The coreceptor CD2 uses plasma membrane microdomains to transduce signals in T cells

The interaction between a T cell and an antigen-presenting cell (APC) can trigger a signaling response that leads to T cell activation. Prior studies have shown that ligation of the T cell receptor (TCR) triggers a signaling cascade that proceeds through the coalescence of TCR and various signaling molecules (e.g., the kinase Lck and adaptor protein LAT [linker for T cell activation]) into microdomains on the plasma membrane. In this study, we investigated another ligand–receptor interaction (CD58–CD2) that facilities T cell activation using a model system consisting of Jurkat T cells interacting with a planar lipid bilayer that mimics an APC. We show that the binding of CD58 to CD2, in the absence of TCR activation, also induces signaling through the actin-dependent coalescence of signaling molecules (including TCR-ζ chain, Lck, and LAT) into microdomains. When simultaneously activated, TCR and CD2 initially colocalize in small microdomains but then partition into separate zones; this spatial segregation may enable the two receptors to enhance signaling synergistically. Our results show that two structurally distinct receptors both induce a rapid spatial reorganization of molecules in the plasma membrane, suggesting a model for how local increases in the concentration of signaling molecules can trigger T cell signaling.     

Cutting edge: in vivo identification of TCR redistribution and polarized IL-2 production by naive CD4 T cells

TCR aggregation at the point of contact with an APC is thought to play an important role in T cell signal transduction. However, this potentially important phenomenon has never been documented during an immune response in vivo. Here we used immunohistology to show that the TCR on naive Ag-specific CD4 T cells in the lymph nodes of mice injected with Ag redistributed to one side of the cell. In cases where the APC could be identified, the TCR was concentrated on the side of the T cell facing the APC. In those T cells that produced IL-2, the TCR and IL-2 localized to the same side of the cell. In vivo IL-2 production depended on costimulatory signaling through CD28, whereas TCR redistribution did not. These results show that Ag-stimulated CD4 T cells produce IL-2 in a polarized fashion and undergo CD28-independent TCR redistribution in vivo.     

Ligation of cytotoxic T lymphocyte antigen-4 to T cell receptor inhibits T cell activation and directs differentiation into Foxp3+ regulatory T cells

Cross-linking of ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) during the early phase of T cell activation attenuates TCR signaling, leading to T cell inhibition. To promote this event, a bispecific fusion protein comprising a mutant mouse CD80 (CD80w88a) and lymphocyte activation antigen-3 was engineered to concurrently engage CTLA-4 and cross-link it to the TCR. Cross-linking is expected to be attained via ligation of CTLA-4 first to MHCII and then indirectly to the TCR, generating a CTLA-4-MHCII-TCR trimolecular complex that forms between T cells and antigen-presenting cells during T cell activation. Treating T cells with this bispecific fusion protein inhibited T cell activation. In addition, it induced the production of IL-10 and TGF-β and attenuated AKT and mTOR signaling. Intriguingly, treatment with the bispecific fusion protein also directed early T cell differentiation into Foxp3-positive regulatory T cells (Tregs). This process was dependent on the endogenous production of TGF-β. Thus, bispecific fusion proteins that engage CTLA-4 and co-ligate it to the TCR during the early phase of T cell activation can negatively regulate the T cell response. Bispecific biologics with such dual functions may therefore represent a novel class of therapeutics for immune modulation. These findings presented here also reveal a potential new role for CTLA-4 in Treg differentiation.     

Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4T cells

CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.     

