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.