Persistent TNF-alpha exposure impairs store operated calcium influx in CD4+ T lymphocytes

Persistent tumour necrosis factor alpha (TNF-alpha) exposure uncouples proximal T-cell receptor (TCR)-signalling events. Here, we demonstrate that chronic TNF-alpha exposure also attenuates signalling distal to the TCR, by specifically inhibiting Ca2+ influx evoked by thapsigargin in CD4+ T-cells. Mitogen-induced Ca2+ responses were impaired in a dose dependent manner, and TCR-induced Ca2+ responses were also significantly reduced. The impairment of Ca2+ influx strongly correlated with poor function as proliferative responses to both mitogen and anti-CD3/CD28 stimulation were suppressed. Our findings show that persistent TNF-alpha exposure of T-cells specifically inhibits store operated Ca2+ influx. This may affect gene activation and contribute to the poor T-cell function in chronic inflammatory disease.     

Resting CD4+ effector memory T cells are precursors of bystander-activated effectors: a surrogate model of rheumatoid arthritis synovial T-cell function

Background

Previously we described a system whereby human peripheral blood T cells stimulated for 8 days in a cytokine cocktail acquired effector function for contact-dependent induction of proinflammatory cytokines from monocytes. We termed these cells cytokine-activated (Tck) cells and found that the signalling pathways elicited in the responding monocytes were identical whether they were placed in contact with Tck cells or with T cells isolated from rheumatoid arthritis (RA) synovial tissue.

Methods

Here, using magnetic beads and fluorescence-activated cell sorting, we extensively phenotype the Tck effector cells and conclude that effector function resides within the CD4⁺CD45RO⁺, CCR7^-, CD49d^high population, and that these cells are derived from the effector memory CD4⁺ T cells in resting blood.

Results

After stimulation in culture, these cells produce a wide range of T-cell cytokines, undergo proliferation and differentiate to acquire an extensively activated phenotype resembling RA synovial T cells. Blocking antibodies against CD69, CD18, or CD49d resulted in a reduction of tumour necrosis factor-α production from monocytes stimulated with CD4⁺CD45RO⁺ Tck cells in the co-culture assay. Moreover, blockade of these ligands also resulted in inhibition of spontaneous tumour necrosis factor-α production in RA synovial mononuclear cell cultures.

Conclusion

Taken together, these data strengthen our understanding of T-cell effector function, highlight the multiple involvement of different cell surface ligands in cell-cell contact and, provide novel insights into the pathogenesis of inflammatory RA disease.     

Thirty-six views of T-cell recognition

While much is known about the signalling pathways within lymphocytes that are triggered during activation, much less is known about how the various cell surface molecules on T cells initiate these events. To address this, we have focused on the primary interaction that drives T-cell activation, namely the binding of a particular T-cell receptor (TCR) to peptide-MHC ligands, and find a close correlation between biological activity and off-rate; that is, the most stimulatory TCR ligands have the slowest dissociation rates. In general, TCRs from multiple histocompatibility complex (MHC) class-II-restricted T cells have half-lives of 1-11s at 25 degrees C, a much narrower range than found with antibodies and suggesting a strong selection for an optimum dissociation rate. TCR ligands with even faster dissociation rates tend to be antagonists. To observe the effects of these different ligands in their physiological setting, we made gene fusions of various molecules with green fluorescent protein (GFP), transfected them into the relevant lymphocytes, and observed their movements during T-cell recognition using multicolour video microscopy. We find that clustering of CD3zeta-GFP and CD4-GFP on the Tcell occurs concomitantly or slightly before the first rise in calcium by the T cell, and that various GFP-labelled molecules on the B-cell side cluster shortly thereafter (ICAM-1, class II MHC, CD48), apparently driven byT-cell molecules. Most of this movement towards the interface is mediated by signals through the co-stimulatory receptors, CD28 and LFA-1, and involves myosin motors and the cortical actin cytoskeleton. Thus, we have proposed that the principal mechanism by which co-stimulation enhances T-cell responsiveness is by increasing the local density of T-cell activation molecules, their ligands and their attendant signalling apparatus. In collaboration with Michael Dustin and colleagues, we have also found that the formation and stability of the TCR-peptide-MHC cluster at the centre of the interaction cap between T and B cells is highly dependent on the dissociation rate of the TCR and its ligand. Thus, we are able to link this kinetic parameter to the formation of a cell surface structure that is linked to and probably causal with respect to T-cell activation.     