Gene Related to Anergy in Lymphocytes (GRAIL) Expression in CD4+ T Cells Impairs Actin Cytoskeletal Organization during T Cell/Antigen-presenting Cell Interactions

GRAIL (gene related to anergy in lymphocytes), is an E3 ubiquitin ligase with increased expression in anergic CD4+ T cells. The expression of GRAIL has been shown to be both necessary and sufficient for the induction of T cell (T) anergy. To date, several subsets of anergic T cells have demonstrated altered interactions with antigen-presenting cells (APC) and perturbed TCR-mediated signaling. The role of GRAIL in mediating these aspects of T cell anergy remains unclear. We used flow cytometry and confocal microscopy to examine T/APC interactions in GRAIL-expressing T cells. Increased GRAIL expression resulted in reduced T/APC conjugation efficiency as assessed by flow cytometry. Examination of single T/APC conjugates by confocal microscopy revealed altered polarization of polymerized actin and LFA-1 to the T/APC interface. When GRAIL expression was knocked down, actin polarization to the T/APC interface was restored, demonstrating that GRAIL is necessary for alteration of actin cytoskeletal rearrangement under anergizing conditions. Interestingly, proximal TCR signaling including calcium flux and phosphorylation of Vav were not disrupted by expression of GRAIL in CD4+ T cells. In contrast, interrogation of distal signaling events demonstrated significantly decreased JNK phosphorylation in GRAIL-expressing T cells. In sum, GRAIL expression in CD4+ T cells mediates alterations in the actin cytoskeleton during T/APC interactions. Moreover, in this model, our data dissociates proximal T cell signaling events from functional unresponsiveness. These data demonstrate a novel role for GRAIL in modulating T/APC interactions and provide further insight into the cell biology of anergic T cells.     

TCR-dependent cell response is modulated by the timing of CD43 engagement

Binding of Ag by the Ag receptor in combination with other stimuli provided by costimulatory receptors triggers the expansion and differentiation of T lymphocytes. However, it is unclear whether the time when costimulatory molecules interact with their counterreceptors with regards to Ag recognition leads to different T cell responses. Provided that the coreceptor molecule CD43 is a very abundant molecule evenly distributed on the membrane of T cell surface protruding 45 nm from the cell, we hypothesized that CD43 is one of the first molecules that interacts with the APC and thus modulates TCR activation. We show that engaging CD43 before or simultaneously with the TCR inhibited Lck-Src homology 2 domain containing phosphatase-1 interaction, preventing the onset of a negative feedback loop on TCR signals, favoring high levels of IL-2, cell proliferation, and secretion of proinflammatory cytokines and chemokines. In contrast, the intracellular signals resulting of engaging the TCR before CD43 were insufficient to induce IL-2 production and cell proliferation. Interestingly, when stimulated through the TCR and CD28, cells proliferated vigorously, independent of the order with which molecules were engaged. These results indicate that CD43 induces a signaling cascade that prolongs the duration of TCR signaling and support the temporal summation model for T cell activation. In addition to the strength and duration of intracellular signals, our data underscore temporality with which certain molecules are engaged as yet another mechanism to fine tune T cell signal quality, and ultimately immune function.     

LFA-1-mediated T cell costimulation through increased localization of TCR/class II complexes to the central supramolecular activation cluster and exclusion of CD45 from the immunological synapse

T cell activation is associated with a dramatic reorganization of cell surface proteins and associated signaling components into discrete subdomains within the immunological synapse in T cell:APC conjugates. However, the signals that direct the localization of these proteins and the functional significance of this organization have not been established. In this study, we have used wild-type and LFA-1-deficient, DO11.10 TCR transgenic T cells to examine the role of LFA-1 in the formation of the immunological synapse. We found that coengagement of LFA-1 is not required for the formation of the central supramolecular activation cluster (cSMAC) region, but does increase the accumulation of TCR/class II complexes within the cSMAC. In addition, LFA-1 is required for the recruitment and localization of talin into the peripheral supramolecular activation cluster region and exclusion of CD45 from the synapse. The ability of LFA-1 to increase the amount of TCR engaged during synapse formation and segregate the phosphatase, CD45, from the synapse suggests that LFA-1 might enhance proximal TCR signaling. To test this, we combined flow cytometry-based cell adhesion and calcium-signaling assays and found that coengagement of LFA-1 significantly increased the magnitude of the intracellular calcium response following Ag presentation. These data support the idea that in addition to its important role on regulating T cell:APC adhesion, coengagement of LFA-1 can enhance T cell signaling, and suggest that this may be accomplished in part through the organization of proteins within the immunological synapse.     

