Sos1 regulates sustained TCR‐mediated Erk activation

The duration and/or the magnitude of Ras‐Erk activation are known to be crucial for cell‐fate decisions. In T cells, sustained Erk activation correlates with differentiation/proliferation, whereas transient Erk activation parallels with unresponsiveness/apoptosis. The mechanism by which Son of sevenless (Sos) proteins and Ras guanyl‐releasing protein 1 (RasGRP1) contribute to dynamics of Erk activation in mature T cells is not yet known. Here, we have assessed this issue using stimuli inducing either transient or sustained TCR signaling and RNA interference mediated suppression of Sos1, Sos2, and RasGRP1 expression in primary human T cells. We found that transient Erk activation depends on RasGRP1 but not on Sos. Conversely, sustained Erk signaling and T‐cell activation depend on both Sos1 and RasGRP1. In summary, our data show for the first time that the two guanine nucleotide exchange factors expressed in T cells are differentially involved in the regulation of the duration of Erk phosphorylation and T‐cell activation.     

Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1

Leukocyte functional antigen 1 (LFA-1), with intercellular adhesion molecule ligands, mediates T cell adhesion, but the signaling pathways and functional effects imparted by LFA-1 are unclear. Here, intracellular phosphoprotein staining with 13-dimensional flow cytometry showed that LFA-1 activation induced phosphorylation of the beta(2) integrin chain and release of Jun-activating binding protein 1 (JAB-1), and mediated signaling of kinase Erk1/2 through cytohesin-1. Dominant negatives of both JAB-1 and cytohesin-1 inhibited interleukin 2 production and impaired T helper type 1 differentiation. LFA-1 stimulation lowered the threshold of T cell activation. Thus, LFA-1 signaling contributes to T cell activation and effects T cell differentiation.     

LFA-1-dependent lipid raft recruitment of DNAM-1 (CD226) in CD4+ T cell

Upon antigen recognition by the TCR, both the leukocyte adhesion molecules DNAM-1 and leukocyte function-associated antigen-1 (LFA-1) associate with lipid rafts and form peripheral supra-molecular activation clusters that surround central-supra-molecular activation clusters at the immunological synapse. The serine residue in the cytoplasmic tail of DNAM-1 is responsible for this association of DNAM-1 with lipid rafts. The TCR-mediated signal also induces physical association of DNAM-1 with LFA-1, for which the serine phosphorylation of DNAM-1 is also responsible. However, how the serine residue is involved in lipid raft recruitment of DNAM-1 has remained unclear. Here, we show that, although the TCR-mediated signal induced the serine phosphorylation of DNAM-1, DNAM-1 did not associate with lipid rafts in CD4+ T cells derived from mice deficient in LFA-1 expression, indicating that lipid raft recruitment of DNAM-1 depends on LFA-1 expression. These results suggest that the serine phosphorylation of DNAM-1 primarily induces physical association of DNAM-1 with LFA-1, which then takes DNAM-1 into lipid raft compartment.     

CD4 Dependence of Activation Threshold and TCR Signalling in Mouse T Lymphocytes

The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4^? mutants obtained from the SR.D10 clone. This non-tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4^? mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4^? clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA-1, or CD45, or with lower p56^lck or p59^fyn activity in the mutant cells. Since inhibition experiments using anti-CD4 antibodies have previously shown that activation of the CD4⁺ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen-plus self MHC may become CD4-independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low-affinity TCR reactivities like those directed at self MHC. Moreover, by using anti-TCR/CD3 antibodies, the authors have confirmed the importance of CD4-associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co-precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56^lck-TCR co-precipitation.     

