Increased Abundances of CD16+ Non-Classical Monocytes Accompany with Elevated Monocytic PD-L1 and CD4+ T Cell Disturbances in Oropharyngeal Cancer.

BACKGROUND: Patients with human papilloma virus (HPV)-related oropharyngeal cancer have a better prognosis than nonvirally associated patients, most likely because of better immune responses. Increased infiltration of T lymphocytes into the oropharyngeal tumor tissue has been observed, but the dynamics of circulating lymphocytes and monocytes are not fully understood. The aim of this study was to understand the population dynamics of circulating monocyte subsets in oropharyngeal cancer (OPC) patients with regard to the clinicopathological parameters and accompanying immunological consequences in view of the CD4/CD8 T cell subset composition, and the expression of checkpoint pathway proteins programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1). MATERIALS AND METHODS: The abundance of circulating monocyte subsets and peripheral blood CD4/CD8 T cells of oropharyngeal cancer patients and their PD-L1 and PD-1 expression levels were analyzed by flow cytometry. RESULTS: The studied oropharyngeal cancer patients revealed heterogeneous individual redistributions of CD14++CD16- (classical), CD14++CD16+ (intermediate), and CD14dim+CD16+ (nonclassical) monocyte subsets compared with healthy donors. These differences in monocyte subset alterations were independent in patients with TNM or HPV status but entailed further immunological consequences. Increased percentages of nonclassical monocytes significantly correlated with increased levels of monocytic PD-L1 expression. We observed significantly decreased levels of CD4+ effector T cells, which were accompanied by increased CD4+ effector memory T cells in OPC patients compared with healthy donors, each having a stronger effect in patients with decreased levels of classical monocytes. CONCLUSION: We conclude that oropharyngeal cancer, as a malignancy from a lymphoid-tissue-rich anatomical region, has a strong systemic impact on the differentiation and regulation of circulating innate and adaptive immune cells. Further comprehensive investigations are required for the possible future usability of the described immunological alterations as bioliquid parameters for prognosis or therapy response prediction.

Impact of Superparamagnetic Iron Oxide Nanoparticles on THP-1 Monocytes and Monocyte-Derived Macrophages.

Superparamagnetic iron oxide nanoparticles (SPIONs) are currently under examination for magnetic particle imaging, which represents a radiation free technology for three-dimensional imaging with high sensitivity, resolution and imaging speed. SPIONs are rapidly taken up by monocytes and other phagocytes which carry them to the site of inflammation. Therefore, the SPION biocompatibility is an essential parameter for a widespread MPI usage. Many improvements are expected from SPION development and its applications for cell visualization, but the impact of MPI optimized dextran coated SPIONs on the cellular characteristics of monocytic cells has been poorly studied up to now. THP-1 monocytes, monocyte-derived macrophages (MDM) as well as peripheral blood monocytes were incubated with MPI-optimized dextran-coated SPIONs of a size between 83.5 and 86 nm. SPION uptake was measured by FITC fluorescence of labeled SPIONs and Prussian blue staining. The activation of monocytes and MDMs was evaluated by CD14, CD11b and CD86 in flow cytometry. The secretion of IL-1ß, and IL-10 was analyzed in supernatants. SPIONs were rapidly taken up by monocytes and monocyte-derived macrophages while no decrease in cell viability was observed. Expression patterns of CD11b, CD14, and CD86 were not affected in THP-1 monocytes and MDMs. Monocyte differentiation in macrophages was hindered during SPION uptake. THP-1 monocytes as well as monocyte-derived macrophages showed significantly increased IL-1ß and decreased IL-10 secretion by tendency after SPION treatment. Dextran-coated SPIONs showed a low cytotoxicity on monocytes but exert undesirable inflammatory side effects that have to be considered for imaging applications.

Reconstitution of Monocyte Subsets and PD-L1 Expression but Not T Cell PD-1 Expression in Obstructive Sleep Apnea Patients upon PAP Therapy.

