A sustained increase of cytosolic Ca in γδ T cells triggered by co-stimulation via TCR/CD3 and LFA-1

We previously reported that co-stimulation with LFA-1 triggered apoptosis in γδ T cells but not in αβ T cells after TCR engagement. We extended our earlier study on TCR/LFA-1 triggered apoptosis to two autoreactive TCR γδ and TCR αβ T cell clones, which were derived from syngeneic mixed lymphocyte culture of BALB/c mice. A γδ T cell clone, KM1, expressed the Vγ4 and Vδ5 genes and CD4-CD8-CD45RB⁺ phenotype; and an αβ T cell clone, BASL1.1, expressed Vβ6 and CD4⁺CD8-CD45RB⁺. Both clones produced Th-1-type cytokines in response to syngeneic BALB/c stimulator cells. KM1 underwent apoptosis upon stimulation with immobilized anti-CD3/LFA-1 mAbs, whereas BASL1.1 could proliferate successfully in response to stimulation with the immobilized mAbs. BASL1.1 was able to down-regulate the increased cytosolic Ca²⁺ after the simultaneous stimulation, but KM1 exhibited a sustained increase of cytosolic Ca²⁺ after stimulation via CD3 and LFA-1. Similar results with respect to the kinetics of cytosolic Ca²⁺ were obtained with normal heterogeneous γδ and αβ T cell populations after co-stimulation via CD3 and LFA-1. Our results suggested that persistently high levels of cytosolic Ca²⁺ might be related to apoptosis in γδ T cell clone triggered by costimulation via CD3 and LFA-1.     

LFA-1–mediated Adhesion Is Regulated by Cytoskeletal Restraint and by a Ca2+-dependent Protease, Calpain

The activity of integrins on leukocytes is kept under tight control to avoid inappropriate adhesion while these cells are circulating in blood or migrating through tissues. Using lymphocyte function-associated antigen-1 (LFA-1) on T cells as a model, we have investigated adhesion to ligand intercellular adhesion molecule-1 induced by the Ca²⁺ mobilizers, ionomycin, 2,5-di-t-butylhydroquinone, and thapsigargin, and the well studied stimulators such as phorbol ester and cross-linking of the antigen-specific T cell receptor (TCR)– CD3 complex. We report here that after exposure of T cells to these agonists, integrin is released from cytoskeletal control by the Ca²⁺-induced activation of a calpain-like enzyme, and adhesive contact between cells is strengthened by means of the clustering of mobilized LFA-1 on the membrane. We propose that methods of leukocyte stimulation that cause Ca²⁺ fluxes induce LFA-1 adhesion by regulation of calpain activity. These findings suggest a mechanism whereby engagement of the TCR could promote adhesion strengthening at an early stage of interaction with an antigen-presenting cell.     

Impact of Exogenous Galectin-9 on Human T Cells: CONTRIBUTION OF THE T CELL RECEPTOR COMPLEX TO ANTIGEN-INDEPENDENT ACTIVATION BUT NOT TO APOPTOSIS INDUCTION*

Galectin-9 (gal-9) is a multifunctional β-galactoside-binding lectin, frequently released in the extracellular medium, where it acts as a pleiotropic immune modulator. Despite its overall immunosuppressive effects, a recent study has reported bimodal action of gal-9 on human resting blood T cells with apoptosis occurring in the majority of them, followed by a wave of activation and expansion of Th1 cells in the surviving population. Our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. One of these events is cytosolic calcium (Ca²⁺) release reported in some murine and human T cells. The aim of this study was to investigate the contribution of Ca²⁺ mobilization to apoptotic and nonapoptotic effects of exogenous gal-9 in human T cells. We found that the T cell receptor (TCR)-CD3 complex and the Lck kinase were required for Ca²⁺ mobilization but not for apoptosis induction in Jurkat cells. These data were confirmed in human CD4⁺ T cells from peripheral blood as follows: a specific Lck chemical inhibitor abrogated Ca²⁺ mobilization but not apoptosis induction. Moreover, Lck activity was also required for the production of Th1-type cytokines, i.e. interleukin-2 and interferon-γ, which resulted from gal-9 stimulation in peripheral CD4⁺ T cells. These findings indicate that gal-9 acts on T cells by two distinct pathways as follows: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis that is independent of this complex.     

