Decreased peripheral blood mononuclear cells (PBMC) activation in patients with head and neck squamous cell carcinoma (HNSCC).

<p>PBMC were isolated from blood collected from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (n = 9), and cultured with or without Pam₃CSK₄ (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 24 h. (<b>A</b>) Thereafter, the supernatants were analyzed for the secreted cytokine profile with Luminex Multiplex Immunoassay, 4 out of 17 investigated cytokines are shown, (<b>B</b>) and the cells were examined for the expression of CD25 and CD98 on CD4 positive T helper (Th) cells with flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. One representative histogram from the healthy controls, AR patients and HNSCC patients is displayed. The open histograms represent LPS stimulated cells, and the light grey histograms are denoting the non-stimulated samples. (<b>C</b>) RNA was extracted from peripheral PBMC, isolated from healthy individuals (n = 8), AR patients (n = 10), and HNSCC patients (n = 7), made into cDNA, and thereafter analyzed for COX-1 and COX-2 mRNA expression with real-time PCR. Data are given in relation to the housekeeping gene β-actin, 100 000×2^−ΔCt, and presented as mean ± SEM. MFI = mean fluorescence intensity; *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001.</p

Clinical tumor (T) and lymph node (N) classification of the head and neck squamous cell carcinoma (HNSCC) patients.

<p>Clinical tumor (T) and lymph node (N) classification of the head and neck squamous cell carcinoma (HNSCC) patients.</p

Increased activation of polymorphonuclear leukocytes (PMN) in patients with head and neck squamous cell carcinoma (HNSCC).

<p>Blood was obtained from healthy controls (n = 10), patients with an ongoing seasonal allergic rhinitis (AR; n = 11) and patients with HNSCC (ELISA n = 9; FACS n = 10). PMN were isolated and cultured in the presence or absence of Pam₃CSK₄ (1 µg/ml), LPS (1 µg/ml), R837 (5 µg/ml) or CpG (0.3 µM) for 4 and 24 h. (<b>A</b>) The cell free supernatants were then analyzed for IL-6 and IL-8 with ELISA, (<b>B</b>) and the CD16 positive cells were investigated for the expression of CD11b and CD69 using flow cytometry. The results are presented as the non-stimulated value minus the TLR stimulated values. Grey colored samples were analyzed on a BD LSRFortessa, whereas the rest of the samples were investigated on a Beckman Coulter Navios flow cytometer. (<b>C</b>) PMN from healthy individuals (n = 9) were incubated with or without culture medium supernatant from the HNSCC cell line FaDu, and stimulated with or without 1 µg/ml LPS for 4 h, and analyzed for IL-6 and IL-8 secretion with ELISA. The basal cytokine levels in the media were subtracted from the concentrations present in the PMN cultures. MFI = mean fluorescence intensity; *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001.</p

Regulatory B cells producing IL-10 are increased in human tumor draining lymph nodes

The contribution of different immune cell subsets, especially T cells, in anti-tumor immune response is well established. In contrast to T cells, the anti-tumor contribution of B cells has been scarcely investigated. B-cells are often overlooked, even though they are important players in a fully integrated immune response and constitute a substantial fraction of tumor draining lymph nodes (TDLNs) known also as Sentinel Nodes. In this project, samples including TDLNs, non-TDLNs (nTDLNs) and metastatic lymph nodes from 21 patients with oral squamous cell carcinoma were analyzed by flow cytometry. TDLNs were characterized by a significantly higher proportion of B cells compared with nTDLNs (P = .0127). TDLNs-associated B cells contained high percentages of naive B cells, in contrary to nTDLNs which contained significantly higher percentages of memory B cells. Patients having metastases in TDLNs showed a significantly higher presence of immunosuppressive B regulatory cells compared with metastasis-free patients (P = .0008). Elevated levels of regulatory B cells in TDLNs were associated with the advancement of the disease. B cells in TDLNs were characterized by significantly higher expression of an immunosuppressive cytokine-IL-10 compared with nTDLNs (P = .0077). Our data indicate that B cells in human TDLNs differ from B cells in nTDLNs and exhibit more naive and immunosuppressive phenotypes. We identified a high accumulation of regulatory B cells within TDLNs which may be a potential obstacle in achieving response to novel cancer immunotherapies (ICIs) in head and neck cancer