Peripheral T-lymphocyte activation by human T-cell leukemia virus type I interferes with the CD2 but not with the CD3/TCR pathway

Human T-cell leukemia virus type I (HTLV-I) is etiologically associated with adult T-cell leukemia, an aggressive lymphoproliferative disorder, and with chronic neurological diseases. In vitro it can infect several types of cells but transforms only human T lymphocytes. We have previously shown that HTLV-I viral particles, even when noninfectious, were able to activate human resting T lymphocytes, suggesting that this activation step may be important in the initiation of the lymphoproliferative process. In the present study, we first demonstrate that in contrast to other mitogenic stimuli, HTLV-I has the unique property to activate human resting T cells in the absence of accessory cells. We then investigate the relationship between HTLV-I-induced T-cell activation and the classical well-known pathways of activation, namely, the CD3/TCR and CD2 pathways. Competitive blocking experiments were performed in which the effects of monoclonal antibodies (MAb) to the CD3/TCR complex or to the CD2 molecule were evaluated on the HTLV-I activation of T cells and compared with that obtained on phytohemagglutinin (PHA)-stimulated cells. It was found that anti-CD3 or -TCR MAb strongly suppress the proliferative response of T cells to PHA, but are significantly less efficient in inhibiting the activation initiated by HTLV-I. By contrast, MAb recognizing specific epitopes of the CD2 molecule inhibit the proliferative response of T cells to PHA or to HTLV-I to the same extent. The results provide evidence that HTLV-I virions interfere mainly with activation via CD2 but not via the CD3/TCR complex. Considering the earlier expression of the CD2 molecule on human T-cell precursors, these observations might be relevant to the characterization of the differentiation stage at which viral infection could interfere with the development and the maturation of T lymphocytes.     

T-cell activation without proliferation in juvenile idiopathic arthritis

A study was done to determine if the differentiation and activation phenotype of T cells in synovial fluid (SF) from patients with juvenile idiopathic arthritis (JIA) is associated with T-cell proliferation in situ. Mononuclear cells were isolated from 44 paired samples of peripheral blood and SF. Differentiation and activation markers were determined on CD4 and CD8 T cells by flow cytometry. Cell-cycle analysis was performed by propidium iodide staining, and surface-marker expression was also assessed after culture of the T cells under conditions similar to those found in the synovial compartment. The majority of the T cells in the SF were CD45RO+CD45RBdull. There was greater expression of the activation markers CD69, HLA-DR, CD25 and CD71 on T cells from SF than on those from peripheral blood. Actively dividing cells accounted for less than 1% of the total T-cell population in SF. The presence or absence of IL-16 in T-cell cultures with SF or in a hypoxic environment did not affect the expression of markers of T-cell activation. T cells from the SF of patients with JIA were highly differentiated and expressed early and late markers of activation with little evidence of in situ proliferation. This observation refines and extends previous reports of the SF T-cell phenotype in JIA and may have important implications for our understanding of chronic inflammation.     

Selective targeting of transforming growth factor-beta1 into TCR/CD28 signalling plasma membrane domains silences T cell activation

TGFβ1 (Transforming Growth Factor-beta1) is a versatile regulator of T cell immune responses. Depending on its context in the immunological environment, TGFβ1 guides T cells toward specific activation programs including T_H17 and regulatory T cell activities. Moreover, TGFβ signals function in immune homeostasis by directly attenuating T cell effector activities. We uncovered a novel context under which TGFβ1 stringently and reversibly silences activation responses of resting human T cells to TCR/CD28 stimulating surfaces:Using ligand-presenting beads, TGFβ1 and TCR/CD28-activating signals were directed into defined plasma membrane domains of T cells. Selective targeting of TGFβ1 cytokine into TCR/CD28 signalling plasma membrane domains held back early response of TCR-proximal tyrosine phosphorylation and bead engulfment at activation sites. Consequently, downstream induction of proliferation and cytokine secretion were stringently attenuated. After extended incubation with TGFβ1-presenting beads, silenced T cells became receptive again to activation by renewed TCR/CD28-stimuli, indicating that the unresponsive state of T cells was reverted and did not reflect long-lasting anergy or decrease in T cell viability. These findings outline a new strategy of physically linking TGFβ1 and TCR-activating functions for the treatment of disease and pathological conditions which are caused by unwanted T cell activity.     