Laminin 5 Promotes Activation and Apoptosis of the T Cells Expressing α3β1 Integrin

By introducing an α3 gene-containing plasmid into a human T cell line Jurkat, we prepared the T cells, which express a high level of the α3β1 integrin, to assess the role of laminin 5 in the skin immune system. The α3β1-expressing T cells adhered to laminin 5 and exhibited spreading. These adhered T cells showed a significant tyrosine phosphorylation of intracellular proteins including p59^fynupon T-cell receptor (TCR) stimulation. Six hours after cross-linking TCR, these cells on laminin 5 secreted a three times higher level of IL-2 than those on a BSA-coated plate. Twenty hours after the stimulation, 48% of the α3β1-expressing T cells on laminin 5 caused apoptosis. The protein level of cyclin D3 and E decreased, while that of p53 increased in these T cells. These data suggest that laminin 5 may play at least two regulatory roles for T cell functions: augmentation of IL-2 production by antigen-stimulated T cells and induction of apoptosis in these T cells.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

Organization of the V Gene Segments in Mouse T-Cell Antigen Receptor α/δ Locus

The mouse T-cell receptor (TCR) α/δ locus was mapped using 17 Vα and 4 Vδ subfamily-specific probes. Four complementary methods were used: (1) an estimate of the V gene repertoire by Southern blot analysis of genomic DNA with subfamily-specific probes; (2) an analysis of V gene segments deleted by TCR gene rearrangements from a panel of T-cell tumors and hybridomas; (3) an analysis of overlapping clusters of cosmid clones; and (4) an analysis of large DNA fragments separated by field-inversion gel electrophoresis. The α/δ locus spans about 1 Mb. The distance between the 3′-most V gene segment (Vδ1) and the δ constant gene (Cδ) is no more than 150 kb. Sixty-six V gene segments have been mapped physically on cosmids. The members of individual Vα gene segment subfamilies are dispersed throughout the locus. In contrast, the Vδ gene segments Vδ1 to 5 are clustered at the 3′ end of the V gene segment cluster. At least two DNA segment duplications, 45 to 80 kb in length, are present in the locus. These data provide information on the evolution of the α/δ locus and on organizational features that might influence the expression of specific V gene segments in γδ cells.     

The Co-Stimulatory Effects of MyD88-Dependent Toll-Like Receptor Signaling on Activation of Murine γδ T Cells

γδ T cells express several different toll-like receptor (TLR)s. The role of MyD88- dependent TLR signaling in TCR activation of murine γδ T cells is incompletely defined. Here, we report that Pam3CSK4 (PAM, TLR2 agonist) and CL097 (TLR7 agonist), but not lipopolysaccharide (TLR4 agonist), increased CD69 expression and Th1-type cytokine production upon anti-CD3 stimulation of γδ T cells from young adult mice (6-to 10-week-old). However, these agonists alone did not induce γδ T cell activation. Additionally, we noted that neither PAM nor CL097 synergized with anti-CD3 in inducing CD69 expression on γδ T cells of aged mice (21-to 22-month-old). Compared to young γδ T cells, PAM and CL097 increased Th-1 type cytokine production with a lower magnitude from anti-CD3- stimulated, aged γδ T cells. Vγ1⁺ and Vγ4⁺ cells are two subpopulations of splenic γδ T cells. PAM had similar effects in anti-CD3-activated control and Vγ4⁺ subset- depleted γδ T cells; whereas CL097 induced more IFN-γ production from Vγ4⁺ subset-depleted γδ T cells than from the control group. Finally, we studied the role of MyD88-dependent TLRs in γδ T cell activation during West Nile virus (WNV) infection. γδ T cell, in particular, Vγ1⁺ subset expansion was significantly reduced in both MyD88- and TLR7- deficient mice. Treatment with TLR7 agonist induced more Vγ1⁺ cell expansion in wild-type mice during WNV infection. In summary, these results suggest that MyD88-dependent TLRs provide co-stimulatory signals during TCR activation of γδ T cells and these have differential effects on distinct subsets.     

