T-cell Receptor Triggering Research Articles

Acute Inhibition of Selected Membrane-Proximal Mouse T Cell Receptor Signaling by Mitochondrial Antagonists

T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide/major histocompatibility complex (MHC) plus lymphocyte function-associated antigen 1 (LFA-1) with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS) platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour) with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin), resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane-proximal TCR signaling event(s). Thus, activation of Akt and PLC-γ1 and entry of extracellular Ca2+ following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption) could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours) on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function.

T cell receptor triggering by force

Antigen recognition through the interaction between the T cell receptor (TCR) and peptide presented by major histocompatibility complex (pMHC) is the first step in T cell-mediated immune responses. How this interaction triggers TCR signalling that leads to T cell activation is still unclear. Taking into account the mechanical stress exerted on the pMHC-TCR interaction at the dynamic interface between T cells and antigen presenting cells (APCs), we propose the so-called receptor deformation model of TCR triggering. In this model, TCR conformational change induced by mechanical forces initiates TCR signalling. The receptor deformation model, for the first time, explains all three aspects of the TCR triggering puzzle: mechanism, specificity, and sensitivity.

Peptide-Major Histocompatibility Complex Dimensions Control Proximal Kinase-Phosphatase Balance during T Cell Activation

T cell antigen recognition requires binding of the T cell receptor (TCR) to a complex between peptide antigen and major histocompatibility complex molecules (pMHC), and this recognition occurs at the interface between the T cell and the antigen-presenting cell. The TCR and pMHC molecules are small compared with other abundant cell surface molecules, and it has been suggested that small size is functionally important. We show here that elongation of both mouse and human MHC class I molecules abrogates T cell antigen recognition as measured by cytokine production and target cell killing. This elongation disrupted tyrosine phosphorylation and Zap70 recruitment at the contact region without affecting TCR or coreceptor binding. Contact areas with elongated forms of pMHC showed an increase in intermembrane distance and less efficient segregation of CD3 from the large tyrosine phosphatase CD45. These findings demonstrate that T cell antigen recognition is strongly dependent on pMHC size and are consistent with models of TCR triggering requiring segregation or mechanical pulling of the TCR.

LFA-1-dependent Ca2+ Entry following Suboptimal T Cell Receptor Triggering Proceeds without Mobilization of Intracellular Ca2+

A surge in cytosolic calcium ion concentration by entry of extracellular Ca2+ is a hallmark of T cell activation. According to store-operated Ca2+ entry mechanism, the Ca2+ entry is preceded by activation of phospholipase C-gamma1 (PLC-gamma1) and the consequent mobilization of intracellular Ca2+. Using membrane vesicles expressing the mouse class I major histocompatibility complex, i.e. Ld plus costimulatory ligands, i.e. B7-1 and intercellular adhesion molecule-1 along with 2C T cell receptor transgenic T cells, we investigated the roles of CD28 and LFA-1 (lymphocyte function-associated antigen-1) in the activation of PLC-gamma1 and Ca2+ signaling. Both CD28 and LFA-1 made significant and comparable contributions to the activation of PLC-gamma1 as gauged by the level of its phosphorylation at tyrosine 783. In contrast, their roles in Ca2+ signaling were quite distinct so that LFA-1/intercellular adhesion molecule-1 interaction exerted a determining role, whereas CD28/B7-1 interaction played only a minimal role. In particular, when the T cells were activated by suboptimal T cell receptor stimulation, LFA-1 played an indispensable role in the Ca2+ signaling. Further experiments using Ca2+-free medium demonstrated that the entry of extracellular Ca2+ was not always accompanied by mobilization of intracellular Ca2+. Thus, intracellular Ca2+ mobilization was hardly detected under the condition that LFA-1 played the indispensable role in the entry of extracellular Ca2+, while a distinct level of intracellular Ca2+ mobilization was readily detected under the condition that LFA-1 played only the supporting role. These results ensure the unique role of LFA-1 in T cell Ca2+ signaling and reveal that LFA-1-dependent Ca2+ entry proceeds via a mechanism separate from store-operated Ca2+ entry.

