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. 2012 Jun;61(6):927-39.
doi: 10.1007/s00262-011-1154-8. Epub 2011 Nov 25.

Characterization of the evolution of immune phenotype during the development and progression of squamous cell carcinoma of the head and neck

Anna-Maria A De Costa  1 , Corinne A Schuyler, David D Walker, M Rita I Young
Affiliations

Characterization of the evolution of immune phenotype during the development and progression of squamous cell carcinoma of the head and neck

Anna-Maria A De Costa et al. Cancer Immunol Immunother. 2012 Jun.

Abstract

While studies have indicated that squamous cell carcinoma of the head and neck (HNSCC) is associated with immune suppression, these studies did not analyze the immune response at the dysplastic stage. The present study utilized a mouse model of 4-nitroquinoline 1-oxide-induced oral carcinogenesis to examine the alterations in immune phenotype at the premalignant and malignant stages of HNSCC. Cervical lymph nodes of HNSCC-bearing mice were found to contain a greater number of cells, including a greater number of conventional (Tconv) and regulatory (Treg) T cells, compared to cervical lymph nodes of control and premalignant lesion-bearing mice, though the Tconv cells appear to be less proliferative and the Treg cells appear to be less suppressive at the HNSCC stage. Premalignant lesion-bearing mouse lymph nodes consist of a greater percentage of Tconv cells expressing markers for activation, memory, and exhaustion compared to both control and HNSCC-bearing mice. Also, lymph nodes' cells from both premalignant lesion-bearing and HNSCC-bearing mice include increased levels of Th1, Tc1, and Th17 cells, with no differences in levels of Th2 cells, compared to control mice. The data show that while there is the expected increase in immunosuppressive Tregs in lymph nodes when HNSCC is present, there is also an unexpected increase in immune populations usually associated with a beneficial antitumor response, including Tconv cells and Th1 and Tc1 cells. In addition, the results demonstrate that the premalignant stage of HNSCC development is associated with a robust immune response involving an increase in inflammatory Th1, Tc1, and Th17 cells.