LFA-3 co-stimulates cytokine secretion by cytotoxic T lymphocytes by providing a TCR-independent activation signal

T cell activation is known to depend not only on efficient antigen recognition and subsequent signaling through TCR, but also on interactions involving multiple adhesion and accessory molecules such as CD28/B7, LFA-1/ICAM-1 and LFA-3/CD2. The present study dissects the role of LFA-3/CD2 interactions in the activation of melanoma-specific CD8⁺ T cell clones. To this end we analyzed the influence of LFA-3 density on melanoma cells on lysis and cytokine production (TNF, IL-2, IFN-γ) by T cells following activation by various amounts of antigenic peptides. Our results indicate that increasing LFA-3 density on melanoma cells variably affects their lysis susceptibility, but systematically and considerably enhances cytokine production by melanoma-specific cytotoxic T lymphocyte (CTL) clones. At any stimulatory antigen density, LFA-3 increased the fraction of responding cells and/or cytokine amounts produced by individual cells, without affecting TCR down-regulation. These results show that CD2 engagement increases cytokine gene activation essentially by providing to T cells a TCR-independent co-activation signal. From a practical point of view, our data demonstrate that the level of LFA-3 expressed on tumors critically affects cytokine production by specific CTL and thus the efficiency of specific immune reactions mediated by these cells.     

DOCK2 is essential for antigen-induced translocation of TCR and lipid rafts,but not PKC-theta and LFA-1, in T cells

DOCK2 is a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City which are known to regulate actin cytoskeleton. DOCK2 is critical for lymphocyte migration, yet the role of DOCK2 in TCR signaling remains unclear. We show here that DOCK2 is essential for TCR-mediated Rac activation and immunological synapse formation. In DOCK2-deficient T cells, antigen-induced translocation of TCR and lipid rafts, but not PKC-theta and LFA-1, to the APC interface was severely impaired, resulting in a significant reduction of antigen-specific T cell proliferation. In addition, we found that the efficacy of both positive and negative selection was reduced in DOCK2-deficient mice. These results suggest that DOCK2 regulates T cell responsiveness through remodeling of actin cytoskeleton via Rac activation.     

Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell-mediated immune responses

HPK1 is a Ste20-related serine-threonine kinase that inducibly associates with the adaptors SLP-76 and Gads after T cell receptor (TCR) signaling. Here, HPK1 deficiency resulted in enhanced TCR-induced phosphorylation of SLP-76, phospholipase C-gamma1 and the kinase Erk, more-persistent calcium flux, and increased production of cytokines and antigen-specific antibodies. Furthermore, HPK1-deficient mice were more susceptible to experimental autoimmune encephalomyelitis. Although the interaction between SLP-76 and Gads was unaffected, the inducible association of SLP-76 with 14-3-3tau (a phosphorylated serine-binding protein and negative regulator of TCR signaling) was reduced in HPK1-deficient T cells after TCR stimulation. HPK1 phosphorylated SLP-76 and induced the interaction of SLP-76 with 14-3-3tau. Our results indicate that HPK1 negatively regulates TCR signaling and T cell-mediated immune responses.     

The protein tyrosine phosphatase PTPN4/PTP-MEG1, an enzyme capable of dephosphorylating the TCR ITAMs and regulating NF-kappaB, is dispensable for T cell development and/or T cell effector functions

T cell receptor signaling processes are controlled by the integrated actions of families of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases). Several distinct cytosolic protein tyrosine phosphatases have been described that are able to negatively regulate TCR signaling pathways, including SHP-1, SHP-2, PTPH1, and PEP. Using PTPase substrate-trapping mutants and wild type enzymes, we determined that PTPN4/PTP-MEG1, a PTPH1-family member, could complex and dephosphorylate the ITAMs of the TCR zeta subunit. In addition, the substrate-trapping derivative augmented basal and TCR-induced activation of NF-kappaB in T cells. To characterize the contribution of this PTPase in T cells, we developed PTPN4-deficient mice. T cell development and TCR signaling events were comparable between wild type and PTPN4-deficient animals. The magnitude and duration of TCR-regulated ITAM phosphorylation, as well as overall protein phosphorylation, was unaltered in the absence of PTPN4. Finally, Th1- and Th2-derived cytokines and in vivo immune responses to Listeria monocytogenes were equivalent between wild type and PTPN4-deficient mice. These findings suggest that additional PTPases are involved in controlling ITAM phosphorylations.     