Interleukin-10 Production by Th1 Cells Requires Interleukin-12-Induced STAT4 Transcription Factor and ERK MAP Kinase Activation by High Antigen Dose

SummaryCD4+ T cells producing interleukin-10 (IL-10) and interferon-γ (IFN-γ) are reported in chronic infections. However, the signals that direct the development of IL-10-producing T helper 1 (Th1) cells are undefined. We showed that development of IL-10-producing Th1 cells required high T cell receptor (TCR) ligation, sustained ERK1 and ERK2 MAP kinases phosphorylation, and IL-12-induced STAT4 transcription factor activation. Repeated TCR triggering led to enhanced IL-10 production by Th1 cells, and continued IL-12 action and high-dose TCR signaling were required for the development and maintenance of IL-10-producing Th1 cells. Although Th1, Th2, and Th17 cells require the activation of distinct STATs for their differentiation, activation of ERK1 and ERK2 was a common requirement for production of IL-10 by all Th cell subsets. IL-10 expression also correlated with c-maf expression. Despite having distinct functions in protection against pathogens, all Th cells share the important task of controlling overexuberant immune responses by means of IL-10 production.

Physical forces can trigger T cell receptor signaling (35.10)

Abstract Engagement of the T cell receptor (TCR) by antigenic peptides presented by the major histocompatibility complex (pMHC) activates specific T cells to control infections. The mechanism of TCR triggering, however, has remained obscure. Here, using micromanipulation of individual T cells, we show that physical forces acting on the TCR can induce calcium mobilization in a Lck-dependent manner. Investigation of artificial antigen-presenting cells (APCs) expressing engineered TCR ligands supports a model in which intercellular tension on the TCR generated during ligand engagement can initiate signaling in T cells. Furthermore, specific interactions between intercellular adhesion molecule-1 (ICAM-1) on APCs and the integrin LFA-1 on T cells may regulate tension to set the threshold for TCR triggering during ligand engagement.

The Influence Of Plasma Membrane Order On TCR Signalling

The signalling events that follow T-cell receptor triggering are mediated by multi-molecular complexes consisting of both membrane-associated and cytosolic proteins. Formation of these complexes is driven by a network of protein-protein and protein-lipid interactions. We study how the selective partitioning of signalling components into separate ordered/raft or disordered phases of the plasma membrane controls the assembly and activity of these signalling complexes.Previous work from our laboratory shows that T cell plasma membrane domains engaged in TCR signalling specifically undergo condensation, and that treatment of Jurkat T-cells with polyunsaturated fatty acids specifically disrupts condensed membrane rafts at TCR activation sites. We have also shown that disruption of membrane order by 7-ketocholesterol leads to reduced recruitment of key TCR signalling components as measured biochemically and by TIRF microscopy of fixed cells. We aim to understand the influence of this biophysical phenomenon on the formation and composition of TCR signalling protein/lipid complexes.To this end, we are currently studying the effects of raft disruption by 7-ketocholesterol and polyunsaturated fatty acids on the kinetic behaviour of TCR signalling components. We employ single molecule real time TIRF microscopy of fluorescently labelled TCR signalling proteins as well as biochemical and proteomic analysis of TCR activation domains upon raft disruption.

RIAM Regulate Spatio-Temporal Distribution of PLC-γ1 and Calcium Mobilization during T Cell Activation

