Figure 4.

Tumour explants, but not spheroids suppress CD8⁺ T cell activity. CD14⁻ PBMCs were cultured with autologous tumour-conditioned Mφs, or control M1/M2/Media Mφs, from either healthy donors or NSCLC patients. Mφ cultures were loaded with 5 μg/mL viral peptide pool (VP stimulated) or DMSO (unstimulated) prior to the addition of T cells. T cell co-cultures were incubated for 6 days. Intracellular cytokine staining of IFNγ and TNFα was performed and analysed by flow cytometry. (A) Representative gating strategy of CD3⁺ cells for flow cytometry analysis of pro-inflammatory cytokine production (++ = double positive staining) by CD4⁺ and CD8⁺ T cells. Summary of the proportion of IFNγ⁺, TNFα⁺, and IFNγ⁺TNFα⁺ cytokine production from CD8⁺ T cells in the presence or absence of (B) M1, M2, and unpolarised media control Mφs (n = 5 healthy donors), (C) Heterotypic tumour spheroids (in the (i) absence and (ii) presence of exogenously added Mφs; n = 3 healthy donors), and (D) tumour explants/explant-conditioned Mφs (n = 4 NSCLC patients). Each symbol represents an individual healthy donor or patient. Values normalised against unpolarised media control Mφs. The p values were obtained from one-way ANOVA followed by post hoc Tukey test. Statistically significant differences were observed compared to all experimental arms. All data were pooled from at least 3 independent experiments and graphs show means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.