Cytokine Production and Proliferation in Mixed Lymphocyte Culture (MLC) May Predict Donor Engraftment in Adult Recipients of Multiple Umbilical Cord Blood (UCB) Unit Transplantation.
Authors: Fanning L. R. , Tary-Lehmann M. , Jaroscak J. , Rubinstein P. , McCormick T. , Swick A. , Koc O. , Lazarus H. , Tse W. , Cooper B. , Finney M. , Masri O. , Kozik M. , Haung Y. , Winter D. , Banks J. , Duam-Woods K. , Laughlin M. J. 
Published: October 2019

Abstract
Limited cell dose within a single UCB graft provides the rationale for multiple UCB unit transplantation. Our single institution phase I study testing the safety and efficacy of multiple UCB unit infusion targeted a nucleated cell dose of minimum ≥ 5x107cells/kg, and patients were transplanted with 3-5 unrelated UCB units. Seven adult patients (median 56 years; range 20–69) with advanced hematologic malignancies (4 AML, 1 ALL, 2 NHL) were enrolled and treated with non-myelablative conditioning including Fludarabine 150mg/m2, Cyclophosphamide 2gm/m2, and ATGAM 60mg/kg. UCB grafts were not T-depleted. All UCB units were 1-2 HLA antigen mismatched with the patient, and HLA matching between units was not required. The patients were transplanted sequentially and received a median infused total nucleated cell dose: 5.4x107/kg (range 4.2–8.9), CD3+: 1.4x107/kg (range 1.4–3.4), and CD34+: 2.2x105/kg (range 1.9–5.3). Three of the 7 patients demonstrated UCB donor engraftment while 3 patients had autologous recovery, and one patient died prior to engraftment (day=56). Mixed lymphocyte culture (MLC) was performed including proliferation and cytokine production in order to evaluate impact of graft-graft and patient-graft immune reactivity on donor engraftment. Cryogenically preserved pre-transplant patient and corresponding UCB graft samples were thawed for MLC with readouts including proliferation (CFSE staining) and cytokine production (cytometric bead assay-CBA)(Becton Dickinson, Franklin Lakes, NJ) including pro-inflammatory TH1 cytokines (IFN-γ, TNF-α, IL-2) and anti-inflammatory TH2 cytokines (IL-10, IL-5, IL-4). We hypothesize that increased proliferation and a strong TH1 response may be detrimental to engraftment which was confirmed by preliminary analysis (n=4). We observed higher rates of proliferation as well as higher TH1 cytokine production within the MLC of patients who did not attain donor engraftment.
Table 1: TH1 Cytokine Output and Proliferation of Patient and Graft Mixed Lymphocyte Cultures
Patient Number Number of UCB Units Donor Engraftment IFNγ(pg/mL) TNFα (pg/mL) IL-2(pg/mL) % Proliferation Pt #1 5 No 940 226 79 16 Pt #2 3 No 234 56 46 20 Pt #3 3 Yes 32 <20 <20 9 Pt #4 5 Yes <20 <20 <20 N/A
TH1 cytokines produced in graft-graft MLCs were also elevated in the non-engrafting patients over those of the engrafting patients. The TH1 response of graft-graft interactions was lower than patient-graft interactions, indicating a bi-directional immune response. PHA positive controls indicate the decreased TH1 cytokine production seen in engrafted patients was not due to an inability to be activated. No trends were identified in the TH2 cytokines measured. These preliminary results suggest that graft-graft immune interactions as well as patient-graft interactions may determine overall donor engraftment. We plan to prospectively test this hypothesis in our ongoing clinical trial. MLCs with TH1 cytokine readouts that are patient and UCB unit specific may predict donor engraftment after multiple unit infusion and may be used as an adjunct to HLA typing.