T cell receptor (TCR) ligation induces rapid polarization of the actin cytoskeleton resulting in the formation of the immunological synapse (IS), recruitment of signaling molecules, and initiation of signaling cascades leading to T cell activation. Specific recruitment, redistribution and organization of signaling molecules in the IS is facilitated by lipid raft microdomains, which provide a scaffold for focal protein assembly. Fyn and ZAP-70 are the most proximal TCR signaling molecules that localize in the IS and are redistributed in the lipid rafts during T cell activation. Currently, it is poorly understood how signals originating from the TCR are linked to specific mechanisms that regulate T cell activation. We have identified RIAM, an adaptor molecule that contains a RA (Ras Association) domain, a PH (Plekstrin Homology) domain and proline-rich motifs. RIAM interacts with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. RIAM also interacts with ADAP/SKAP-55 and, thereby, is recruited to the plasma membrane during T cell activation. We have previously determined that, during TCR ligation by antigen, RIAM localizes at the IS and the lipid rafts and serves as a substrate for Fyn and ZAP-70. Because of these properties, we examined whether RIAM might be involved in regulating the molecular and functional outcome of T cell activation. Using RIAM-knock down (KD) T cells in which endogenous RIAM was depleted by siRNA, we determined that RIAM was necessary for IL-2 transcription and RIAM-KD cells had impaired capacity for IL-2 production in response to stimulation with SEE-loaded APC or to TCR/CD3-plus-CD28 crosslinking. However, despite the impaired IL-2 production, analysis of TCR-proximal signaling events did not show impairment of ZAP-70 phosphorylation or formation of the LAT signalosome comprised of phosphorylated PLC-γ1, SLP-76 and Vav1. TCR triggering of both control and RIAM-KD cells also resulted in similar phosphorylation of PLC-γ1. Activation of PLC-γ1 leads to the generation of InsP3 and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PtdIns (4,5)P2). InsP3 binds to InsP3 receptors and triggers Ca2+ release from intracellular stores. Strikingly, TCR triggering of RIAM-KD cells resulted in markedly reduced upregulation of InsP3 compared to that in control T cells. Consistent with the defective upregulation of InsP3, calcium flux of RIAM-KD cells was dramatically impaired. This event was due to the impaired InsP3-mediated calcium release from the endoplasmic reticulum and not due to impaired store content or impaired calcium release-activated calcium (CRAC) channel entry as determined by using the Ca2+ ATPase blocker thapsigargin, which resulted in abundant calcium release in RIAM-KD cells. To analyze the consequences of deregulated InsP3 production and to investigate whether RIAM is specifically involved in PLC-γ1-mediated processes we evaluated activation of several signaling events on which PLC-γ1 activation has distinct effects. Whereas activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 that are PLC-γ1 and Ca2+-dependent, was impaired in the absence of RIAM, activation of p38 and IKK was unaltered compared to control T cells. These results are consistent with a specific role of RIAM in InsP3-mediated Ca2+ release and indicate that deletion of RIAM does not result in a generalized defect in TCR-mediated signaling. Activation of PLC-γ1 at the cell membrane for proper generation of InsP3 requires appropriate docking and positioning of PLC-γ1. For this reason, we examined whether RIAM interacted with PLC-γ1 and regulated its subcellular localization after T cell activation. Detailed analysis by in vivo co-precipitation experiments in cell lysates and by in vitro association assays of purified proteins revealed a direct RIAM-PLC-γ1 interaction that was mediated via the SH3 domain of PLC-γ1. Furthermore, subcellular fractionation into cytoplasmic and cytoskeletal fractions revealed that PLC-γ1 translocated to the cytoskeleton upon T cell activation and this event was abrogated in RIAM-KD cells. These results indicate a novel and unexpected role of RIAM in T cell responses that involves regulation of spatio-temporal distribution and activation of PLC-γ1, leading to generation of InsP3 and Ca2+ mobilization after T cell receptor triggering.

Do T cells need endogenous peptides for activation?

T cells are sensitive to small numbers of antigenic peptide-MHC ligands that are distributed among an excess of endogenous peptide-MHC complexes on the surface of antigen-presenting cells. Although there are accumulating data that indicate a role for these endogenous peptide-MHC complexes in T-cell receptor triggering, whether they are necessary, and the nature of their function, is controversial. In this Opinion article, I argue that endogenous peptide-MHC complexes are required for T-cell stimulation and that their mechanism of action differs between CD4(+) and CD8(+) T cells.

Coronin-1A Links Cytoskeleton Dynamics to TCRαβ-Induced Cell Signaling

Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of αβT cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca2+ influx and phosphorylation and degradation of the inhibitor of NF-κB (IκB). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts αβT cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages.

A Novel Antibody–4-1BBL Fusion Protein for Targeted Costimulation in Cancer Immunotherapy

Costimulation is an essential step in T-cell activation and hence, represents an important aspect in cancer immunotherapy. 4-1BB, a member of the tumor necrosis factor receptor family, has gained particular interest as a costimulatory molecule. Here, we investigated the potential of a targeted activation of 4-1BB-mediated costimulation at the tumor site by generating a recombinant antibody-cytokine fusion protein composed of a single-chain antibody fragment (scFv36) specific for the tumor stromal antigen fibroblast activation protein (FAP) and the extracellular domain of the 4-1BB ligand (4-1BBL). The scFv36-4-1BBL fusion protein is a homotrimeric molecule that binds specifically to FAP and the receptor 4-1BB. T-cell costimulation was demonstrated by interferon-gamma release of peripheral blood mononuclear cells cocultured with FAP-expressing HT1080 cells upon T-cell receptor triggering by monoclonal anti-CD3 antibody. Costimulatory activity of the scFv36-4-1BBL fusion protein was concentration dependent, ligand-specific, and substantially constrained to FAP-expressing target cell binding. Furthermore, scFv36-4-1BBL enhanced T-cell activation when the bispecific antibody scDb33CD3 (specific for FAP and CD3) was used as primary stimulus. Thus, target cell-dependent costimulation with scFv36-4-1BBL constitutes a new option to enhance T-cell activation by bispecific antibodies or antigen-dependent T-cell receptor triggering and should be useful to improve T cell-mediated antitumor responses.

