Myeloid-derived suppressor cells impede T cell functionality and promote Th17 differentiation in oral squamous cell carcinoma.

Oral tumor microenvironment is characterized by chronic inflammation signified with infiltrating leukocytes and soluble mediators which cause immune suppression. However, how immunosuppressive cells like myeloid-derived suppressor cells (MDSCs) maintain the immunosuppressive tumor microenvironment and influence T cell function in oral squamous cell carcinoma (OSCC) patients remains poorly understood. In the present study, we found that percentages of MDSCs were higher in oral cancer patients compared to healthy individuals and correlated with cancer stage. Monocytic MDSCs (M-MDSCs) were prevalent in the periphery, while granulocytic/polymorphonuclear subset dominated the tumor compartment. M-MDSCs suppressed the lymphocyte proliferation and decreased the CD3-ζ (zeta) chain expression and interferon gamma production. The percentage of M-MDSCs in peripheral blood correlated inversely with CD3-ζ chain expression in T cells of these patients. Interleukin 6 (IL-6)-induced phosphorylated STAT3-regulated programmed cell death ligand 1, CCAAT/enhancer-binding proteins alpha and beta and Interleukin 10 expression in MDSCs. MDSCs inhibited TGF-ß-driven generation of induced regulatory T cells in vitro. M-MDSCs secreted interleukins IL-6, IL-1ß, IL-23 and PGE2 and facilitated T-helper 17 (Th17) cell differentiation which utilizes nitric oxide synthase and cyclooxygenase 2 enzyme activity. Interestingly, OSCC patients showed increased levels of Th17 cells in peripheral blood and tumor tissue. Thus, increased frequency of MDSCs, Th17 cells and decreased expression of CD3-ζ chain portray T cell tolerance and chronic inflammatory state facilitating tumor growth.

TCR crosslinking promotes Crk adaptor protein binding to tyrosine-phosphorylated CD3ζ chain.

T cell antigen receptor (TCR) binding of a peptide antigen presented by antigen-presenting cells (APCs) in the context of surface MHC molecules initiates signaling events that regulate T cell activation, proliferation and differentiation. A key event in the activation process is the phosphorylation of the conserved tyrosine residues within the CD3 chain immunoreceptor tyrosine-based activation motifs (ITAMs), which operate as docking sites for SH2 domain-containing effector proteins. Phosphorylation of the CD3ζ ITAMs renders the CD3 chain capable of binding the ζ-chain associated protein 70 kDa (ZAP70), a protein tyrosine kinase that is essential for T cell activation. We found that TCR/CD3 crosslinking in Jurkat T cells promotes the association of Crk adaptor proteins with the transiently phosphorylated CD3ζ chain. Pull down assays using bead-immobilized GST fusion proteins revealed that the Crk-SH2 domain mediates binding of phospho-CD3ζ. Phospho-CD3ζ binding is selective and is mediated by the three types of Crk, including CrkI, CrkII, and CrkL, but not by other SH2 domain-containing adaptor proteins, such as Grb2, GRAP and Nck. Crk interaction with phospho-CD3ζ is rapid and transient, peaking 1 min post TCR/CD3 crosslinking. The results suggest the involvement of Crk adaptor proteins in the early stages of T cell activation in which Crk might help recruiting effector proteins to the vicinity of the phospho-CD3ζ and contribute to the fine-tuning of the TCR/CD3-coupled signal transduction pathways.

Extracellular 2'5'-oligoadenylate synthetase 2 mediates T-cell receptor CD3-ζ chain down-regulation via caspase-3 activation in oral cancer.

Decreased expression of CD3-ζ chain, an adaptor protein associated with T-cell signalling, is well documented in patients with oral cancer, but the mechanistic justifications are fragmentary. Previous studies in patients with oral cancer have shown that decreased expression of CD3-ζ chain was associated with decreased responsiveness of T cells. Tumours are known to induce localized as well as systemic immune suppression. This study provides evidence that oral tumour-derived factors promote immune suppression by down-regulating CD3-ζ chain expression. 2'5'-Oligoadenylate synthetase 2 (OAS2) was identified by the proteomic approach and our results established a causative link between CD3-ζ chain down-regulation and OAS2 stimulation. The surrogate situation was established by over-expressing OAS2 in a HEK293 cell line and cell-free supernatant was collected. These supernatants when incubated with T cells resulted in down-regulation of CD3-ζ chain, which shows that the secreted OAS2 is capable of regulating CD3-ζ chain expression. Incubation of T cells with cell-free supernatants of oral tumours or recombinant human OAS2 (rh-OAS2) induced caspase-3 activation, which resulted in CD3-ζ chain down-regulation. Caspase-3 inhibition/down-regulation using pharmacological inhibitor or small interfering RNA restored down-regulated CD3-ζ chain expression in T cells induced by cell-free tumour supernatant or rh-OAS2. Collectively these results show that OAS2 leads to impairment in CD3-ζ chain expression, so offering an explanation that might be applicable to the CD3-ζ chain deficiency observed in cancer and diverse disease conditions.

