskott Aldrich Syndrome and Comparison with Mismatched Allogeneic Hematopoietic Stem Cell Transplantation

Background. Wiskott Aldrich Syndrome (WAS) is a rare primary immunodeficiency associated with thrombocytopenia, eczema, severe infectious and autoimmune complications, and lymphomas. Mismatched allogeneic hematopoietic stem cell transplantation (HSCT) is an alternative for patient lacking an HLA-matched donor but is associated with an increased frequency of complications. Moreover low lymphoid and myeloid chimerism is related to a higher rate of autoimmunity and thrombocytopenia. Recent gene therapy (GT) trials showed that gene-corrected autologous CD34+ cells infusion could be an appropriate therapeutic approach for these patients. It has been recently shown that B cell homeostasis is altered in WAS. As the B cell reconstitution participates to the restoration of immunological competence, a comprehensive study of this compartment after GT and the comparison with mismatched allogeneic HSCT is crucial.

Objective. To perform a longitudinal study of B cell reconstitution in WAS patients after lentiviral vector-mediated GT, compared to mismatched allogeneic HSCT.

Methods. Five patients (age 0.8-15.5 years) underwent GT at our center since 2011(follow-up 1.5-4.2 years) after near-myeloablative and immunosuppressive conditioning regimen with (n=3) or without (n=2) anti-CD20 administration. Patient 2 (P2) died 7 months after GT from a pre-existing infectious complication. Eleven patients undergoing mismatched allogeneic HSCT (age 0.6-10.9 years) at the same center were studied (follow-up 5.1-14.7 years). Longitudinal B cell assessment included B cell count before and after treatment, and the following subsets: switched memory (SM, CD19⁺ CD27⁺ IgD^-), marginal zone (MZ, CD19⁺ CD27⁺ IgD⁺), naives (CD19⁺ CD27^- IgD⁺), transitional (CD19⁺ CD27^- IgD⁺ CD24^high CD38^high), circulating plasma cells (CD19⁺ CD27⁺ IgD^- CD27^high CD38^high) and CD21^low B cells (CD19⁺ CD21^low CD38^-), a subset abnormally expanded in WAS. Quantification of the B cell replication history was assessed through k-deleting recombination excision circles (KRECs). Analyses were compared to age-matched controls. WAS protein (WASP) expression and vector copy number (VCN) were measured in sorted B cells.

Results. All alive GT patients show stable engraftment of functionally corrected lymphoid cells, without adverse events. Transduced B cells number and WASP expression increased progressively after GT. Absolute B cell count attained normal values in all the patients, and correlates with WASP expression and VCN in B cells. IgM levels are below normal ranges in four patients. P3 and P4 attained a B-cell phenotype within normal ranges; P3 discontinued intravenous immunoglobulin (IvIg) replacement. No expansion of CD21^low B cells was observed. P1 and P5 (follow-up 18 months) present a variable defect in SM, naives and/or MZ B cells. P1 recently developed autoimmune manifestations; no significant changes were observed concomitantly. A defect in B cell lymphopoiesis was observed before GT as measured by KRECs analysis, normalizing after GT (P1, P3 and P4). Several complications were recorded in patients undergoing mismatched allogeneic HSCT, including dysimmunity, arthritis, developmental deficit and infections. Total B cell count normalized in eight patients, IgM levels were low in three. Among patients with available information, four still remain under IvIg replacement. Four patients developed a mixed lymphoid and myeloid chimerism, variably associated with low B cell count, low IgM and IvIg replacement. A complete B cell assessment for these patients is ongoing.

Conclusions. B cell transgene expression is obtained after lentiviral vector-mediated GT in WAS patients and is associated with improved B cell lymphopoiesis. A correct B cell phenotype is observed in two patients who did not receive rituximab prior GT. The question whether this is related to the treatment will need a longer follow-up to be answered. Patients undergoing mismatched allogeneic HSCT present a higher frequency of complications. Although a higher proportion of these patients discontinued IvIg replacement, B cell reconstitution is not optimal. Analysis of patients in particular with mixed chimerism will provide important information in the setting of GT. The analysis of B cell reconstitution after GT and mismatched allogeneic HSCT deserves particular attention in the assessment of immunological reconstitution.