Polarized release of RANTES by cytotoxic T cells paints tumor targets and enhances apoptotic cell removal

The release of regulated on activation normal T-cell expressed and secreted (RANTES) from CD8+ lymphocytes has been shown to be dependent on T-cell receptor triggering by major histocompatability complex class I/peptide complex engagement. We characterized the secretion of RANTES by human leukocyte antigen-A2-restricted tyrosinase-specific cytotoxic T lymphocyte (CTL) in the context of human melanoma cell killing. CTL contact with tumor cell targets elicited a vectorial release of the chemokine RANTES resulting in the selective deposition of RANTES on target cells but not on nontarget bystander cells or the CTL. RANTES on the surface of apoptotic cells enhanced their phagocytosis by murine macrophages. This effect appeared unique to RANTES as the related chemokines macrophage inflammatory protein (MIP) -1alpha, MIP-1beta, and monocyte chemoattractant protein-1 did not significantly affect uptake and were mediated through chemokine receptor CCR1. Oligomerization of RANTES, at least at the level of a tetramer, was required for the enhanced phagocytosis. These results suggest that the role of RANTES in inflammatory disorders might not be restricted to inducing leukocyte infiltration but could also extend to potent macrophage modulation, directly regulating inflammation.

Priming of T cells to Fas-mediated proliferative signals by interleukin-7

AbstractT-cell depletion associated with HIV infection or cytoreductive therapies triggers potential T-cell regenerative mechanisms such as peripheral T-lymphocyte expansion to weak antigenic stimuli and the increased availability of interleukin-7 (IL-7), a cytokine with potent antiapoptotic and proliferative activities. Deleterious mechanisms also associated with lymphopenia, such as increased Fas expression and apoptosis of T cell, however, may result in opposing effects. In this study, we show that Fas molecules, primarily associated with T-cell depletion in lymphopenic settings, may also contribute to compensatory T-cell expansion through transmitting costimulatory signals to suboptimally activated T cells. Proliferation of T lymphocytes in response to concomitant Fas and T-cell receptor (TCR) triggering was shown to be increased in HIV-infected individuals compared with noninfected controls. As IL-7 levels are often elevated in lymphopenic individuals in association with increased Fas expression, we analyzed whether IL-7 would influence Fas-mediated proliferative signals in T cells. We show that IL-7 is able to increase the efficacy of Fas to induce proliferation of suboptimally activated T cells. Thus, high IL-7 levels associated with lymphopenic conditions may simultaneously induce sensitivity to Fas-mediated apoptosis in nonactivated T cells and increase Fas-induced costimulatory signals in T cells recognizing low-affinity antigens.

Retraction

The release of regulated on activation normal T-cell expressed and secreted (RANTES) from CD8+ lymphocytes has been shown to be dependent on T-cell receptor triggering by major histocompatability complex class I/peptide complex engagement. We characterized the secretion of RANTES by human leukocyte antigen-A2-restricted tyrosinase-specific cytotoxic T lymphocyte (CTL) in the context of human melanoma cell killing. CTL contact with tumor cell targets elicited a vectorial release of the chemokine RANTES resulting in the selective deposition of RANTES on target cells but not on nontarget bystander cells or the CTL. RANTES on the surface of apoptotic cells enhanced their phagocytosis by murine macrophages. This effect appeared unique to RANTES as the related chemokines macrophage inflammatory protein (MIP) -1alpha, MIP-1beta, and monocyte chemoattractant protein-1 did not significantly affect uptake and were mediated through chemokine receptor CCR1. Oligomerization of RANTES, at least at the level of a tetramer, was required for the enhanced phagocytosis. These results suggest that the role of RANTES in inflammatory disorders might not be restricted to inducing leukocyte infiltration but could also extend to potent macrophage modulation, directly regulating inflammation.