Regulation of sustained actin dynamics by the TCR and costimulation as a mechanism of receptor localization

The localization of receptors, signaling intermediates, and cytoskeletal components at the T cell/APC interface is thought to be a major determinant of efficient T cell activation. However, important questions remain open. What are the dynamics of the T cell cytoskeleton as a potential mediator of such localization? How are they regulated by the TCR and costimulatory receptors? Do they actually mediate receptor localization? In this study, we have addressed these questions. Even under limiting T cell activation conditions, actin accumulated immediately and transiently at the T cell/APC interface, the microtubule organizing center reoriented toward it. In contrast, sustained (>5 min) actin accumulation in highly dynamic patterns depended on an optimal T cell stimulus: high concentrations of the strong TCR ligand agonist peptide/MHC and engagement of the costimulatory receptors CD28 and LFA-1 were required in an overlapping, yet distinct, fashion. Intact sustained actin dynamics were required for interface accumulation of TCR/MHC in a central pattern and for efficient T cell proliferation, as established using a novel approach to selectively block only the sustained actin dynamics. These data suggest that control of specific elements of actin dynamics by TCR and costimulatory receptors is a mechanism to regulate the efficiency of T cell activation.     

Cutting edge: B7/CD28 interactions regulate cell cycle progression independent of the strength of TCR signaling

The role of B7/CD28 signals in Ag-induced cell cycle progression of CD4(+) T cells was examined using the technique of CFSE dye dilution and flow cytometry. In wild-type T cells, proliferation was directly related to the concentration of Ag available to the APC. Consistent with this, the rate of G(0)-->G(1) cell cycle progression varied with the concentration of Ag. However, cell division by T cell blasts occurred at a constant rate, independent of Ag concentration. G(0)-->G(1) phase progression by CD28-deficient CD4(+) T cells or wild-type T cells cultured in the presence of neutralizing anti-B7 mAbs was slowed, confirming that a synergy does exist between TCR and CD28 signaling in the initial activation of the T cells. However, unlike the TCR, the strength of CD28 stimulation was also shown to play a unique role in controlling the rate of cell division by T cell blasts.     

Dual effects of Sprouty1 on TCR signaling depending on the differentiation state of the T cell

Sprouty (Spry) is known to be a negative feedback inhibitor of growth factor receptor signaling through inhibition of the Ras/MAPK pathway. Several groups, however, have reported a positive role for Spry involving sequestration of the inhibitory protein c-Cbl. Thus, Spry may have various functions in the regulation of receptor-mediated signaling depending on the context. In the immune system, the function of Spry is unknown. In this study, we investigated the role of Spry1 in T cell activation. Spry1, among the four mammalian homologs, was specifically induced by TCR signaling of CD4(+) murine T cells. In fully differentiated Th1 clones, overexpressed Spry1 inhibited TCR signaling and decreased IL-2 production while reducing expression with specific siRNA transfection had the opposite effect, increasing IL-2 production. In contrast, in naive T cells, Spry1 overexpression enhanced TCR signaling, and increased proliferation and IL-2 production, while siRNA transfection again had the opposite effect, reducing IL-2 production following activation. The enhancing effect in naive cells was abrogated by preactivation of the T cells with Ag and APC, indicating that the history of exposure to Ag is correlated with a hierarchy of T cell responsiveness to Spry1. Furthermore, both the NF-AT and MAPK pathways were influenced by Spry1, implying a different molecular mechanism from that for growth factor receptor signaling. Thus, Spry1 uses a novel mechanism to bring about differential effects on TCR signaling through the same receptor, depending on the differentiation state of the T cell.     