Obstructive sleep apnea (OSA) is characterized by nocturnal breathing intermissions resulting in oxidative stress and eventually, a low-grade systemic inflammation. The study aimed to investigate the impact of positive airway pressure (PAP) therapy on the inflammatory milieu as measured by monocyte and T cell phenotypic alterations. Participants were assessed for their OSA severity before PAP therapy and about six months later, including patient-reported outcome and therapy usage by telemetry readout. The distributions of the CD14/CD16-characterized monocyte subsets as well as the CD4/CD8-characterized effector T cell subsets with regard to their PD-1 and PD-L1 expression were analyzed by flow cytometry from blood samples. Data of 25 patients revealed a significant reconstitution of the monocyte subset distribution and a decrease in PD-L1 expression on pan-monocytes and CD8+ T cells without an association to initial AHI and overweight. The PD-1 expression was still increased on T cell subsets, especially on CD4+ TH17/22 cells. We conclude that PAP therapy might have a rapid effect on the monocyte phenotype and overall PD-L1 expression levels. However, T cell immune alterations especially on TH17/22 cells persist longer, indicating an ongoing disturbance of the adaptive immune system.

Spatial Distribution of Immune Cells in Head and Neck Squamous Cell Carcinomas.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) have a very moderate response rate to immune checkpoint inhibitor (ICI) treatment compared to other cancer types. Lacking predictive markers for treatment response, we analyzed the immune status of HNSCC and assessed the spatial distribution of immune cells. MATERIALS AND METHODS: Via assessing hematoxylin-eosin (H&E) stains, we divided HNSCCs by the immune cell distribution in hot, cold, and excluded tumors. For each group, each with 10 tumors, we performed serial immunohistochemical (IHC) staining of the immune cell markers, checkpoint molecules, and immune regulators. RESULTS: The spatial distributions were different for each immune cell type, allocating regulatory T cells (Tregs) and CD11b cells predominantly in the stroma. CD4 and CD8 cells were present either in the tumor stroma or between cancer cells. Interestingly, the expressions of PD-1 (programmed cell death 1 receptor) and PD-L1 (programmed death-ligand 1) were higher in hot tumors in comparison to cold and excluded tumors. The expression of pSMAD [indicating active transforming growth factor beta (TGF-ß)] was higher in excluded tumors. CONCLUSION: Different immune cell distribution patterns within tumors might be crucial for ICI treatment response since hot tumors have the highest expressions of PD-1 and PD-L1. TGF-ß might be a key regulator for immune cell distribution and a promising therapeutic target that determines the formation of hot or excluded immune patterns.

Measurement of leukocyte-platelet aggregates (LPA) by FACS: a comparative analysis.

In addition to their role in hemostasis and coagulation platelets bear critical roles in modulation of the innate and adaptive immune system. Upon platelet activation in response to tissue injury, bacterial or viral infections, they secrete many soluble factors or directly interact with leukocytes. An increase of leukocyte-platelet aggregates (LPA) has been described for many pathological conditions. Nevertheless, a standardized method for the reliable measurement of PLAs is not securely established. This methodical study provides a comparison of four different protocols from the literature and summarizes major pitfalls of measuring and interpreting leukocyte-platelet aggregates. The different techniques vary in the workup of the blood samples, applying variable washing and centrifugation steps or the use of erythrocyte lysis. All samples were finally analyzed by flow cytometry. Leukocyte subsets were stained with specific antibodies and platelet aggregates were identified by additional expression of CD41. The different procedures generated very heterogeneous data from the same blood sample which highlight the abundance of error measuring LPA. The most reproducible technique turned out to be a two-color whole blood flow cytometry assay with erythrocyte lysis and without washing or centrifugation steps avoiding platelet activation and artificial aggregate formation to achieve data mirroring the true situation in peripheral blood.

Redistribution of Monocyte Subsets in Obstructive Sleep Apnea Syndrome Patients Leads to an Imbalanced PD-1/PD-L1 Cross-Talk with CD4/CD8 T Cells.

