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.

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.

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.

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 CD14^dim+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.