Rituximab Infusion after Allogeneic HCT Delays B Cell Reconstitution and Alloimmunity.

B cells are implicated in the pathophysiology of chronic graft-vs-host disease (cGVHD) and anti-B cell rituximab is effective cGVHD therapy. We have treated 31 MCL and CLL patients with a nonmyeloablative transplant preparative regimen consisting of total lymphoid irradiation (TLI, 80 cGy in 10 fractions, days -11 to -1) and anti-thymocyte globulin (ATG, 1.5mg/kg/day, days -11 to -7, total 7.5mg/kg) followed by rituximab 375mg/m2 on days 56, 63, 70, and 77 after transplant. Primary GVHD prophylaxis was mycophenolic acid and cyclosporine tapered off by 6 months. Thus far, two patients with MCL have died of disease progression before rituximab infusion and the remaining 29 are alive. Here we study B cell reconstitution in 12 patients with more than one year of follow-up. We used Hi-D FACS technology to distinguish common lymphoid progenitors (CD34⁺CD117⁺), early B cell progenitors (CD34⁺CD10⁺CD19⁺), pre B cells (CD3⁻CD19⁺CD10⁺CD34⁻), immature B cells (CD3⁻CD19⁺CD10⁺CD20⁺CD5⁻IgM⁺), mature and memory B cells (CD3⁻CD19⁺CD20⁺CD27⁺), and plasma cells (CD138⁺CD38⁺). Peripheral B cells (CD19⁺CD20⁺) remained undetectable 6 and 9 months after transplant. Peripheral blood CD19+ cells were first detected in 3/9 patients at 1 year and 6/6 patients at 1.5 years. The majority of recovering peripheral blood B cells expressed a memory phenotype (CD19⁺CD27⁺, n–=6). Bone marrow aspirates collected 180 and 365 days post transplant showed CD34⁺CD117⁺ lymphoid progenitors (n=4) are increased after rituximab and then decline from 13–20% of cells 180 days post transplant to 3–8% of cells 365 days post transplant. Control patients transplanted using a TLI-ATG regimen without rituximab infusion show a lymphoid progenitor cell frequency of 4–7% (n=3). CD19⁺CD10⁺ immature progenitor B cells accumulated after rituximab, comprised 3–6% of lymphoid cells in the bone marrow 90 and 180 days after HCT (n=4), and with time were replaced by mature B cells lacking CD10 expression. IgM and/or IgD expressing mature cells (that usually express CD20) were rarely detected in bone marrow until peripheral CD19+ B cell recovery. As expected the frequency of CD19⁺CD27⁺ mature memory B cells was very low at 0.3–1.5% (n=4). Finally, CD38⁺CD138⁺ plasma cells accounted for 0.5–2% of bone marrow before and after rituximab. In summary, B cells recover from increased proportion of lymphoid progenitors with reconstitution recapitulating B cell ontogeny. No adverse infusion events occurred with rituximab and infectious complications reflected usual transplant incidence including CMV and VZV reactivation, influenza B, aspergillus and pseudomonal bacteremia. Plasma IgG levels increased from the patient’s peritransplant baseline to 110% at 9 months, 158% at 12 months, and 124% at 18months. At 6, 9, and 12 months, EBV titer was 76%, 104%, and 103% relative to pretransplant patient titers demonstrating protective antibodies are maintained despite rituximab therapy presumably secreted from long-lived CD20 negative plasma cells. Thus far, no allogeneic antibody responses have developed in the five male with female donors against H-Y antigens and suggest post-HCT rituximab prevent or diminish allogeneic B cell responses. This first trial of rituximab treatment 2 months after allogeneic HCT was well tolerated, patients maintained protective humoral immunity, and peripheral blood B cells reconstituted 12–18 months after transplant.