LFA-1 overexpression and T cell autoreactivity: mechanisms

Overexpressing LFA-1 (CD11a/CD18) on antigen specific CD4+ T cells makes the cells proliferate to normally subthreshold stimuli, including self-Ia molecules without specific antigen. The mechanisms by which this occurs are unknown, but potentially include transmission of an increased costimulatory signal, overstabilization of normally low affinity TCR-Ia interactions, or both. A role for increased costimulatory signaling was tested by culturing control and CD18-transfected antigen-specific T cells clones with anti-CD3 and anti-CD11a. Minimal calcium fluxes were detected, but increased protein tyrosine phosphorylation was observed in the transfectants. However, the proliferative response to graded amounts of these antibodies were identical in the transfectants and controls, suggesting that increased signaling alone was insufficient to cause the increased responsiveness. To test for overstabilization, transfected and control clones were cultured with syngeneic M? with or without antigen. The transfected but not control cells downregulated TCR expression in response to M? alone, thus demonstrating successful TCR signaling to a low affinity interaction. These results indicate that LFA-1 overexpression permits TCR signal transmission to a normally subthreshold stimulus presented by M?, consistent with overstabilization. LFA-1 overexpression also causes increased tyrosine phosphorylation, but this alone is not sufficient to cause a proliferative response to low level stimuli.     

Requirement of the serine at residue 329 for lipid raft recruitment of DNAM-1 (CD226)

Upon antigen recognition by the TCR, leukocyte function-associated antigen-1 (LFA-1) physically associates with the leukocyte adhesion molecule DNAM-1 (CD226), for which the serine phosphorylation at residue 329 (S329) of DNAM-1 plays a critical role. The TCR-mediated signal also induces the formation of the immunological synapse (IS), in which lipid raft-associated molecules, including LFA-1, DNAM-1, protein kinase C, Fyn and others, are recruited, resulting in efficient signal transduction for T cell activation. However, the molecular mechanisms of lipid raft recruitment of many associated molecules have remained unclear. Here, we demonstrate that, while both wild-type (WT) and mutant DNAM-1 at S329 were polarized at the IS, the WT, but not mutant, DNAM-1 associated with lipid rafts at the peripheral supra-molecular activation clusters. We also demonstrate that the association of DNAM-1 with lipid rafts was necessary for the tyrosine phosphorylation of DNAM-1, which is essential for LFA-1-mediated co-stimulatory signaling for naive T cell proliferation and differentiation.     

Bam32: a novel mediator of Erk activation in T cells

Bam32 (B lymphocyte adapter molecule of 32 kDa) is an adapter protein expressed in some hematopoietic cells including B and T lymphocytes. It was previously shown that Bam32-deficient mice have defects in various aspects of B cell activation including B cell receptor (BCR)-induced Erk activation, BCR-induced proliferation and T-independent antibody responses. In this study, we have examined the role of Bam32 in T cell activation using Bam32-deficient mice. By comparing CD4(+) T cells from lymph nodes of wild-type and Bam32-deficient mice, we found that Bam32 was required for optimal TCR-induced Erk activation, cytokine production, proliferation and actin-mediated spreading of CD4(+) T cells. These results indicate a novel pathway to Erk activation in T cells involving the adapter protein Bam32.     

