Comparative immune phenotypic analysis of cutaneous Squamous Cell Carcinoma and Intraepidermal Carcinoma in immune-competent individuals: proportional representation of CD8+ T-cells but not FoxP3+ Regulatory T-cells is associated with disease stage

Andrew Freeman, Jennifer A Bridge, Pirashanthini Maruthayanar, Nana H Overgaard, Ji-Won Jung, Fiona Simpson, Tarl W Prow, H Peter Soyer, Ian H Frazer, Michael Freeman, James W Wells, Andrew Freeman, Jennifer A Bridge, Pirashanthini Maruthayanar, Nana H Overgaard, Ji-Won Jung, Fiona Simpson, Tarl W Prow, H Peter Soyer, Ian H Frazer, Michael Freeman, James W Wells

Abstract

Squamous Cell Carcinoma (SCC) is a type of non-melanoma skin cancer prevalent in immune-suppressed transplant recipients and older individuals with a history of chronic sun-exposure. SCC itself is believed to be a late-stage manifestation that can develop from premalignant lesions including Intraepidermal Carcinoma (IEC). Notably, while SCC regression is rare, IEC typically regresses in response to immune modifying topical treatments, however the underlying immunological reasons for these differential responses remain unclear. This study aimed to define whether IEC and SCC are associated with distinct immune profiles. We investigated the immune cell infiltrate of photo-damaged skin, IEC, and SCC tissue using 10-colour flow cytometry following fresh lesion digest. We found that IEC lesions contain higher percentages of CD3+ T-cells than photo-damaged skin, however, the abundance of CD3-CD56+ Natural Killer (NK) cells, CD11c+HLA-DR+ conventional Dendritic Cells (cDC), BDCA-2+HLA-DR+ plasmacytoid DC (pDC), FoxP3+ Regulatory T-cells (T-reg), Vα24+Vβ11+ invariant NKT-cells, and γδ Tcells did not alter with disease stage. Within the total T-cell population, high percentages of CD4+ T-cells were associated with SCC, yet CD8+ T-cells were less abundant in SCC compared with IEC. Our study demonstrates that while IEC lesions contain a higher proportion of T-cells than SCC lesions in general, SCC lesions specifically display a lower abundance of CD8+ T-cells than IEC. We propose that differences in CD8+ T-cell abundance contribute critically to the different capacity of SCC and IEC to regress in response to immune modifying topical treatments. Our study also suggests that a high ratio of CD4+ T-cells to CD8+ T-cells may be a immunological diagnostic indicator of late-stage SCC development in immune-competent patients.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Representative photomicrographs of each of…
Figure 1. Representative photomicrographs of each of the three analysis groups.
(A) Moderate to severe photo-damage in the upper dermis, (B) IEC, and (C) moderately differentiated SCC. Images presented are H&E stains, scale bar = 100 µm.
Figure 2. Gating strategy for analysis of…
Figure 2. Gating strategy for analysis of immune cell populations in tumour lesions by flow cytometry.
Age-matched sun-exposed skin or tumour lesions were excised and digested as outlined in Materials & Methods. Samples were stained with antibody and analysed by flow cytometry using the gating strategy shown in (A). (B) Ratio of Keratin 10+ Keratin 14− cells (suprabasal keratinocytes) to Keratin 10+ Keratin 14+ cells (basal keratinocytes) in the live CD45− population as determined by intracellular staining. (C) Comparison of viable immune cell extraction showing similar extraction efficacy between sample types. Live CD45+ cells as a percentage of the total live cell population is shown (skin; n = 6, IEC; n = 9, SCC; n = 13). ns: not significant. SSC-A: Side Scatter-Area, FSC-A: Forward Scatter-Area, TOF: Time of flight.
Figure 3. T-cells, NK cells, and DC…
Figure 3. T-cells, NK cells, and DC detected in photo-damaged skin and lesional skin of patients with IEC and SCC.
Fresh biopsies were digested to release cells and subsequently stained with antibodies to determine the representation of immune subpopulations by flow cytometry. (A) CD3+ T-cells (skin; n = 6, IEC; n = 9, SCC; n = 13), (B) CD3−CD56+ NK cells (skin; n = 6, IEC; n = 8, SCC; n = 13), (C) CD11c+HLA-DR+ cDC (includes both CD11bhi and CD11b−/lo populations, (skin; n = 6, IEC; n = 7, SCC; n = 7), (D) BDCA-2+HLA-DR+ pDC (skin; n = 6, IEC; n = 7, SCC; n = 7), (E) remaining CD11b+ myeloid cell populations (skin; n = 6, IEC; n = 7, SCC; n = 7), and (F) remaining immune cells (skin; n = 6, IEC; n = 6, SCC; n = 7). All data displayed are % of total CD45+ immune cell population. ns: not significant.
Figure 4. IEC and SCC lesions display…
Figure 4. IEC and SCC lesions display distinct patterns of CD4+ and CD8+ T-cell infiltration.
Fresh biopsies were digested to release cells and subsequently stained with antibodies to determine the representation of T-cell subpopulations by flow cytometry; (A) CD4+ T-cells (skin; n = 6, IEC; n = 9, SCC; n = 10), (B) FoxP3+ T-regs (skin; n = 6, IEC; n = 7, SCC; n = 8), (C) CD8+ T-cells (skin; n = 6, IEC; n = 8, SCC; n = 13), (D) TCRγδ+ T-cells (skin; n = 6, IEC; n = 9, SCC; n = 11), and (E) Vα24+Vβ11+ invariant Natural Killer T-cells (iNKT; (skin; n = 6, IEC; n = 9, SCC; n = 12). All data displayed are % of total CD3+ T-cells with the exception of (B) which represents the % of total CD3+CD4+ T-cells. ns: not significant.
Figure 5. SCC lesions display both an…
Figure 5. SCC lesions display both an increased CD4/CD8 ratio and an increased T-reg/CD8 ratio when compared with IEC lesions.
Absolute CD3+CD4+ T-cell, CD3+CD4+FoxP3+ T-cell, and CD3+CD8+ T-cell numbers within each lesion were determined by flow cytometry and used to determine the proportion of (A) CD3+CD4+ T-cells to CD3+CD8+ T-cells (skin; n = 6, IEC; n = 9, SCC; n = 11), and (B) CD3+CD4+FoxP3+ T-cells to CD3+CD8+ T-cells (skin; n = 6, IEC; n = 7, SCC; n = 9). ns: not significant.

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