Accumulation of Metal-Specific T Cells in Inflamed Skin in a Novel Murine Model of Chromium-Induced Allergic Contact Dermatitis

Chromium (Cr) causes delayed-type hypersensitivity reactions possibly mediated by accumulating T cells into allergic inflamed skin, which are called irritants or allergic contact dermatitis. However, accumulating T cells during development of metal allergy are poorly characterized because a suitable animal model is not available. This study aimed to elucidate the skewing of T-cell receptor (TCR) repertoire and cytokine profiles in accumulated T cells in inflamed skin during elucidation of Cr allergy. A novel model of Cr allergy was induced by two sensitizations of Cr plus lipopolysaccharide solution into mouse groin followed by single Cr challenge into the footpad. TCR repertoires and nucleotide sequences of complementary determining region 3 were assessed in accumulated T cells from inflamed skin. Cytokine expression profiles and T-cell phenotypes were determined by qPCR. CD3+CD4+ T cells accumulated in allergic footpads and produced increased T helper 1 (Th1) type cytokines, Fas, and Fas ligand in the footpads after challenge, suggesting CD4+ Th1 cells locally expanded in response to Cr. Accumulated T cells included natural killer (NK) T cells and Cr-specific T cells with VA11-1/VB14-1 usage, suggesting metal-specific T cells driven by invariant NKT cells might contribute to the pathogenesis of Cr allergy.     

T-cell activation without proliferation in juvenile idiopathic arthritis

A study was done to determine if the differentiation and activation phenotype of T cells in synovial fluid (SF) from patients with juvenile idiopathic arthritis (JIA) is associated with T-cell proliferation in situ. Mononuclear cells were isolated from 44 paired samples of peripheral blood and SF. Differentiation and activation markers were determined on CD4 and CD8 T cells by flow cytometry. Cell-cycle analysis was performed by propidium iodide staining, and surface-marker expression was also assessed after culture of the T cells under conditions similar to those found in the synovial compartment. The majority of the T cells in the SF were CD45RO⁺CD45RB^dull. There was greater expression of the activation markers CD69, HLA-DR, CD25 and CD71 on T cells from SF than on those from peripheral blood. Actively dividing cells accounted for less than 1% of the total T-cell population in SF. The presence or absence of IL-16 in T-cell cultures with SF or in a hypoxic environment did not affect the expression of markers of T-cell activation. T cells from the SF of patients with JIA were highly differentiated and expressed early and late markers of activation with little evidence of in situ proliferation. This observation refines and extends previous reports of the SF T-cell phenotype in JIA and may have important implications for our understanding of chronic inflammation.     

The role of B7 costimulation in T-cell immunity.

CD4+ T cells are considered to be the major controlling element of the adaptive immune response. They recognize foreign peptides by interaction of the T cell receptor (TCR) with peptide complexed to major histocompatibility complex (MHC) class II molecules on the surface of antigen presenting cells (APC). Once activated, CD4+ T cells orchestrate the various phases of the immune response. They are responsible for the production of numerous cytokines, which activate specific immune effector cell populations including B cells, eosinophils, mast cells and macrophages. Not surprisingly, the activation of CD4+ T cells needs to be tightly regulated and is subject to finely tuned control mechanisms. The requirement for a second or 'costimulatory' signal, in addition to the antigenic signal, provides a key element for the exquisite control of T cell activation. One of the major signalling pathways responsible for delivery of this costimulatory signal is induced by interaction of CD28 on T cells with B7 molecules found only on APC. The present review outlines our current understanding of the physiological role of B7 costimulatory signals in regulating CD4+ T cell responses.     

Alloreactive CD4 T cell activation in vivo: an autonomous function of the indirect pathway of alloantigen presentation

Activation of alloreactive CD4 T cells occurs via the direct and indirect pathways of alloantigen presentation. A novel TCR/alloantigen transgenic system was designed that permitted in vivo visualization of CD4 T cell priming through these pathways. When both pathways of alloantigen presentation were intact, CD4 T cell activation in response to cardiac allografts was rapid and systemic by day 4 after transplantation, in contrast to that seen in response to skin allografts, which was delayed until 10-12 days after transplantation. Despite this systemic CD4 T cell activation in response to cardiac allografts, there was a paucity of activated graft-infiltrating CD4 T cells at 4 days posttransplantation. This finding suggests that the initial priming of alloimmune CD4 T cell responses occurs within draining lymphoid organs. Furthermore, alloantigens derived from cardiac allografts failed to promote thymic negative selection of developing thymocytes expressing the alloreactive TCR clonotype. In the absence of a functional direct pathway, the kinetics of activation, anatomic localization, and effector function of alloreactive CD4 T cells remained unchanged. Overall, the present study defines the anatomic and temporal characteristics of CD4 T cell alloimmune responses and demonstrates that CD4 T cell priming via the indirect pathway proceeds optimally in the absence of the direct pathway of alloantigen presentation.     

