Study design and sample collection

This is a single institute, prospective study enrolling patients with hematologic malignancies between 2012 and 2015. Written informed consent was obtained in accordance with Declaration of Helsinki for research under protocols approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute. The patients were eligible if they had a 6/6 HLA-identical sibling donor and appropriate end-organ function: cardiac function with ejection fraction >40%, pulmonary function with DLCO adjusted for hemoglobin and ventilation >50%, renal function with estimated GFR >15ml/min, and liver function with total bilirubin < 5 times upper limited normal or AST/ALT less than 10 times upper limited normal. The patients received either an ex-vivo CD3⁺/CD19 cell depleted, CD34⁺ cell negatively selected graft (CD3/19D, n=20, NIH protocol: 12-H-0028, {"type":"clinical-trial","attrs":{"text":"NCT01517035","term_id":"NCT01517035"}}NCT01517035) or an ex-vivo CD34⁺ cell positively selected graft (CD34S, n=22, NIH protocol: 13-H-0144, {"type":"clinical-trial","attrs":{"text":"NCT01866839","term_id":"NCT01866839"}}NCT01866839). G-CSF mobilized peripheral blood stem cells (PBSC) were used as a source of graft for both cohorts. For the CD34S study cohort, PBSC was processed to select CD34 cells using the Miltenyi CliniMACS® CD34 magnetic selection system. For CD3/19D study cohort, the PBSC graft was manipulated to deplete CD3⁺T cells and CD19⁺B cells using Miltenyi CliniMACS CD3 and CD19 Reagent System and CD34⁺ selected product was combined for the final product. Both cohorts targeted CD34+ dose at minimum of 3 ×10⁶/kg and a CD3+ add-back target dose ranging 5 ×10⁴ to 1 ×10⁶/kg. Most patients received a dose of 5 ×10e⁴ CD3⁺/kg. However, those who received reduced dose TBI or those who were not treated for acute leukemia received a dose of 5 ×10⁵ CD3⁺/kg. All cell products were cryopreserved until transplant day 0 when the products were thawed according to the institutional cell processing protocol. The detailed strategies of ex-vivo graft manipulation are described in Supplementary material. Both cohorts were treated with the same conditioning regimen of cyclophosphamide, fludarabine, and total body irradiation (600–1200 cGy, based upon age). Low dose cyclosporine was used as single-agent GVHD prophylaxis targeting trough level of 100–200 ng/ml until day 21. Antithymocyte globulin was not used and prophylactic donor lymphocyte infusions were not given.

Peripheral blood mononuclear cells and plasma samples were collected from donors before transplant and from the recipient at days 14, 30, 60, and 100 post-transplant. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation, then cryopreserved and stored in liquid nitrogen until further use. Plasma samples were isolated with centrifugation from EDTA containing tube and stored in −80°C.