Lck Mediates Signal Transmission from CD59 to the TCR/CD3 Pathway in Jurkat T Cells

The glycosylphosphatidylinositol (GPI)-anchored molecule CD59 has been implicated in the modulation of T cell responses, but the underlying molecular mechanism of CD59 influencing T cell signaling remained unclear. Here we analyzed Jurkat T cells stimulated via anti-CD3ε- or anti-CD59-coated surfaces, using time-resolved single-cell Ca²⁺ imaging as a read-out for stimulation. This analysis revealed a heterogeneous Ca²⁺ response of the cell population in a stimulus-dependent manner. Further analysis of T cell receptor (TCR)/CD3 deficient or overexpressing cells showed that CD59-mediated signaling is strongly dependent on TCR/CD3 surface expression. In protein co-patterning and fluorescence recovery after photobleaching experiments no direct physical interaction was observed between CD59 and CD3 at the plasma membrane upon anti-CD59 stimulation. However, siRNA-mediated protein knock-downs of downstream signaling molecules revealed that the Src family kinase Lck and the adaptor molecule linker of activated T cells (LAT) are essential for both signaling pathways. Furthermore, flow cytometry measurements showed that knock-down of Lck accelerates CD3 re-expression at the cell surface after anti-CD59 stimulation similar to what has been observed upon direct TCR/CD3 stimulation. Finally, physically linking Lck to CD3ζ completely abolished CD59-triggered Ca²⁺ signaling, while signaling was still functional upon direct TCR/CD3 stimulation. Altogether, we demonstrate that Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells, and propose that CD59 may act via Lck to modulate T cell responses.     

Regulation and Function of the CD3γ DxxxLL Motif: A Binding Site for Adaptor Protein-1 and Adaptor Protein-2 in Vitro

Several receptors are downregulated by internalization after ligand binding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction. One way T cells regulate TCR expression is by phosphorylation/dephosphorylation of the TCR subunit clusters of differentiation (CD)3γ. Thus, phosphorylation of CD3γ serine 126 (S126) causes a downregulation of the TCR. In this study, we have analyzed the CD3γ internalization motif in three different systems in parallel: in the context of the complete multimeric TCR; in monomeric CD4/CD3γ chimeras; and in vitro by binding CD3γ peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3γ D¹²⁷xxxLL^131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4/ CD3γ molecules independently of S126. An acidic amino acid is required at position 127 and a leucine (L) is required at position 131, whereas the requirements for position 132 are more relaxed. The spacing between aspartic acid 127 (D127) and L131 is crucial for the function of the motif in vivo and for AP binding in vitro. Furthermore, we provide evidence indicating that phosphorylation of CD3γ S126 in the context of the complete TCR induces a conformational change that exposes the DxxxLL sequence for AP binding. Exposure of the DxxxLL motif causes an increase in the TCR internalization rate and we demonstrate that this leads to an impairment of TCR signaling. On the basis of the present results, we propose the existence of at least three different types of L-based receptor sorting motifs.     

High Affinity of Interaction between Superantigen and T Cell Receptor Vβ Molecules Induces a High Level and Prolonged Expansion of Superantigen-reactive CD4+ T Cells

In mice implanted with an osmotic pump filled with the superantigen (SAG) staphylococcal enterotoxin A (SEA), the Vβ3⁺CD4⁺ T cells exhibited a high level of expansion whereas the Vβ11⁺CD4⁺ T cells exhibited a mild level of expansion. In contrast, in mice implanted with an osmotic pump filled with SE-like type P (SElP, 78.1% homologous with SEA), the Vβ11⁺CD4⁺ T cells exhibited a high level of expansion while the Vβ3⁺CD4⁺ T cells exhibited a low level of expansion, suggesting that the level of the SAG-induced response is determined by the affinities between the TCR Vβ molecules and SAG. Analyses using several hybrids of SEA and SElP showed that residue 206 of SEA determines the response levels of Vβ3⁺CD4⁺ and Vβ11⁺CD4⁺ T cells both in vitro and in vivo. Analyses using the above-mentioned hybrids showed that the binding affinities between SEA and the Vβ3/Vβ11 β chains and between SEA-MHC class II-molecule complex and Vβ3⁺/Vβ11⁺ CD4⁺ T cells determines the response levels of the SAG-reactive T cells both in vitro and in vivo.     