Trogocytosis is a gateway to characterize functional diversity in melanoma-specific CD8+ T cell clones

Trogocytosis, the transfer of membrane patches from target to immune effector cells, is a signature of tumor-T cell interaction. In this study, we used the trogocytosis phenomenon to study functional diversity within tumor-specific T cell clones with identical TCR specificity. MART-1(26-35)-specific CD8 T cell clones, which differed in their trogocytosis capacity (low [2D11], intermediate [2G1], high [2E2]), were generated from melanoma patients. Functional evaluation of the clones showed that the percentage of trogocytosis-capable T cells closely paralleled each clone's IFN-γ and TNF-α production, lysosome degranulation, and lysis of peptide-pulsed targets and unmodified melanoma. The highly cytotoxic 2E2 clone displayed the highest TCR peptide binding affinity, whereas the low-activity 2D11 clone showed TCR binding to peptide-MHC in a CD8-dependent manner. TCR analysis revealed Vβ16 for clones 2E2 and 2G1 and Vβ14 for 2D11. When peptide-affinity differences were bypassed by nonspecific TCR stimulation, clones 2E2 and 2D11 still manifested distinctive signaling patterns. The high-activity 2E2 clone displayed prolonged phosphorylation of ribosomal protein S6, an integrator of MAPK and AKT activation, whereas the low-activity 2D11 clone generated shorter and weaker phosphorylation. Screening the two clones with identical TCR Vβ by immunoreceptor array showed higher phosphorylation of NK, T, and B cell Ag (NTB-A), a SLAM family homophilic receptor, in clone 2E2 compared with 2G1. Specific blocking of NTB-A on APCs markedly reduced cytokine production by CD8 lymphocytes, pointing to a possible contribution of NTB-A costimulation to T cell functional diversity. This finding identifies NTB-A as a potential target for improving anti-cancer immunotherapy.     

Selective defect in antigen-induced TCR internalization at the immune synapse of CD8 T cells bearing the ZAP-70(Y292F) mutation

Cbl proteins have been implicated in ligand-induced TCR/CD3 down-modulation, but underlying mechanisms are unclear. We analyzed the effect of mutation of a cbl-binding site on ZAP-70 (ZAP-Y292F) on dynamics, internalization, and degradation of the TCR/CD3 complex in response to distinct stimuli. Naive CD8 T cells expressing the P14 transgenic TCR from ZAP-Y292F mice were selectively affected in TCR/CD3 down-modulation in response to antigenic stimulation, whereas neither anti-CD3 Ab-, and PMA-induced TCR down-modulation, nor constitutive receptor endocytosis/cycling were impaired. We further established that the defect in TCR/CD3 down-modulation in response to Ag was paralleled by an impaired TCR/CD3 internalization and CD3zeta degradation. Analysis of T/APC conjugates revealed that delayed redistribution of TCR at the T/APC contact zone was paralleled by a delay in TCR internalization in the synaptic zone in ZAP-Y292F compared with ZAP-wild-type T cells. Cbl recruitment to the synapse was also retarded in ZAP-Y292F T cells, although F-actin and LFA-1 redistribution was similar for both cell types. This study identifies a step involving ZAP-70/cbl interaction that is critical for rapid internalization of the TCR/CD3 complex at the CD8 T cell/APC synapse.     

New insights to the functional role of the T cell-antigen presenting cell immunological synapse

Three innovative and complementary morphological approaches were employed to study the T cell/antigen presenting cell (APC) interaction: (i) high resolution three-dimensional confocal microscopy of the T cell-APC contact site; (ii) time lapse video recording in living T cells of [Ca2+]I and changes in distribution of various GFP fusion proteins with TCR/CD3-zeta complex associated- and other signaling components; (iii) measurement of lateral TCR mobility and that of recruited signaling components using techniques based on fluorescence recovery after photo-bleaching. These approaches were combined with biochemical and functional experiments to investigate two principal issues: (A) Recruitment and the three-dimensional arrangement of receptors and signaling components at the contact site between human CD4+ T lymphocytes and APCs, (B) Structure of the immunological synapse formed at the contact site between cytotoxic T lymphocytes (CTLs) and target cells. We discuss evidence indicating that TCR engagement and triggering can occur in the absence of large-scale molecular segregation into the T cell-APC contact site. Taken together our results indicate that although not required for TCR engagement and triggering, formation of the IS is important to reinforce TCR-mediated signal transduction and achieve full T cell activation.     

