Visceral adipose tissue secretome from early and late-stage oesophageal cancer patients differentially affects effector and regulatory T cells

Figures

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  Fig. 1. ACM derived from OGJ patients upregulates CD27 co-stimulatory marker and promotes differentiation into a central memory-like T cell phenotype. Healthy donor PBMCs were activated for 2 days using plate bound anti-CD3 and anti-CD28 in the absence (n = 12) or presence of ACM generated from OGJ patients with early-stage tumours (n = 14) or late-stage tumours (n = 14). CD3+, CD3+CD4+ and CD3+CD8+ cells were assessed for the expression of T cell activation markers: CD62L, CD69, CD27, CD45RA and CD45RO by flow cytometry (A). The percentage of naïve (CD45RA+CD27+), central memory (CD45RA−CD27+), effector memory (CD45RA−CD27−) and terminally differentiated effector memory (CD45RA+CD27−) T cells was also determined by flow cytometry (B). Paired non-parametric t test and data presented as percentages ± SEM *p < 0.05, **p < 0.01

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  Fig. 2. ACM derived from OGJ patients significantly upregulates TIGIT on the surface of T cells. Healthy donor PBMCs were activated for 2 days using plate bound anti-CD3 and anti-CD28 in the absence (n = 12) or presence of ACM generated from OGJ patients with early-stage tumours (n = 14) and late-stage tumours (n = 14). CD3+, CD3+CD4+ and CD3+CD8+ cells were assessed for the expression of inhibitory IC receptors including PD-1, TIGIT, TIM-3, LAG-3, A2aR, CTLA-4 and KLRG-1 (A) and IC ligands including PD-L1, PD-L2 and CD160 (B) by flow cytometry. Paired, non-parametric t-test, *p < 0.05, **p < 0.01

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  Fig. 3. ACM derived from OGJ patients promotes a Th1-like phenotype and upregulates CTLA-4 expression on the surface of Th-1 like cells. The effect of early-stage ACM (n = 9) and late-stage ACM (n = 8) on the frequency of Th1-like CD3+CD4+ and CD3+CD8+ cells was assessed by flow cytometry and compared with untreated cells (n = 10). The effect of early-stage ACM (n = 9) and late-stage ACM (n = 8) on the expression of TIGIT, CTLA-4, PD-1 and PD-L1 was assessed on the surface of CD3+CD4+Th1-like and CD3+CD8+Th1-like cells by flow cytometry (B). The effect of ACM on the secretion of Th1-like cytokine IFN-γ was also assessed in CD3+CD4+ and CD3+CD8+ cells by flow cytometry (C). The effect of single agent nivolumab, single agent atezolizumab, single agent ipilimumab, dual nivolumab-atezolizumab or dual nivolumab-ipilimumab in the absence (n = 6) or presence of early-stage ACM (n = 6) and late-stage ACM (n = 6) on the secretion of anti-tumour cytokine IFN-γ was assessed in CD3+CD4+ and CD3+CD8+ cells by flow cytometry (D). Th1-like cells were characterised as CXCR3+CCR6− cells. Representative dot plot shown displaying frequency of CD4+ Th1-like cells (A). Paired, non-parametric t test and data…

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  Fig. 4. ACM derived from OGJ patients increases the frequency of pro-inflammatory-like Th-17 cells and downregulates ICs on their surface. The effect of early-stage ACM (n = 9) and late-stage ACM (n = 8) on the frequency of Th1/17-like and Th17-like CD3+CD4+ and CD3+ CD8+ cells was assessed by flow cytometry and compared with untreated cells (n = 10) (A). The effect of early-stage ACM (n = 9) and late-stage ACM (n = 8) on the expression of TIGIT, CTLA-4, PD-1 and PD-L1 was assessed on the surface of CD4+Th1/17-like and CD8+Th17-like cells (B) CD4+Th17-like cells and CD8+Th17-like cells (C) by flow cytometry and compared with untreated cells (n = 10). Th1/17-like cells were characterised as CXCR3+CCR6+ cells and Th17-like cells were characterised as CXCR3−CCR6+ cells. Representative dot plot shown displaying frequency of CD4+ Th1/17-like cells and Th17-like cells (A). Paired, non-parametric t test and data presented as percentages ± SEM. *p < 0.05 and **p < 0.01

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  Fig. 5. ICBs attenuates the ACM-enhanced pro-inflammatory T cell cytokine signature. The effect of early-stage (n = 9) ACM and late-stage ACM (n = 8) derived from OGJ patients on the secretion of pro-inflammatory cytokines IL-17A/F and TNF-α was assessed in CD3+CD4+ and CD3+CD8+ cells by flow cytometry and compared with untreated cells (n = 10) (A). The effect of single agent nivolumab, single agent atezolizumab, single agent ipilimumab, dual nivolumab-atezolizumab or dual nivolumab-ipilimumab in the absence (n = 6) or presence of early-stage ACM (n = 6) and late-stage ACM (n = 6) on the secretion of pro-inflammatory cytokines IL-17A/F (B) and TNF-α (C) was also assessed in CD3+CD4+ and CD3+CD8+ cells by flow cytometry. Paired, non-parametric t test and data presented as percentages ± SEM. *p < 0.05 and **p < 0.01. Abbreviation: niv nivolumab, atez atezolizumab, ipi ipilimumab

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  Fig. 6. ACM derived from OGJ patients increases IL-10 production by T cells, an effect which is attenuated by ICB. The effect of early-stage ACM (n = 6) and late-stage ACM (n = 6) on the frequency of CD4+ Treg cells was assessed by flow cytometry (A). The effect of early-stage ACM (n = 6) and late-stage ACM (n = 6) on the expression of TIGIT, CTLA-4, PD-1 and PD-L1 was assessed on the surface of CD4+ Treg cells by flow cytometry (B). The effect of ACM on the secretion of anti-inflammatory regulatory cytokine IL-10 was also assessed in CD3+CD4+ and CD3+CD8+ cells by flow cytometry (C). The effect of single agent nivolumab, single agent atezolizumab, single agent ipilimumab, dual nivolumab-atezolizumab or dual nivolumab-ipilimumab in the absence or presence of early-stage ACM (n = 6) and late-stage ACM (n = 6) on the secretion of anti-inflammatory regulatory cytokine IL-10 was also assessed in CD3+, CD3+CD4+ and CD3+CD8+ cells by flow cytometry (D). CD4+ Treg cells were characterised as the frequency of CD4+CD127LOWCD25HIGH that were FOXP3+. Representative dot plots displayed in (A) showing gating strategy for Treg cells. Paired, non-parametric t test and data presented as percentages ±…