Renal Cell Carcinoma-Infiltrating CD3low Vγ9Vδ1 T Cells Represent Potentially Novel Anti-Tumor Immune Players

Due to the highly immunogenic nature of renal cell carcinoma (RCC), the tumor microenvironment (TME) is enriched with various innate and adaptive immune subsets. In particular, gamma-delta (γδ) T cells can act as potent attractive mediators of adoptive cell transfer immunotherapy because of their unique properties such as non-reliance on major histocompatibility complex expression, their ability to infiltrate human tumors and recognize tumor antigens, relative insensitivity to immune checkpoint molecules, and broad tumor cytotoxicity. Therefore, it is now critical to better characterize human γδ T-cell subsets and their mechanisms in RCCs, especially the stage of differentiation. In this study, we aimed to identify γδ T cells that might have adaptive responses against RCC progression. We characterized γδ T cells in peripheral blood and tumor-infiltrating lymphocytes (TILs) in freshly resected tumor specimens from 20 RCC patients. Furthermore, we performed a gene set enrichment analysis on RNA-sequencing data from The Cancer Genome Atlas (TCGA) derived from normal kidneys and RCC tumors to ascertain the association between γδ T-cell infiltration and anti-cancer immune activity. Notably, RCC-infiltrating CD3^low Vγ9Vδ1 T cells with a terminally differentiated effector memory phenotype with up-regulated activation/exhaustion molecules were newly detected as predominant TILs, and the cytotoxic activity of these cells against RCC was confirmed in vitro. In an additional analysis of the TCGA RCC dataset, γδ T-cell enrichment scores correlated strongly with those for CTLs, Th1 cells, “exhausted” T cells, and M1 macrophages, suggesting active involvement of γδ T cells in anti-tumor rather than pro-tumor activity, and Vδ1 cells were more abundant than Vδ2 or Vδ3 cells in RCC tumor samples. Thus, we posit that Vγ9Vδ1 T cells may represent an excellent candidate for adoptive immunotherapy in RCC patients with a high risk of relapse after surgery.     

A Novel Mechanism of Soluble HLA-G Mediated Immune Modulation: Downregulation of T Cell Chemokine Receptor Expression and Impairment of Chemotaxis

Background

In recent years, many immunoregulatory functions have been ascribed to soluble HLA-G (sHLA-G). Since chemotaxis is crucial for an efficient immune response, we have investigated for the first time the effects of sHLA-G on chemokine receptor expression and function in different human T cell populations.

Methodology/Principal Findings

T cell populations isolated from peripheral blood were stimulated in the presence or absence of sHLA-G. Chemokine receptors expression was evaluated by flow cytometry. sHLA-G downregulated expression of i) CCR2, CXCR3 and CXCR5 in CD4⁺ T cells, ii) CXCR3 in CD8⁺ T cells, iii) CXCR3 in Th1 clones iv) CXCR3 in TCR Vδ2γ9 T cells, and upregulated CXCR4 expression in TCR Vδ2γ9 T cells. sHLA-G inhibited in vitro chemotaxis of i) CD4⁺ T cells towards CCL2, CCL8, CXCL10 and CXCL11, ii) CD8⁺ T cells towards CXCL10 and CXCL11, iii) Th1 clones towards CXCL10, and iv) TCR Vδ2γ9 T cells towards CXCL10 and CXCL11. Downregulation of CXCR3 expression on CD4+ T cells by sHLA-G was partially reverted by adding a blocking antibody against ILT2/CD85j, a receptor for sHLA-G, suggesting that sHLA-G downregulated chemokine receptor expression mainly through the interaction with ILT2/CD85j. Follicular helper T cells (T_FH) were isolated from human tonsils and stimulated as described above. sHLA-G impaired CXCR5 expression in T_FH and chemotaxis of the latter cells towards CXCL13. Moreover, sHLA-G expression was detected in tonsils by immunohistochemistry, suggesting a role of sHLA-G in local control of T_FH cell chemotaxis. Intracellular pathways were investigated by Western Blot analysis on total extracts from CD4+ T cells. Phosphorylation of Stat5, p70 s6k, β-arrestin and SHP2 was modulated by sHLA-G treatment.

Conclusions/Significance

Our data demonstrated that sHLA-G impairs expression and functionality of different chemokine receptors in T cells. These findings delineate a novel mechanism whereby sHLA-G modulates T cell recruitment in physiological and pathological conditions.     