TCR Triggering by the pMHC Complex: Valency, Affinity, and Dynamics

The interaction between the T cell receptor (TCR) and a peptide-loaded major histocompatibility complex (pMHC) is one of the most-studied interactions in immunology, and yet the precise mechanism by which this system operates is still not fully understood. One key issue is whether TCR triggering minimally requires monomeric pMHC complexes or higher-order multimers (two or more pMHCs). Any model of TCR triggering must explain the high sensitivity, specificity, and dynamic range of ligand responsiveness that this receptor system exhibits. Most models of TCR triggering have not fully appreciated the dynamic aspects of TCR triggering. TCR triggering happens very quickly, and the properties of sensitivity and specificity can be explained by a model that accounts for the interaction dynamics of such a receptor system. In this paper, it is proposed that the important parameter in TCR triggering is the immobilization of the TCR-pMHC complex in the plasma membrane. Whether this involves monomeric or multimeric pMHCs may depend on the affinity of the TCR for the pMHC.

Pulling on the TCR

SCIENCE SIGNALING The exact mechanism by which peptide-loaded major histocompatibility complex (pMHC) activates the T cell receptor (TCR) has been controversial. Effective TCR activation requires presentation by antigen-presenting cellssoluble pMHC cannot activate T cells. In the presence of endogenous pMHC, however, very low concentrations of agonist pMHC are required to activate TCR signaling, as are costimulatory interactions. Ma et al . anchored pMHCs to lipid bilayers or plastic surfaces and found that in the absence of nonagonist pMHC, a minimum of 1 to 10 monomeric agonist pMHCs (MCC-loaded pMHC) per T cell was sufficient to stimulate TCR signaling in T cells interacting with the artificial substrates. Furthermore, the T cell response (calcium signal) was not enhanced by the addition of nonagonist pMHC with the agonist pMHC or under conditions where the nonagonist pMHC and agonist pMHC were close enough to function as a dimer. When murine B cell lymphoma cells were loaded with various endogenous peptides and the MCC peptide, the production of interleukin-2 by the T cells was the same whether a costimulatory endogenous pMHC was present or not. In further experiments, adhesion was shown to be necessary for TCR triggering, as was the actin cytoskeleton. The authors propose a model whereby the agonist pMHC-TCR interaction leads to receptor deformation and activation when the cytoskeleton provides a pulling force on the complex. Endogenous pMHC-TCR interactions would be insufficiently strong to provide the necessary activation signal, and the complex would dissociate when pulled on by the cytoskeleton. NRG PLoS Biol. 6 , e43 (2008).

Pulling on the TCR

The exact mechanism by which peptide-loaded major histocompatibility complex (pMHC) activates the T cell receptor (TCR) has been controversial. Various models have been proposed to account for the requirement of presentation by antigen-presenting cells (APCs) for effective TCR activation and the lack of activation by soluble pMHC, the ability of very low concentrations of agonist pMHC in the presence of endogenous pMHC to activate TCR signaling, and the apparent necessity of costimulatory interactions for effective activation. Ma et al . anchored pMHCs to either a fluid lipid bilayer or plastic surfaces and found that in the absence of nonagonist pMHC a minimum of 1 to 10 monomeric agonist pMHC (MCC-loaded pMHC) per T cell was sufficient to stimulate TCR signaling in T cells interacting with the artificial substrates. Furthermore, the T cell response (calcium signal) was not enhanced by the addition of nonagonist pMHC with the agonist pMHC or under conditions where the nonagonist pMHC and agonist pMHC were close enough to function as a dimer (by coupling to the same streptavidin molecule before covalent attachment to the artificial surface). To confirm that endogenous nonagonist pMHC did not enhance T cell activation in a more physiological situation, the authors loaded murine B cell lymphoma cells with various endogenous peptides and the MCC peptide and found that the production of interleukin-2 by the T cells was the same whether a previously reported costimulatory endogenous pMHC was present or not. The authors used another lipid bilayer system to which T cells would not bind unless the adhesion molecule ICAM was incorporated and found that adhesion was necessary for TCR triggering by MCC-loaded pMHC. When the T cells were treated with cytochalasin to depolymerize actin, the T cells failed to respond to MCC-loaded lymphoma cells or MCC-loaded pMHC bilayers or plastic surfaces, suggesting that the actin cytoskeleton is necessary for TCR triggering. The authors discuss how their results can be reconciled with the reports, suggesting that endogenous pMHC enhance T cell response to low-abundance agonist pMHC and propose a receptor deformation model for TCR activation. In their model, the agonist pMHC-TCR interaction leads to receptor deformation and activation when the cytoskeleton provides a pulling force on the complex. Endogenous pMHC-TCR interactions would be insufficiently strong to provide the necessary activation signal, and the complex would dissociate when "pulled on" by the cytoskeleton. Z. Ma, K. A. Sharp, P. A. Janmey, T. H. Finkel, Surface-anchored monomeric agonist pMHCs alone trigger TCR with high sensitivity. PLoS Biol. 6 , e43 (2008). [PubMed]

