Brefeldin a and monensin

All cells from each individual tumor-draining lymph node (dLN) or 10⁶ total splenocytes were plated in 2 mL media and restimulated with PMA (250 ng/mL) and ionomycin (1 μM) in the presence of monensin and brefeldin A (Biolegend). After three hours, the cells were washed and permeabilized before being stained for intracellular IFNγ for flow cytometry.

Proliferation assays were performed in 96‐well U‐bottom plates (Greiner Bio‐One). BMDCs (pulsed with or without lysates) were co‐cultured with 10⁵ CD3⁺ cells at different ratios (1:1, 10:1, and 100:1 CD3:BMDC) in RPMI 1640 medium as described above. The CD3⁺ cells were previously labeled with CellTrace Violet per the manufacturer's directions (Thermo Fisher Scientific). After 6 days of co‐culture, flow cytometry was used to measure CD3⁺ T‐cell proliferation. Supernatants were collected from cells co‐cultured for 24 h, and assayed by ELISA for interferon‐γ (Legend Max, BioLegend™).
Cytotoxicity assays were performed using 10⁵ rat splenic CD3⁺ cells co‐cultured in 96‐well U‐bottom plates with 10⁴ BMDCs in RPMI 1640, supplemented as described above. On day 7, the CD3⁺ cells were collected and incubated for 18 h in flat‐bottom 96‐well plates, along with target cells, at effector‐to‐target ratios ranging from 20:1 to 0.625:1. The plates were assayed for lysis by LDH assay (Thermo Scientific Pierce). CD107a expression was measured essentially as described by Betts et al (2003). BMDCs were added to CD3⁺ cells, co‐cultured for 1 h in the presence of CD107a antibody, and incubated for another 5 h in the presence of monensin and brefeldin A (BioLegend). The cells were fixed and stained with anti‐CD4, anti‐CD8, and secondary to CD107a antibodies and analyzed by flow cytometry.

Fifteen-day-old CTRL and MAT C57BL/6J infant mice were infected with Vac-OVA (1 × 10⁴ PFU i.p.) and orally treated with E. coli 0111:B4-derived LPS (50 μg orogastric; InvivoGen) beginning on the day of the infection and continuing every other day for 10 days. Mice were monitored daily for weight loss and appearance of illness. Eleven days after infection, mice were euthanized by CO₂ inhalation. Spleens were mechanically disrupted to obtain single cell suspensions and then treated with ACK buffer to lyse red blood cells. For the detection of cytokines, the splenocytes were cultured for 5 h in RPMI-10 with SIINFEKL peptide (5 μM; New England Peptide), phorbol 12-myristate 13-acetate (PMA) (10 ng/ml), and ionomycin (1 μg/ml) in the presence of brefeldin A and monensin (BioLegend).