Effect of STm-delta-aroA on CD4+ T cell activation. Effect of STm-delta-aroA on CD4+ T cell activation

The experiment was designed to test how tumour conditoned media from STm-delta-aroA infected tumour organoids would modulate T cell activation. Using splenocytes from Nr4a3-Tocky Great Smart-17A mice, T cells were activated with soluble anti-CD3 and anti-CD28 antibodies. At 4 and 16 h live CD4+ T cells were FACS sorted and RNA extracted. Analysis revealed that T cells exposed to STm-delta-aroA conditioned tumor medium failed to upregulate metabolic pathways associated with canonical T cell activation. Overall design: Tumour organoids (colonic and small intestinal) derived from APCmin mice were cultured in Matrigel and infected with either an attenuated STm mutant (aroA, containing a deletion in the shikimate pathway) or left non-infected (non-treated) for 2 hours. After 2 hours, the organoids were washed twice with PBS and then re-cultured in fresh media containing gentamicin, in order to kill any extracellular bacteria. Tumours were incubated at 37C for 48hours, after which time the tumour-conditioned media (TCM) was aspirated and pooled. Meanwhile, splenocytes from Nr4a3-Tocky-GS mice were isolated and seeded into a 96-well plate. Splenocytes (1x10^6/well) were cultured in 100ul TCM from non-treated or STm-infected tumours for 4 and 16h, with 1 ug/mL a-CD3 and 5 ug/mL a-CD28 antibodies added to each well to induce T cell activation. Live CD4+ T cells were then FACS-sorted and RNA extracted. Libraries were made using Lexogen's Quant-Seq 3' mRNA FWD UMI kits according to the manufacturer's instructions. Libraries were pooled and sequenced on a NextSeq 500 machine.