Surface-Anchored Monomeric Agonist pMHCs Alone Trigger TCR with High Sensitivity

At the interface between T cell and antigen-presenting cell (APC), peptide antigen presented by MHC (pMHC) binds to the T cell receptor (TCR) and initiates signaling. The mechanism of TCR signal initiation, or triggering, remains unclear. An interesting aspect of this puzzle is that although soluble agonist pMHCs cannot trigger TCR even at high concentrations, the same ligands trigger TCR very efficiently on the surface of APCs. Here, using lipid bilayers or plastic-based artificial APCs with defined components, we identify the critical APC-associated factors that confer agonist pMHCs with such potency. We found that CD4+ T cells are triggered by very low numbers of monomeric agonist pMHCs anchored on fluid lipid bilayers or fixed plastic surfaces, in the absence of any other APC surface molecules. Importantly, on bilayers, plastic surfaces, or real APCs, endogenous pMHCs did not enhance TCR triggering. TCR triggering, however, critically depended upon the adhesiveness of the surface and an intact T cell actin cytoskeleton. Based on these observations, we propose the receptor deformation model of TCR triggering to explain the remarkable sensitivity and specificity of TCR triggering.

Interleukin 2: from immunostimulation to immunoregulation and back again

Interleukin 2 (IL-2) was one of the first cytokines to be discovered. However, the complex role of IL-2 and its receptor in the regulation of immune responses is only now emerging. This review explores the various signals triggered by IL-2 and discusses their translation into biological function. A model is outlined that accommodates the seemingly contradictory functions of IL-2, and explains how one cytokine can be an essential T-cell growth and differentiation factor and yet also be indispensable to maintain peripheral tolerance.

Requirements for Retroviral Targeting of a Suicide Gene to Alloreactive Memory Stem T Cells for Adoptive Immunotherapy of Leukemia.

In a phase I/II clinical trial investigating the prophylactic infusion of suicide gene-modified donor T cells in the context of haploidentical hemopoietic cell transplantation (haplo-HCT) for the treatment of high-risk leukemia, we observed a rapid and effective immune reconstitution. After activation with anti-CD3 antibodies, genetic modification of donor T cells was accomplished with a retroviral vector encoding for the Herpes Simplex thymidine kinase (TK). In vitro before infusion and in vivo at immune reconstitution, TK+ cells displayed an effector memory (EM) phenotype (CD45RA–CD62L−, CD28±CD27+, IL-2±IFN–γ+). The graft-versus-leukemia (GvL) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. Gene targeting with retroviral vectors is limited to memory T cells. Central memory (CM) T cells (CD45RA–CD62L+, CD28+CD27+, IL-2+IFN-γ±) share many characteristics with stem cells, namely the ability to self-renew and to differentiate into a progeny of effector cells. EM TK+ cells have a reduced alloreactivity. Recently, it has been proposed that alloreactivity may be confined to memory T cells with stem cell-features. Since alloantigens are the target not only of graft-versus-host disease (GvHD), but also of the GvL effect, crucial to the success of the strategy is the suicide gene-modification of this subset of memory T cells. We found that addition of CD28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as IL-7 and IL-15, generates central memory (CM) TK+ cells. CM TK+ cells are highly alloreactive, both in vitro and in vivo in a humanized animal model of GvHD based on the grafting of human skin onto NOD/scid mice. Interestingly, CM TK+ cells express the IL7Rα, a marker associated with the stem cell-features of memory T cells. Moreover, IL7Rα expression is maintained after stimulation with alloantigens. Stimulation of CM, but not of EM TK+ cells with autologous dendritic cells pulsed with restricted peptides from the minor histocompatibility alloantigen (mHag) HA-1 or H-Y efficiently induces mHag-specific effector T cells that lyse natural ligand expressing HLA-A2+ targets. TK+ mHag-specific effector T cells also lysed mHag+HLA-A2+ leukemic cells and, when infused in conditioned NOD/scid mice harboring human leukemia, significantly delayed disease progression. Altogether, these data suggest that optimal T-cell receptor triggering and homeostatic cytokines are required for retroviral targeting of a suicide gene to alloreactive memory stem T cells and warrant their use for a safe and powerful GvL effect.