Dendritic cell-T cell co-culture

After euthanasia, bone marrow was collected from the femurs of male ApoE^−/− and ApoE^−/−CD40^−/−. Briefly, bones were cleaned of any muscle and other tissue then collected in serum-free RPMI 1640 (Gibco, Thermo Fisher Scientific). The femur was cut near the knee joint and centrifuged at 9300 × g for 1 min at 4 °C. The bone marrow containing pellet was washed with PBS and centrifuged at 300 × g for 5 min at 4 °C. Cells were incubated with a red blood cell lysis buffer containing 150 mM ammonium chloride (Sigma Aldrich) and 10 mM sodium bicarbonate (Sigma Aldrich) at pH 7.4 for 1 min on ice. After incubation, cells were washed with RPMI1640 plus Glutamax (Life Technologies) media supplemented with 10% fetal bovine serum (FBS, Life Technologies), 100 U/ml Pencillin and 100 mg/ml Streptomycin (P/S, Life Technologies, and 50 uM ß-mercapatoethanol (Sigma Aldrich), and 20 ng/ml recombinant GM-CSF (Peprotech) termed R10 media. 20 million per animal were plated in 100 mm non-tissue culture treated dishes in 20 ml of R10 media. After 3 days, 20 ml of R10 media was added. On Day 6, 20 ml of culture media was removed centrifuged at 300 × g for 5 min at 4 °C to recover non-adherent cells. After centrifugation, the supernatant was aspirated, and cells were resuspended in fresh R10 media supplemented with 10 ng/mL recombinant IL-4 (Biolegend). On Day 7, the cell culture supernatant was aspirated to obtain the non-adherent DCs. Adherent cells including the bone marrow derived macrophages were not used for co-culture. Cells were centrifuged as described earlier and resuspended at a concentration of 1 million cells/ml in R10 media supplemented with 5 ng/ml IL-4 and 1 ug/ml LPS to upregulate MHCII and CD40 expression. After overnight stimulation, cells were washed with PBS to remove residual cytokines. Dendritic cells (DC) were resuspended in R10 media with 10 ug/ml 323–339 OVA peptide (Sigma Aldrich) for 1 h at 37 °C.

To isolate naïve T cells, the spleen and axillary, brachial, and inguinal lymph nodes were harvested from OT-II mice after euthanasia. Harvested spleens and lymph nodes were torn apart manually using forceps and subsequently filtered through a 70 µm stranger in order to prepare single cell suspensions. To lyse the red blood cells, single cell splenic suspensions were first incubated with a red blood cell lysis buffer containing 150 mM ammonium chloride (Sigma Aldrich) and 10 mM sodium bicarbonate (Sigma Aldrich) at pH 7.4 for 2 min on ice. After incubation, cells were washed with PBS and counted (TC-20, Biorad). 50 million cells were aliquoted and centrifuged at 400 x g for 5 min at 4 °C. Naïve T cells were isolated according to the manufacturer’s instructions using the mouse naïve CD4 + T cell isolation kit (Miltenyi Biotec). After isolation, T cells were resuspended with RPMI1640 plus Glutamax (Life Technologies) media supplemented with 10% fetal bovine serum (FBS, Life Technologies), 100 U/ml Pencillin and 100 mg/ml Streptomycin (P/S, Life Technologies), 1× MEM non-essential amino acids (Sigma Aldrich), 1 mM sodium pyruvate (Sigma Aldrich) and 50 µM 2-mercaptoethanol (Sigma Aldrich). 100,000 naïve T cells were aliquoted in duplicate in a round bottom, tissue culture treated 96 well plate with 20,000 stimulated DCs (1:5 ratio). Dendritic cells (DC) and T cells were cultured together for 72 h before cells were prepared for flow cytometry as described in the flow cytometry section. Culture supernatant was frozen at −80 °C until it was used following manufacturer’s instruction using a multiplex bead-based assay (Mouse Th1/Th2/Th2/Th17/Th22/Treg 17-plex panel, Thermo Fisher Scientific).