Table 1.
Patient characteristics.
Fig 1.
Perineural tumour contains both CD8 and non-CD8 T cells.
Freshly excised tumour tissue and blood from each patient was processed into a single cell suspension, stained with antibodies and analysed via flow cytometry. (A) An example of the gating strategy used to distinguish CD8 and non-CD8 T cells within tumour and a blood sample from the same patient. (B) The percentage of live, CD45+CD3+ T cells within the tumour or blood cells from the cohort of patients is expressed as the mean and SEM. (C) Amongst CD45+CD3+ T cells in the blood and tumour tissue, the ratio of CD8+ to non-CD8 cells was calculated for individual patients. * indicates p<0.05
Fig 2.
The fraction of T cells, NKT cells and NK cells is significantly different between blood and tumour from patients with perineural SCC.
Freshly excised tumour and matched blood samples were stained with antibodies against various immune cell markers and analysed by flow cytometry. (A) An example of the gating strategy used to analyse different immune cell subsets within blood and tumour. (B) The percentage of CD3+ (T cells), TCRVa24+ (NKT cells), CD3-CD56+ (NK cells), CD19+ (B cells) and CD3+CD56+ (activated T cells/NKT cells) cells amongst live CD45+ cells was plotted for both tumour and blood samples for individual patients. Lines connect the matched blood and tumour sample from each individual patient. * indicates p<0.05
Fig 3.
The proportion of CD8 T cells expressing PD-1, Tim-3 and CTLA-4 is elevated in tumour relative to the blood.
Upper plots are an example of antibody staining for each indicated receptor after gating on live, CD45+, CD3+, CD8+ cells. Shaded plots represent negative control staining. The lower graphs summarise the percentage of PD-1, Tim-3 and CTLA-4+ cells (after subtraction of negative control percentage staining) for individual patients. The lines connect the matched blood and tumour sample from each individual patient. * indicates p<0.05.
Fig 4.
The proportion of non-CD8 T cells expressing Tim-3 and CTLA-4, but not PD-1, is elevated in tumour relative to the blood.
Upper plots are an example of antibody staining for each indicated receptor after gating on live, CD45+, CD3+, CD8+ cells. Shaded plots represent negative control staining. The lower graphs summarise the percentage of PD-1, Tim-3 and CTLA-4+ cells (after subtraction of negative control percentage staining) for individual patients. The lines connect the matched blood and tumour sample from each individual patient. * indicates p<0.05.
Fig 5.
Limited co-expression of PD-1, Tim-3 and CTLA-4 in the blood and tumour of a patient with expression of all three receptors.
Gated CD8 T cell populations (live, CD45+, CD3+, CD8+ cells) from the blood (upper plots) and matching tumour sample (lower plots) were analysed for co-expression of PD-1, Tim-3 and CTLA-4 using flow cytometry.
Fig 6.
PD-1 and PD-L1 can share a close proximity within the perineural tumour tissue.
Tonsil tissue from a healthy patient (upper panel) and perineural tissue samples from two perineural SCC patients (middle and lower panels) were stained with either PD-L1 (left images) or PD-1 (right images) antibodies using immunohistochemistry. Brown colour represents positive staining. Patient 1 was considered to have limited PD-1 staining cells with isolated areas of PD-L1 expression. Patient 2 had high PD-1 staining in isolated patches and light/moderate staining for PD-L1. Boxed areas have been enlarged in the lower figures. Arrows in the bottom panels indicate tumour cells. Scale bars– 100um (tonsil), 50um (Patient 1 and Patient 2), 20um (Patient 2 enlarged).