Inflammatory Response, Immunosuppression and Arginase Activity after Cardiac Surgery Using Cardiopulmonary Bypass.

Background: Major surgeries suppress patients' cellular immunity for several days, but the mechanisms underlying this T-cell dysfunction are not well understood. A decreased L-Arginine (L-Arg) level may inhibit T-cell function. Arginase 1 (Arg 1) is induced after traumatic injury, leading to molecular changes in T cells, including decreased expression of cell surface T-cell receptors (TCRs) and a loss in CD3ζ chain expression. In this study, we examined the temporal patterns of CD3ζ expression and Arg 1 activity in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB). Methods: We determined the CD3ζ chain expression; the Arg 1 activity; and the leukocyte, neutrophil and lymphocyte levels of patients on the day before surgery and at 24, 48 and 72 h after surgery. Results: Fifty adult patients scheduled for elective cardiac surgery with CPB were eligible for enrolment. Arginase activity was significantly increased between the day before surgery and at 24, 48 and 72 h after surgery (p < 0.01), and CD3ζ expression was significantly decreased between the day before surgery and at 24, 48 and 72 h after surgery (p < 0.001). We observed significant leukocytosis, neutrophilia and lymphopenia after surgery. Conclusions: The decreased CD3ζ chain expression could be due to the increased Arg 1 activity secondary to the activation of neutrophils in cardiac surgery under CPB. These findings could explain the limited immune-system-mediated organ damage resulting from systemic inflammatory response to major cardiac surgery with CPB.

Inhibition of Notch signalling has ability to alter the proximal and distal TCR signalling events in human CD3+ αß T-cells.

The Notch signalling pathway is an important regulator of T cell function and is known to regulate the effector functions of T cells driven by T cell receptor (TCR). However, the mechanism integrating these pathways in human CD3+ αß T cells is not well understood. The present study was carried out to investigate how Notch and TCR driven signalling are synchronized in human αß T cells. Differential expression of Notch receptors, ligands, and target genes is observed on human αß T cells which are upregulated on stimulation with α-CD3/CD28 mAb. Inhibition of Notch signalling by GSI-X inhibited the activation of T cells and affected proximal T cell signalling by regulating CD3-ζ chain expression. Inhibition of Notch signalling decreased the protein expression of CD3-ζ chain and induced expression of E3 ubiquitin ligase (GRAIL) in human αß T cells. Apart from affecting proximal TCR signalling, Notch signalling also regulated the distal TCR signalling events. In the absence of Notch signalling, α-CD3/CD28 mAb induced activation and IFN-γ production by αß T cells was down-modulated. The absence of Notch signalling in human αß T cells inhibited proliferative responses despite strong signalling through TCR and IL-2 receptor. This study shows how Notch signalling cooperates with TCR signalling by regulating CD3-ζ chain expression to support proliferation and activation of human αß T cells.

Crk adaptor proteins regulate CD3ζ chain phosphorylation and TCR/CD3 down-modulation in activated T cells.

T cell receptor (TCR) recognition of a peptide antigen in the context of MHC molecules initiates positive and negative cascades that regulate T cell activation, proliferation and differentiation, and culminate in the acquisition of effector T cell functions. These processes are a prerequisite for the induction of specific T cell-mediated adaptive immune responses. A key event in the activation of TCR-coupled signaling pathways is the phosphorylation of tyrosine residues within the cytoplasmic tails of the CD3 subunits, predominantly CD3ζ. These transiently formed phosphotyrosyl epitopes serve as docking sites for SH2-domain containing effector molecules, predominantly the ZAP70 protein tyrosine kinase, which is critical for signal propagation. We found that CrkI and CrkII adaptor proteins also interact with CD3ζ in TCR activated-, but not in resting-, T cells. Crk binding to CD3ζ was independent of ZAP70 and also occurred in ZAP70-deficient T cells. Binding was mediated by Crk-SH2 domain interaction with phosphotyrosine-containing motifs on CD3ζ, via a direct physical interaction, as demonstrated by Far-Western blot. CrkII binding to CD3ζ could also be demonstrated in a heterologous system, where coexpression of a catalytically active Lck was used to phosphorylate the CD3ζ chain. TCR activation-induced Crk binding to CD3ζ resulted in increased and prolonged phosphorylation of CD3ζ, as well as ZAP70 and LAT, suggesting a positive role for CrkI/II binding to CD3ζ in regulation of TCR-coupled signaling pathways. Furthermore, Crk-dependent increased phosphorylation of CD3ζ coincided with inhibition of TCR downmodulation, supporting a positive role for Crk adaptor proteins in TCR-mediated signal amplification.