Lymphocyte Culture Test, Mixed

Bone marrow aspirate was collected aseptically from the sternum of 10 horses by using 11-gauge Jamshidi bone marrow biopsy needles under standing sedation with local anesthesia. For each harvest, a total of 120 ml of aspirate was collected into 60-ml syringes containing 25,000 units of heparin each. Three horses underwent a second aspirate collection 2 months after the first, for a total of 13 aspirates (six ELA-A2, six ELA-A3, one ELA-A9 haplotypes). Bone marrow aspirates were purified via Ficoll-Paque Plus (Amersham Biosciences) gradient centrifugation, as previously described [32 (link)] and plated onto 100-mm tissue-culture plates in low glucose (1 g/dl) DMEM media (Gibco) containing 10% FBS, 2 mM l-glutamine, penicillin (100 units/ml), streptomycin (100 μg/ml), and basic fibroblastic growth factor (bFGF, 1 ng/ml).
MSCs were expanded over one passage, such that cryopreserved passage 2 (P2) stocks were obtained for each aspirate. P2 MSCs were later thawed and cultured through P8 to examine potential MHC class II expression changes over time and to compare MHC class II expression with that previously described for mid- to late-passage cells [17 -19 (link)]. At each passage, MSC stocks were cryopreserved for immunophenotyping and mixed leukocyte reactions (MLRs). Throughout culturing, media were exchanged every 48 hours. Cells were passaged 1:3 at approximately 80% subconfluency by using Accumax cell-dissociation solution (Innovative Cell Technologies, Inc., San Diego, CA, USA) and plated at a density of approximately 1 × 10⁴ cells/cm². Cells to be cryopreserved were pelleted after dissociation, resuspended in freeze media (MSC media as described earlier with 10% FBS and 10% dimethyl sulfoxide), and frozen at either 5 × 10⁶ cells/cryovial for expansion and flow cytometry or 1 × 10⁶ cells/cryovial for use in MLR experiments [21 (link)].

Buffy coats from healthy donors were obtained according to protocols accepted by the institutional review board at the University of Bonn (local ethics votes no. 288/13). Human monocytes, B cells, T cells, and NK cells (Table S1) were purified from peripheral blood mononuclear cells by MACS in accordance with the manufacturer’s instructions. Macrophages (M^b, baseline) were generated from monocytes by stimulation with GM-CSF or M-CSF for 72 hr and further activated with 28 stimulation conditions (Table S1). DCs were generated by GM-CSF in presence of IL-4 for 72 hr followed by further stimulation with uLPS, TNF+PGE₂, or αCD40 mAbs+TNF. Cells were phenotypically assessed by flow cytometry using cell lineage and activation markers. Expression of STAT4 was measured by immunoblotting. CXCL5 and IL-1α in cell culture supernatants were assessed by ELISA following the manufacturer’s instructions. The influence of macrophages on T cell activation was measured in an allogeneic mixed lymphocyte reaction where CD3⁺ T cells were stimulated with CD3+CD28 mAbs in presence or absence of differentially activated macrophages. T cell proliferation was assessed 72 hr later using the carboxyfluorescein succinimidyl ester dilution method.

Purified pDCs were cultured in RPMI-1640 GlutaMax supplemented with 10% fetal calf serum (FCS) and 100U/ml of Penicillin-Streptomycin at 37°C in the presence of 5% CO2. pDCs were stimulated (in 96-well plates at 5x10⁴ cells/ml) by CpG (4 µg/mL) (Miltenyi Biotec), R848 (10 µg/mL) (In vivogen, Toulouse, France) or recombinant IL-3 (40 ng/mL) (R&D systems). After 24h, pDCs were stained for phenotypic analysis, or collected for the mixed lymphocyte reaction (MLR), or their supernatants were collected for cytokine measurement. For MLR experiment, activated pDCs were washed once with PBS and then co-cultured for 6 days in 96-well plates with allogenic T cells stained with 5μM of CFSE (Biolegend) (pDC: T cell ratio 1:5). Then, T cells were stained for phenotypic analysis or their supernatants were collected for cytokine measurement.