T cell proliferation using mixed lymphocyte reaction
assay
Mononuclear cells were prepared as described above. Stimulator cells
were extracted and re-suspended in enriched media for irradiation. Cells
were irradiated at 3000 rad. For responder cells, T cell enrichment was
performed using the Easy Sep T cell enrichment antibody mix (1 μL per
106 cells), magnetic beads and Easy Sep Purple Magnet
separation (Stemcell Technologies). MLRs were applied in the following
combinations: maternal T cell responders vs irradiated cord blood cells,
cord blood T cell responders vs irradiated maternal cells, maternal T
cell responders vs irradiated neonatal cells and neonatal T cell
responders vs irradiated maternal cells. For the setup of neonatal MLRs,
all corresponding maternal cells had undergone prior cryopreservation
and were thawed as described above. In selected maternal, cord blood and
neonatal samples (n = 6 each), the T cell enriched suspensions were
split between non-CD25-depleted and CD25-depleted samples. Suspensions
that were retained for CD25-depletion were depleted using CD25
MicroBeads with magnetic MACS microcolumn separation (Miltenyi Biotec,
Bergisch Gladbach, Germany).
To trace proliferation of
responder T cells in the assay, 1 μL of CellTrace Violet dye
(Invitrogen) was added per 106 cells and incubated at
37 degrees for 20 minutes. 2 x 105 irradiated
stimulator mononuclear cells were added at a 2:1 ratio to each responder
sample of 1 x 105 in a 96-well round-bottom plate in
enriched media. Each sample was run in duplicate. Positive control
samples were established with the addition of 5 μL of CD3/CD28 activator
Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA) instead of
stimulator cells. Negative controls were established in enriched media
only. To each sample and positive control, 10 U of IL-2 cytokine was
added. Samples were incubated for 5 days. At day 3, 150 μL of media per
well was replaced with fresh media. Each sample was harvested and washed
with PBS (Sigma Aldrich). Surface staining was performed using
APC-Cy7-conjugated CD3, BV510-conjugated CD4 and PerCP-Cy5.5-conjugated
CD8 (all from Biolegend). Samples were washed and then re-suspended. 1
μL of propidium iodide (Biolegend) was added for live/dead
discrimination to each sample immediately before flow cytometry was
performed on an LSRII flow cytometer equipped with blue, red and violet
lasers (BD Biosciences). At least 20,000 cells were recorded per sample.
During the gating process, doublets were first excluded based on FSC-A
and FSC-H characteristics. Lymphocytes were then identified based on
FSC-A and SSC-A characteristics. Dead cells were excluded based on
positivity in the ECD channel. Further gating was performed within CD3+
cells. Flow cytometry data was analyzed using the FlowJo software
package.