Absence of both Sos‐1 and Sos‐2 in peripheral CD4+ T cells leads to PI3K pathway activation and defects in migration

Sos‐1 and Sos‐2 are ubiquitously expressed Ras‐guanine exchange factors involved in Erk‐MAP kinase pathway activation. Using mice lacking genes encoding Sos‐1 and Sos‐2, we evaluated the role of these proteins in peripheral T‐cell signaling and function. Our results confirmed that TCR‐mediated Erk activation in peripheral CD4⁺ T cells does not depend on Sos‐1 and Sos‐2, although IL‐2‐mediated Erk activation does. Unexpectedly, however, we show an increase in AKT phosphorylation in Sos‐1/2dKO CD4⁺ T cells upon TCR and IL‐2 stimulation. Activation of AKT was likely a consequence of increased recruitment of PI3K to Grb2 upon TCR and/or IL‐2 stimulation in Sos‐1/2dKO CD4⁺ T cells. The increased activity of the PI3K/AKT pathway led to downregulation of the surface receptor CD62L in Sos‐1/2dKO T cells and a subsequent impairment in T‐cell migration.     

Interleukin-10 is crucial for maintenance but not for developmental induction of peripheral T cell tolerance

To asses the requirement of interleukin (IL)-10 for peripheral CD4 T cell tolerance, the IL-10 knockout (KO) was introduced into a T cell receptor-transgenic mouse model (TCR1) specific for SV40 T antigen (Tag). IL-10-deficient TCR1-transgenic mice failed to establish antigen-specific T cell tolerance following sequential injections with Tag peptide. Nevertheless, IL-10 was not required for the establishment of CD4 T cell tolerance in double transgenic RT2/TCR1 mice in which Tag is expressed endogenously under control of the insulin promoter. However, in contrast to stable anergy in wild-type RT2/TCR1 mice, tolerant T cells in RT2/TCR1/Il-10KO mice could be driven into vigorous proliferation by exogenous antigenic stimulation in vivo. The observed reactivation of anergic T cell populations in IL-10-deficient mice was only seen after in vivo but not in vitro peptide priming, reflecting an important regulatory function of IL-10 in the context of the living organism. Taken together, these results demonstrate that IL-10 is required to maintain T cell tolerance following exposure to enhanced antigenic stimuli but is not essential for the induction of self-tolerance.     

Actin cytoskeleton-dependent down-regulation of early IgE-mediated signaling in human basophils

Two regions of down-regulation of FcepsilonRI [high-affinity immunogloublin E (IgE) receptor] signaling have been localized recently in basophils. An early down-regulatory step is located proximal to syk and appears responsible for a transient syk phosphorylation in antigen-stimulated basophils. A second, more distal region appears responsible for the transient activation of the ras-extracellular-regulated kinase (Erk) pathway when syk phosphorylation is sustained in anti-IgE-stimulated basophils. As the actin cytoskeleton has been demonstrated to inhibit the early FcepsilonRI signaling in rat basophilic leukemia cells, we explored the hypothesis that the actin cytoskeleton was responsible for the transience of syk phosphorylation in antigen-stimulated basophils. The inhibition of F-actin polymerization with latrunculin A induced a sustained syk phosphorylation in basophils stimulated with an optimal dose of the antigen benzyl penicilloyl-human serum albumin. However, in the presence of latrunculin A, Erk phosphorylation remained transient after stimulation with the antigen or anti-IgE. Latrunculin A also increased downstream events such as histamine release, leukotriene C(4) release, and the intracellular calcium signal, although some of these effects were not specific for an immunologic stimulus. Our results suggest that the actin cytoskeleton is responsible for down-regulation of FcepsilonRI signaling at a point located proximal to syk phosphorylation. Moreover, the fact that latrunculin A did not result in sustained Erk phosphorylation supports the presence of a second down-regulatory step between syk and Erk that cannot be overcome by a sustained early signal.     

