B-cell reconstitution and rituximab quantification. (A) Blood CD19⁺CD5⁻CD23⁻ B-cell quantification. Blood samples collected from HLA identical donors (○), and study subjects (●) before TLI-ATG conditioning and 56 days after alloHCT were FACS analyzed to quantify CD19⁺ B cells. Recipient MCL and CLL cancer cells were excluded from this donor B-cell quantification by excluding CD5⁺ or CD23⁺ cells. Blood CD19⁺CD5⁻CD23⁻ B-cell quantification performed 90 and 180 days after alloHCT showed limited donor B-cell recovery after rituximab. For comparison, donor B-cell recovery 56 days after TLI-ATG alloHCT was determined in 19 patients who never received rituximab (▴) to control for passive transmission of rituximab infused before HCT. (B) Rituximab infused 6 months or less before HCT is detected by ELISA at transplantation. Blood collected immediately before conditioning was measured by ELISA for rituximab concentration (μg/mL; y-axis). Each pre-HCT rituximab level was related to the number of months since their last rituximab infusion (x-axis). (C) CLL decreases following rituximab infusion 56 days after alloHCT. Immunophenotyping detected persistent CD19⁺CD5⁺CD23⁺ CLL cells 56 days after HCT in most CLL patients. Ten of 16 (63%) patients had > 10 CD19⁺CD5⁺CD23⁺ cells/μL of peripheral blood. After rituximab infusion, only 4 of 20 had CLL detected by flow cytometry on day 90. Twelve MCL patients had negligible CD19⁺CD5⁺CD23⁻ cells in the blood measured on both days 56 and 90.