Obstructive sleep apnea syndrome (OSAS) represents a substantial disease of recurrent sleep fragmentation, leading to intermittent hypoxia and subsequent diseases such as cardiovascular, metabolic, or cognitive dysfunctions. In addition, OSAS is considered as low-grade systemic inflammation, which is associated with a higher incidence of cancer, severity of infections, and an overall immune dysregulation. This research project aims to comprehensively investigate the interplay of wholesome sleep and the immune functions of circulating monocytes and T cells in OSAS patients, which are known to be affected by oxidative stress. We studied the distribution of the CD14/CD16 characterized monocyte subsets in peripheral blood as well as their PD-L1 expression and complex formation with T cells. Furthermore, a detailed analysis of T cell subsets with regard to their PD-1 and PD-L1 expression was performed. Data revealed a decrease of classical monocytes accompanied by an increase of both CD16+ monocyte subsets in OSAS patients that was positively correlated with the body mass index. OSAS patients revealed an increased PD-1 and PD-L1 expression in T cells and monocytes, respectively, which was linked to the severity of monocyte subset alterations. The complex formation of monocytes and T cells was also elevated in OSAS patients, which indicates a deregulated PD-1/PD-L1 cross-talk between these cells. Our data show for the first time, to our knowledge, massive alterations of peripheral monocyte subsets in response to OSAS and its accompanying phenomena.

Platelet Induced Functional Alteration of CD4+ and CD8+ T Cells in HNSCC.

Platelets (PLT) are the second most abundant cell type in human blood and exert various immune-regulatory functions under both physiological and pathological conditions. In fact, immune cell regulation via platelets has been demonstrated in several studies within the past decade. However, the exact mechanisms behind T cell regulation remain poorly understood. We questioned whether the formation of aggregates of platelets and T cells has an impact on T-cell functions. In the present study, we stimulated PBMC cultures with anti-CD3 and anti-CD28 mABs and cultured them at a PLT: PBMC ratio of 1:1 or 100:1. After 24, 48, and 72 h, PD-1, PD-L1 expression, and proliferation were analyzed on T cells using flow cytometry. Cytokine production was measured in PHA stimulated CD4 cells after 6 h. We found a significant platelet-mediated decrease in PD-1 and PD-L1 expression, proliferation, as well as IFN-γ and TNF-α production. Perturbations also at least partially remained after spatial separation of PLTs from PBMCs in Transwell-assays. T cell-platelet aggregates showed similar levels of activation markers, proliferation, and secreted cytokines as their non-complexed counterparts. Results indicate a platelet mediated regulation of T cells via direct and indirect contact, but only mediocre effects of the complex formation itself.

Non-specific activation of CD8α-characterised γδ T cells in PBL cultures of different chicken lines.

Avian γδ T lymphocytes still represent an enigmatic cell population not only regarding their functions but also their activation requirements and subset differentiation. To find a suitable method for non-specific stimulation and multiplication of CD8α-characterised γδ T-cell subsets in peripheral blood lymphocyte (PBL) cultures, PBLs of four different chicken lines (WLA, BLA, R11, L68) were treated with a range of commonly used non-specific reagents (PMA, PHA, ConA) and interleukins (IL-2, IL-12, IL-15), and the CD8α(-), CD8α(hi)ß(+) and CD8αα(hi+) γδ T-cell subsets examined in relation to activation (CD25 expression) and proliferation by flow cytometry. The culture of avian PBLs with PMA led to an increase of CD25-expression intensity, whereas IL-2 induced proliferation of γδ T-cells. The combinational application of IL-2 plus PMA functioned synergistically and resulted in significantly enhanced numbers of CD25(+) cells, with simultaneous significant increase of the CD25-expression intensity and proliferation in all γδ T-cell subsets of all chicken lines. The four chicken lines revealed only sporadically differences with frequently highest proliferation rates in PBLs of WLA. In conclusion, PMA in combination with IL-2 is the best suitable additive for avian PBL cultures in order to multiply, activate and maintain CD8α-characterised γδ T-cell subsets of different chicken lines.