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Figures

Fig. 1
Fig. 1
Established HNSCC is associated with an increase in numbers of regulatory and conventional T cells in cervical lymph nodes of affected mice. Representative results (a) and graphical representation (b) of flow cytometric staining of cervical lymph node cells from control, premalignant lesion-bearing (PM), and HNSCC-bearing mice (HNSCC) with at least 3 mice per group. HNSCC-bearing mouse cervical lymph nodes exhibit an increased percentage of CD4+Foxp3+ cells compared to both other groups. *P < 0.05. Total numbers of populations (c) were determined by multiplying the percent positive by the number of cervical lymph node cells for each individual mouse. HNSCC-bearing mice have greatly increased numbers of both conventional and regulatory CD4+ and CD8+ lymphocytes compared to control and premalignant lesion-bearing mice. *P < 0.05; **P < 0.01; ***P < 0.001. The CD4+ conventional T cell to regulatory T-cell ratio of both premalignant lesion-bearing and HNSCC-bearing mice is significantly decreased compared to control (d). *P < 0.05. Representative results and graphical representation (e) of flow cytometric analysis of CFSE-stained CD4+CD25− conventional T cells from control, premalignant lesion-bearing mouse, and HNSCC-bearing mouse lymph nodes (at least 3 mice per group) after three days culture with no stimulation (unstim) or stimulation with anti-CD3/anti-CD28 beads (stim). Cell proliferation was measured by percentage of cells with diluted CFSE. Stimulated conventional T cells from HNSCC-bearing mice proliferated significantly less than stimulated control mouse conventional T cells. *P < 0.05. Cytometric bead array analysis (f) of CD4+CD25− conventional T-cell supernatant revealed a decrease in IL-2 release from stimulated HNSCC-bearing mouse conventional T cells compared to stimulated control mouse conventional T cells. *P < 0.05. Representative results and graphical representation (g) of flow cytometric analysis of CFSE-stained control CD4+CD25− conventional T cells after coincubation at a 1:1 ratio for three days with control mouse, premalignant lesion-bearing mouse, or HNSCC-bearing mouse (at least 3 mice per group) CD4+CD25+ regulatory T cells in the presence of anti-CD3/anti-CD28 beads. HNSCC-bearing mouse regulatory T cells suppressed control conventional T-cell proliferation significantly less than control mouse regulatory T cells. *P < 0.05
Fig. 2
Fig. 2
Premalignant lesion-bearing mouse and HNSCC-bearing mouse cervical lymph nodes exhibit an increase in activated conventional T cells compared to control mouse cervical lymph nodes. Representative results (a) and graphical representation (b) of flow cytometric analysis of cervical lymph node cells from control, premalignant lesion-bearing (PM), and HNSCC-bearing mice (HNSCC) with at least 3 mice per group. Premalignant lesion-bearing mice have an increased percentage of CD25+ helper T cells (CD4+Foxp3−) compared to control and HNSCC-bearing mice, while HNSCC-bearing mice have an increased percentage of CD25+ helper T cells and cytotoxic T cells (CD8+Foxp3−) compared to control. *P < 0.05; ***P < 0.001. Total numbers of populations (c) were determined by multiplying percent positive by number of cervical lymph node cells for each individual mouse. HNSCC-bearing mice have an increased total number of CD25+ and CD69+ helper T cells and cytotoxic T cells compared to control and premalignant lesion-bearing mice, while premalignant lesion-bearing mice have an increased number of CD25+ helper T cells compared to control. *P < 0.05; **P < 0.01; ***P < 0.001
Fig. 3
Fig. 3
Premalignant lesion-bearing mouse cervical lymph nodes consist of a greater percentage of CD44high memory conventional T cells compared to control and HNSCC-bearing mice. Representative results (a) and graphical representation (b) of flow cytometric analysis of cervical lymph node cells from control, premalignant lesion-bearing (PM), and HNSCC-bearing mice (HNSCC) with at least 3 mice per group. Premalignant lesion-bearing mice have an increased percentage of CD44high helper T cells (CD4+Foxp3−) compared to control and an increased percentage of CD44high cytotoxic T cells (CD8+Foxp3−) compared to control and HNSCC-bearing mice. *P < 0.05; **P < 0.01. Total numbers of populations (c) were determined by multiplying percent positive by number of cervical lymph node cells for each individual mouse. HNSCC-bearing mice have an increased total number of CD44high helper T cells compared to control and premalignant lesion-bearing mice and an increased number of CD44high cytotoxic T cells compared to control mice. **P < 0.01; ***P < 0.001
Fig. 4
Fig. 4
Premalignant lesion-bearing mouse cervical lymph nodes contain an increased percentage of KLRG1+ helper T cells, while HNSCC-bearing mouse cervical lymph nodes contain a greater total number of PD-1+ conventional T cells. Representative results (a) and graphical representation (b) of flow cytometric analysis of cervical lymph node cells from control, premalignant lesion-bearing (PM), and HNSCC-bearing mice (HNSCC) with at least 3 mice per group. Cervical lymphocytes from premalignant lesion-bearing mice consist of a greater percentage of KLRG1+ helper T cells (CD4+Foxp3−) compared to both control and HNSCC, while cervical lymphocytes from HNSCC-bearing mice consist of a lower percentage of KLRG1+ helper T cells than control. *P < 0.05; **P < 0.01. Total numbers of populations (c) were determined by multiplying percent positive by number of cervical lymph node cells for each individual mouse. HNSCC-bearing mice have an increased total number of PD-1+ helper T cells compared to control and premalignant lesion-bearing mice. Both HNSCC-bearing mice and premalignant lesion-bearing mice have an increased number of PD-1+ cytotoxic T cells (CD8+Foxp3−) compared to control. *P < 0.05; **P < 0.01
Fig. 5
Fig. 5
Th1/Tc1 cells and related cytokines are increased in cervical lymph nodes of premalignant lesion-bearing and HNSCC-bearing mice. Representative results (a) and graphical representation (b) of flow cytometric analysis of control, premalignant lesion-bearing mouse, and HNSCC-bearing mouse cervical lymph node cells after stimulation for 4 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin A, with at least 9 mice per group. Both premalignant lesion-bearing mice and HNSCC-bearing mice have a greater percentage of Th1 (CD4+IFN-γ+) and Tc1 (CD8+IFN-γ+) cells compared to controls. *P < 0.05; **P < 0.01. Total numbers of populations (c) were determined by multiplying percent positive by number of cervical lymph node cells for each individual mouse. HNSCC-bearing mouse cervical lymph nodes consist of a greater total number of Th1 and Tc1 cells compared to controls. Premalignant lesion-bearing mouse cervical lymph nodes consist of a greater number of Tc1 cells compared to controls. *P < 0.05. Cytometric bead array analysis (d) of control, premalignant lesion-bearing mouse, and HNSCC-bearing mouse cervical lymph node cells after stimulation for 4 h with PMA and ionomycin, with at least 9 mice per group. Cervical lymph node cells from both premalignant lesion-bearing mice and HNSCC-bearing mice release an increased amount of Th1/Tc1-related cytokines (IFN-γ and IL-2) and chemokines (RANTES, MIP-1α and β) compared to control. *P < 0.05; **P < 0.01. Cytometric bead array analysis (e) of control, premalignant lesion-bearing mouse, and HNSCC-bearing mouse tongue epithelium lysate, with normalization to 100 μg of protein (as determined by BCA protein assay), with at least 10 mice per group. While HNSCC mouse tongue epithelium lysate contained a decreased level of IL-2 compared to control, HNSCC mouse tongue epithelium lysate contained a significantly increased level of RANTES and MIP-1β and a trend toward an increased level of IFN-γ and MIP-1α compared to control tongue epithelium lysate. While premalignant tongue epithelium lysate had a trend toward a decreased level of IL-2 compared to control, premalignant mouse tongue epithelium lysate contained a significantly increased level of IFN-γ and RANTES and a trend toward an increased level of MIP-1α and β compared to control tongue epithelium lysate. *P < 0.05
Fig. 6
Fig. 6
Th17 cells are increased in cervical lymph nodes of premalignant lesion-bearing and HNSCC-bearing mice. Representative results (a) and graphical representation (b) of flow cytometric analysis of control, premalignant lesion-bearing mouse, and HNSCC-bearing mouse cervical lymph node cells after stimulation for 4 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin A, with at least 9 mice per group. Both premalignant lesion-bearing and HNSCC-bearing mice have a greater percentage of Th17 (CD4+IL-17A+) cells than controls, and premalignant lesion-bearing mice have a greater percentage of Th17 cells compared to HNSCC-bearing mice as well. *P < 0.05; ***P < 0.001. Total numbers of populations (c) were determined by multiplying percent positive by number of cervical lymph node cells for each individual mouse. Premalignant lesion-bearing mouse and HNSCC-bearing mouse cervical lymph nodes consist of a greater total number of Th17 cells compared to controls. *P < 0.05; **P < 0.01. Cytometric bead array analysis (d) of control, premalignant lesion-bearing mouse, and HNSCC-bearing mouse cervical lymph node cells after stimulation for 4 h with PMA and ionomycin, with at least 9 mice per group. Cervical lymph node cells from both premalignant lesion-bearing mice release an increased amount of IL-17A compared to control. **P < 0.01
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