Mechanisms involved in α6β1-integrin-mediated Ca signalling

Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting α6β1-integrin-mediated Ca²⁺ signalling. Both Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores and capacitative Ca²⁺ entry via Ca²⁺ channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of α6β1-integrin-mediated Ca²⁺ signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca²⁺-releasing second messengers d-myo-inositol 1,4,5-trisphosphate (InsP₃) and cADPR.     

The histone demethylase Kdm6b regulates the maturation and cytotoxicity of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes

Intestinal intraepithelial lymphocytes (IELs) are distributed along the length of the intestine and are considered the frontline of immune surveillance. The precise molecular mechanisms, especially epigenetic regulation, of their development and function are poorly understood. The trimethylation of histone 3 at lysine 27 (H3K27Me3) is a kind of histone modifications and associated with gene repression. Kdm6b is an epigenetic enzyme responsible for the demethylation of H3K27Me3 and thus promotes gene expression. Here we identified Kdm6b as an important intracellular regulator of small intestinal IELs. Mice genetically deficient for Kdm6b showed greatly reduced numbers of TCRαβ⁺CD8αα⁺ IELs. In the absence of Kdm6b, TCRαβ⁺CD8αα⁺ IELs exhibited increased apoptosis, disturbed maturation and a compromised capability to lyse target cells. Both IL-15 and Kdm6b-mediated demethylation of histone 3 at lysine 27 are responsible for the maturation of TCRαβ⁺CD8αα⁺ IELs through upregulating the expression of Gzmb and Fasl. In addition, Kdm6b also regulates the expression of the gut-homing molecule CCR9 by controlling H3K27Me3 level at its promoter. However, Kdm6b is dispensable for the reactivity of thymic precursors of TCRαβ⁺CD8αα⁺ IELs (IELPs) to IL-15 and TGF-β. In conclusion, we showed that Kdm6b plays critical roles in the maturation and cytotoxic function of small intestinal TCRαβ⁺CD8αα⁺ IELs.Subject terms: Epigenetics, Gene regulation, Immunological disorders, T cells     

MECHANISMS OF THE INDUCTION OF APOPTOSIS IN HUMAN HEPATOMA CELLS BY TUMOUR NECROSIS FACTOR-α

Tumour necrosis factor alpha (TNF-α) at 20 ng/ml induced apoptosis in human hepatoma cellsin vitro . The effect of TNF-α-induced apoptosis was exacerbated by the hypoxanthine-xanthine oxidase (HX/XO) system and cycloheximide (CHX), but alleviated by superoxide dismutase (SOD), suggesting that TNF-α-induced apoptosis may be due to oxidative stress, and independent of protein synthesis. TNF-α elevated free Ca²⁺concentration, triggered lipid peroxidation and decreased the expression of bcl-2 protein. The findings suggest that TNF-α-induced apoptosis may be involved in stimulating Ca²⁺-dependent endonuclease activity and increasing membrane lipid peroxidation. Bcl-2 may play a pivotal role in serving as a Ca²⁺regulator or antioxidant, preventing lipid peroxidation in the process.     

Induction of γδT cells from HSC‐enriched BMCs co‐cultured with iPSC‐derived thymic epithelial cells

T cells bearing γδ antigen receptors have been investigated as potential treatments for several diseases, including malignant tumours. However, the clinical application of γδT cells has been hampered by their relatively low abundance in vivo and the technical difficulty of inducing their differentiation from hematopoietic stem cells (HSCs) in vitro. Here, we describe a novel method for generating mouse γδT cells by co‐culturing HSC‐enriched bone marrow cells (HSC‐eBMCs) with induced thymic epithelial cells (iTECs) derived from induced pluripotent stem cells (iPSCs). We used BMCs from CD45.1 congenic C57BL/6 mice to distinguish them from iPSCs, which expressed CD45.2. We showed that HSC‐eBMCs and iTECs cultured with IL‐2 + IL‐7 for up to 21 days induced CD45.1⁺ γδT cells that expressed a broad repertoire of Vγ and Vδ T‐cell receptors. Notably, the induced lymphocytes contained few or no αβT cells, NK1.1⁺ natural killer cells, or B220⁺ B cells. Adoptive transfer of the induced γδT cells to leukemia‐bearing mice significantly reduced tumour growth and prolonged mouse survival with no obvious side effects, such as tumorigenesis and autoimmune diseases. This new method suggests that it could also be used to produce human γδT cells for clinical applications.