Recruitment of the actin-binding protein HIP-55 to the immunological synapse regulates T cell receptor signaling and endocytosis

Actin cytoskeleton dynamics critically regulate T cell activation. We found that the cytoplasmic adaptor HIP-55, a Src/Syk-kinases substrate and member of the drebrin/Abp1 family of actin-binding proteins, localized to the T cell-antigen-presenting cell (APC) contact site in an antigen-dependent manner. Using green fluorescent protein fusion proteins, both Src homology 3 (SH3) and actin binding domains were found necessary for recruitment at the T cell-APC interface. HIP-55 was not implicated in conjugate formation and actin polymerization but regulated distal signaling events through binding and activation of hematopoietic progenitor kinase 1 (HPK1), a germinal center kinase (GCK) family kinase involved in negative signaling in T cells. Using RNA interference and overexpression experiments, the HIP-55-HPK1 complex was found to negatively regulate nuclear factor of activated T cell (NFAT) activation by the T cell antigen receptor. Moreover, we show that HIP-55, which partly co-localized with early endocytic compartments, promoted both basal and ligand-dependent T cell receptor (TCR) down-modulation, resulting in a decreased TCR expression. SH3 and actin-depolymerizing factor homology domains were required for this function. As controls, the expression of CD28 and the glycosylphosphatidylinositol-linked protein CD59 was not affected by HIP-55 overexpression. These results suggest that, in addition to binding to HPK1, HIP-55 might negatively regulate TCR signaling through down-regulation of TCR expression. Our findings show that HIP-55 is a key novel component of the immunological synapse that modulates T cell activation by connecting actin cytoskeleton and TCRs to gene activation and endocytic processes.     

Peptide-specific intercellular transfer of MHC class II to CD4+ T cells directly from the immunological synapse upon cellular dissociation

The transfer of membrane proteins from APC to T cells was initially described in the 1970s, and subsequent work has described two mechanisms of transfer: APC-derived exosomes and direct transfer of small packets, while cells remain conjugated. Using fibroblast APC expressing a GFP-tagged I-E(k) molecule with covalently attached antigenic peptide, we observed a third mechanism in live cell imaging: T cells spontaneously dissociating from APC often capture MHC:peptide complexes directly from the immunological synapse. Using two I-E(k)-restricted murine TCR transgenic T cells with different peptide specificity, we show in this study that the MHC transfer is peptide specific. Using blocking Abs, we found that MHC:peptide transfer in this system requires direct TCR-MHC:peptide interactions and is augmented by costimulation through CD28-CD80 interactions. Capture of the GFP-tagged MHC:peptide complexes correlates with an activated phenotype of the T cell, elevated CD69 with down-modulated TCR. The transferred MHC:peptide molecules transferred to the T cell are associated with molecules that imply continued TCR signaling; p56(lck), phosphotyrosine, and polarization of the actin cytoskeleton.     

CD28 delivers a costimulatory signal involved in antigen-specific IL-2 production by human T cells.

CD4+ T cells require two signals to produce maximal amounts of IL-2, i.e., TCR occupancy and an unidentified APC-derived costimulus. Here we show that this costimulatory signal can be delivered by the T cell molecule CD28. An agonistic anti-CD28 mAb, but not IL-1 and/or IL-6, stimulated T cell proliferation by tetanus toxoid-specific T cells cultured with Ag-pulsed, costimulation-deficient APC. Furthermore, the ability of B cell tumor lines to provide costimulatory signals to purified T cells correlated well with expression of the CD28 ligand B7/BB-1. Finally, like anti-CD28 mAb, autologous human APC appeared to stimulate a cyclosporine A-resistant pathway of T cell activation. Together, these results suggest that the two signals required for IL-2 production by CD4+ T cells can be transduced by the TCR and CD28.     