Differential SLP-76 expression and TCR-mediated signaling in effector and memory CD4 T cells

We present in this study novel findings on TCR-mediated signaling in naive, effector, and memory CD4 T cells that identify critical biochemical markers to distinguish these subsets. We demonstrate that relative to naive CD4 T cells, memory CD4 T cells exhibit a profound decrease in expression of the linker/adapter molecule SLP-76, while effector T cells express normal to elevated levels of SLP-76. The reduced level of SLP-76 is memory CD4 T cells is coincident with reduced phosphorylation overall, yet the residual SLP-76 couples to a subset of TCR-associated linker molecules, leading to downstream mitogen-activated protein (MAP) kinase activation. By contrast, effector CD4 T cells strongly phosphorylate SLP-76, linker for activation of T cells, and additional Grb2-coupled proteins, exhibit increased associations of SLP-76 to phosphorylated linkers, and hyperphosphorylate downstream Erk1/2 MAP kinases. Our results suggest distinct coupling of signaling intermediates to the TCR in naive, effector, and memory CD4 T cells. Whereas effector CD4 T cells amplify existing TCR signaling events accounting for rapid effector responses, memory T cells engage fewer signaling intermediates to efficiently link TCR triggering directly to downstream MAP kinase activation.     

2B4 (CD244) signaling via chimeric receptors costimulates tumor-antigen specific proliferation and in vitro expansion of human T cells

Regulatory NK cell receptors can contribute to antigen-specific adaptive immune responses by modulating T cell receptor (TCR)-induced T cell activation. We investigated the potential of the NK cell receptor 2B4 (CD244) to enhance tumor antigen-induced activation of human T cells. 2B4 is a member of the CD2 receptor subfamily with both activating and inhibitory functions in NK cells. In T cells, its expression is positively associated with the acquisition of a cytolytic effector memory phenotype. Recombinant chimeric receptors that link extracellular single-chain Fv fragments specific for the tumor-associated surface antigens CD19 and G_D2 to the signaling domains of human 2B4 and/or TCRζ were expressed in non-specifically activated peripheral blood T cells by retroviral gene transfer. While 2B4 signaling alone failed to induce T cell effector functions or proliferation, it significantly augmented the antigen-specific activation responses induced by TCRζ. 2B4 costimulation did not affect the predominant effector memory phenotype of expanding T cells, nor did it increase the proportion of T cells with regulatory phenotype (CD4+CD25^hiFoxP3+). These data support a costimulatory role for 2B4 in human T cell subpopulations. As an amplifier of TCR-mediated signals, 2B4 may provide a powerful new tool for immunotherapy of cancer, promoting sustained activation and proliferation of gene-modified antitumor T cells.     

TRPM8 channel augments T-cell activation and proliferation

The transient receptor potential melastatin 8 (TRPM8) is an ion channel that has been widely studied as a cold-sensitive nociceptor. However, its importance in nonneuronal cells is mostly unexplored. Here, we describe the presence and functional significance of endogenous TRPM8, a nonselective Ca²⁺-channel in T cell functions. The major pool of TRPM8 resides at the T cell surface and its surface accumulation significantly increases in activated T cells. TRPM8 activation synergizes with T-cell receptor (TCR) stimulation to increase CD25, CD69 levels and enhances secretion of proinflammatory cytokine tumor necrosis factor. However, TRPM8 inhibition does not restrict TCR stimulation mediated activation of T cells, indicating that unlike the heat-sensitive TRPV1 and TRPV4 channels, the cold-sensitive TRPM8 channel may be dispensable during T-cell activation, at least in mice. In this study, we demonstrate that TRPM8 promotes TCR-induced intracellular calcium increase. TRPM8 activation is beneficial for T-cell activation and differentiation into effector cells. TRPM8 inhibition during the T-cell activation process may lead to altered phenotype and reduced proliferation, without affecting cell viability. These results collectively establish TRPM8 as a functional calcium channel whose activation may be utilized for mounting an effective immune response. The findings of this study will be relevant to the regulation and response of T cells during cell-mediated immunity. These results will likely further our understanding on the role of ion channels in T-cell activation.