Methods for MLR – Supplementary Information for Fanning et al, “ALLOGENEIC TRANSPLANTATION OF
MULTIPLE UMBILICAL CORD BLOOD UNITS IN ADULTS: ROLE OF PRE-TRANSPLANT MIXED
LYMPHOCYTE REACTION TO PREDICT HOST vs. GRAFT REJECTION”
Cells
With informed consent, peripheral blood from UCB transplant recipients was collected prior to administration of
conditioning regimen. All patient and graft cells were either were used immediately or cryopreserved individually
with 10% v/v dimethylsulfoxide in fetal bovine serum (FBS) and stored at -180ºC for subsequent study.
Mixed Lymphocyte Reactions
Frozen cells, both patient and grafts, were thawed using high protein thaw mix (25% FBS in Dulbecco’s phosphate
buffered saline) and treated with 50U Benzonase/mL (EM Industries, Inc., Gibbstown, NJ) in complete RPMI
(RPMI+10%FBS+1%L-glutamine) to prevent cellular clumping.
Cells were evaluated in modified mixed
lymphocyte reactions (MLR) including cell proliferation and secreted cytokine analyses as described previously(2628). Briefly, cells were either left non-stained (stimulators) or treated (responders) with 10μM carboxyfluorescein
dacetate, succinimidyl ester (CFSE) for evaluation of cellular proliferation. UCB units were pooled and plated in 96
well round bottom culture plates with corresponding patient cells. Cultures were set-up with 1x105 cells/well and all
assays included appropriate negative controls (patient peripheral blood mononuclear cells alone) and positive control
cultures (PHA 10μg/mL).
MLR were cultured for 72 hr after which cells were removed from the plate for
proliferative studies measured by flow cytometry (CSFE analyses of specified gated CD3 + T and CD56+ NK cells)
and 150uL of supernatant was removed for cytokine analyses.
CFSE cell cycling and cytokine secretion analyses
At 72 hr MLR culture, cells were surface stained for CD3 and CD56 (BD Pharmingen) and fixed for flow cytometric
analysis. The cells were evaluated on a FacScan (Becton-Dickinson), and results were analyzed using WinList
software (Verity Software; Topsham, ME). Specified CD3 + T and CD56+ NK lymphocyte populations were gated
and surface CFSE quantified to measure cell cycling in these specified lymphocyte populations. MLR supernatant at
72 hr culture was evaluated using cytometric bead assays (CBA, BD-Biosciences) following standard procedure (BD
Biosciences). Briefly, supernatant samples were incubated with antibody conjugated beads and fluorochrome and
immediately analyzed on an LSR1 flow cytometer (BD Biosciences). Results were then plotted to determine
cytokine protein (IL-2, IL-4, IL-5, IL-10, TNFα and IFNγ) concentrations using a standard curve. Reported data
outline supernatant cytokine concentrations secreted in MLR cultures incorporating UCB grafts and patient PBMC
minus that secreted in supernatant from controls incorporating patient PBMC alone.