Cytoskeletal Polarization of T Cells Is Regulated by an Immunoreceptor Tyrosine-based Activation Motif–dependent Mechanism

Abstract. Binding of a T cell to an appropriate antigen-presenting cell (APC) induces the rapid reorientation of the T cell cytoskeleton and secretory apparatus towards the cell–cell contact site in a T cell antigen receptor (TCR) and peptide/major histocompatibility complex–dependent process. Such T cell polarization directs the delivery of cytokines and cytotoxic mediators towards the APC and contributes to the highly selective and specific action of effector T cells. To study the signaling pathways that regulate cytoskeletal rearrangements in T lymphocytes, we set up a conjugate formation assay using Jurkat T cells as effectors and cell-sized latex beads coated with various antibodies as artificial APCs. Here, we report that beads coated with antibodies specific for the TCR-CD3 complex were sufficient to induce T cell polarization towards the bead attachment site, as judged by reorientation of the microtubule-organizing center (MTOC) and localized actin polymerization. Thus, these cytoskeletal changes did not depend on activation of additional coreceptors. Moreover, single subunits of the TCR complex, namely TCR-ζ and CD3ε, were equally effective in inducing cytoskeletal polarization. However, mutagenesis of the immunoreceptor tyrosine-based activation motifs (ITAMs), present three times in TCR-ζ and once in CD3ε, revealed that the induction of cytoskeletal rearrangements required the presence of at least one intact ITAM. In agreement with this result, lack of functional Lck, the protein tyrosine kinase responsible for ITAM phosphorylation, abolished both MTOC reorientation and polarized actin polymerization. Both inhibitor and transient overexpression studies demonstrated that MTOC reorientation could occur in the absence of Ras activation. Our results suggest that APC-induced T cell polarization is a TCR-mediated event that is coupled to the TCR by the same signaling motif as TCR-induced gene activation, but diverges in its distal signaling requirements.     

Blockade of T cell activation using a surface-linked single-chain antibody to CTLA-4 (CD152)

CTLA-4 (CD152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by engaging CTLA-4 has been limited by sharing of its natural ligands with the costimulatory protein CD28. In the present study, a CTLA-4-specific single-chain Ab (scFv) was developed and expressed on the cell surface to promote selective engagement of this regulatory molecule. Transfectants expressing anti-CTLA-4 scFv at their surface bound soluble CTLA-4 but not soluble CD28. Coexpression of anti-CTLA-4 scFv with anti-CD3epsilon and anti-CD28 scFvs on artificial APCs reduced the proliferation and IL-2 production by resting and preactivated bulk T cells as well as CD4+ and CD8+ T cell subsets. Importantly, expression of anti-CTLA-4 scFv on the same cell surface as the TCR ligand was essential for the inhibitory effects of CTLA-4-specific ligation. CTLA-4-mediated inhibition of tyrosine phosphorylation of components of the proximal TCR signaling apparatus was similarly dependent on coexpression of TCR and CTLA-4 ligands on the same surface. These findings support a predominant role for CTLA-4 function in the modification of the proximal TCR signal. Using T cells from DO11.10 and 2C TCR transgenic mice, negative regulatory effects of selective CTLA-4 ligation were also demonstrated during the stimulation of Ag-specific CD4+ and CD8+ T cells by MHC/peptide complexes. Together these studies demonstrate that selective ligation of CTLA-4 using a membrane-bound scFv results in attenuated T cell responses only when coengaged with the TCR during T cell/APC interaction and define an approach to harnessing the immunomodulatory potential of CTLA-4-specific ligation.     

Immunological synapse formation licenses CD40-CD40L accumulations at T-APC contact sites

The maintenance of tolerance is likely to rely on the ability of a T cell to polarize surface molecules providing "help" to only specific APCs. The formation of a mature immunological synapse leads to concentration of the TCR at the APC interface. In this study, we show that the CD40-CD154 receptor-ligand pair is also highly concentrated into a central region of the synapse on mouse lymphocytes only after the formation of the TCR/CD3 c-SMAC. Concentration of this ligand was strictly dependent on TCR recognition, the binding of ICAM-1 to T cell integrins and the presence of an intact cytoskeleton in the T cells. This may provide a novel explanation for the specificity of T cell help directing the help signal to the site of Ag receptor signal. It may also serve as a site for these molecular aggregates to coassociate and/or internalize alongside other signaling receptors.     

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.