Antigen-specific and nonspecific deletion of immature cortical thymocytes caused by antigen injection

Analysis of antigen-induced negative selection of thymocytes in T cell receptor (TCR)-transgenic mice is complicated by the presence of an antigen-responsive peripheral T cell compartment. Our experiments address the question of whether and how peripheral T cell activation can affect immature thymocytes. Following three daily injections of peptide antigen into mice expressing a peptide-specific transgenic TCR and deficient for TAP1, we and others have found profound deletion of the CD4⁺CD8⁺ (DP) thymocyte subset. However, our work shows that even though mature CD8⁺ T cells are inefficiently selected in TAP1-deficient mice, there was a striking degree of peripheral expansion and activation of CD8⁺ peripheral T cells. Furthermore, when cells from TCR-transgenic mice were adoptively transferred, we found that deletion of non-transgenic DP thymocytes occurred in Thy-1-congenic and even more efficiently in TAP1-deficient recipients after repeated peptide injection resulting in peripheral T cell activation. In the adoptive transfer experiments the degree of deletion of immature bystander thymocytes was decreased upon blocking of TNF. These data show that deletion of DP thymocytes can result from excessive peripheral T cell activation and identify TNF as an important effector molecule for this process. When steps are taken to avoid peripheral T cell activation, peptide antigen can induce TCR-mediated thymocyte deletion, presumably in the thymus cortex, since injection of TAP1-deficient TCR-transgenic mice resulted in deletion of immature DP thymocytes prior to detectable peripheral T cell expansion and activation. This effect was not blocked by inhibiting tumor necrosis factor activity. In addition, DP depletion was seen in the absence of peripheral T cell activation when antibody-mediated depletion of CD8⁺ T cells was performed. Our work clearly shows that two mechanisms for deletion of DP thymocytes exist: deletion induced by antigen presentation in the thymus and deletion as a consequence of repeated stimulation of mature peripheral T cells.     

T regulatory cells maintain intestinal homeostasis by suppressing γδ T cells

Immune tolerance against enteric commensal bacteria is important for preventing intestinal inflammation. Deletion of phosphoinositide-dependent protein kinase 1 (Pdk1) in T?cells via Cd4-Cre induced chronic inflammation of the intestine despite the importance of PDK1 in T?cell activation. Analysis of colonic intraepithelial lymphocytes of PDK1-deficient mice revealed markedly increased CD8α(+) T?cell receptor (TCR)γδ(+) T?cells, including an interleukin-17 (IL-17)-expressing population. TCRγδ(+) T?cells were responsible for the inflammatory colitis as shown by the fact that deletion of Tcrd abolished spontaneous colitis in the PDK1-deficient mice. This dysregulation of intestinal TCRγδ(+) T?cells was attributable to a reduction in the number and functional capacity of PDK1-deficient T regulatory (Treg) cells. Adoptive transfer of wild-type Treg cells abrogated the spontaneous activation and proliferation of intestinal TCRγδ(+) T?cells observed in PDK1-deficient mice and prevented the development of colitis. Therefore, suppression of intestinal TCRγδ(+) T?cells by Treg cells maintains enteric immune tolerance.     

T-T cell interactions are mediated by adhesion molecules.

The mechanism by which T cells signal other T cells is not well defined. This was investigated by studying the ability of circulating T cells to induce the proliferation of autologous T cell clones. Peripheral blood T cells activated by cross-linking of the CD3/T cell receptor complex, which increased the expression of cell adhesion molecules LFA-1, LFA-3 and ICAM-1, induced the proliferation of autologous T cell clones. Irradiated antigen-activated peripheral blood T cells could also induce the proliferation of T cell clones which could not recognize that antigen. T-T cell activation required cell contact, was not major histocompatibility complex (MHC) restricted and was blocked by monoclonal antibodies directed against adhesion molecules CD2 and LFA-3 but was not blocked by antibody to class II MHC determinants. As CD2 is the natural ligand for LFA-3, increased expression of T cell surface adhesion molecules LFA-1, ICAM-1 and particularly LFA-3 during an inflammatory response may rapidly recruit T cells that are activated through the CD2 pathway. These results allow a simplified model to explain how relatively few antigen/MHC-specific T cells can recruit large numbers of non-antigen-specific T cells in the generation of an inflammatory response and postulates a novel role of the CD2 molecule in T cell immune function.