Aggregation of Membrane Proteins by Cytosolic Cross-Linkers: Theory and Simulation of the LAT-Grb2-SOS1 System

Ligand-induced receptor aggregation is a well-known mechanism for initiating intracellular signals but oligomerization of distal signaling molecules may also be required for signal propagation. Formation of complexes containing oligomers of the transmembrane adaptor protein, linker for the activation of T cells (LAT), has been identified as critical in mast cell and T cell activation mediated by immune response receptors. Cross-linking of LAT arises from the formation of a 2:1 complex between the adaptor Grb2 and the nucleotide exchange factor SOS1, which bridges two LAT molecules through the interaction of the Grb2 SH2 domain with a phosphotyrosine on LAT. We model this oligomerization and find that the valence of LAT for Grb2, which ranges from zero to three, is critical in determining the nature and extent of aggregation. A dramatic rise in oligomerization can occur when the valence switches from two to three. For valence three, an equilibrium theory predicts the possibility of forming a gel-like phase. This prediction is confirmed by stochastic simulations, which make additional predictions about the size of the gel and the kinetics of LAT oligomerization. We discuss the model predictions in light of recent experiments on RBL-2H3 and Jurkat E6.1 cells and suggest that the gel phase has been observed in activated mast cells.     

Construction and functional characterization of a fully human anti-CD19 chimeric antigen receptor (huCAR)-expressing primary human T cells

Although remarkable results have been attained by adoptively transferring T cells expressing fully murine and/or humanized anti-CD19 chimeric antigen receptors (CARs) to treat B cell malignancies, evidence of human anti-mouse immune responses against CARs provides a rationale for the development of less immunogenic CARs. By developing a fully human CAR (huCAR), these human anti-mouse immune responses are likely eliminated. This, perhaps, not only increases the persistence of anti-CD19 CAR T cells—thereby reducing the risk of tumor relapse—but also facilitates administration of multiple, temporally separated doses of CAR T cells to the same recipient. To these ends, we have designed and constructed a second-generation fully human anti-CD19 CAR (or huCAR19) containing a fully human single-chain variable fragment (ScFv) fused with a CD8a hinge, a 4-1BB transmembrane domain and intracellular T cell signaling domains of 4-1BB and CD3z. T cells expressing this CAR specifically recognized and lysed CD19⁺ target cells produced cytokines and proliferated in vitro. Moreover, cell volume data revealed that our huCAR construct cannot induce antigen-independent tonic signaling in the absence of cognate antigen. Considering our results, our anti-CD19 huCAR may overcome issues of transgene immunogenicity that plague trials utilizing CARs containing mouse-derived ScFvs. These results suggest that this huCAR19 be safely and effectively applied for adaptive T cell immunotherapy in clinical practice.     

Quantum dot fluorescence characterizes the nanoscale organization of T cell receptors for antigen

Changes in the clustering of surface receptors modulate cell responses to ligands. Hence, global measures of receptor clustering can be useful for characterizing cell states. Using T cell receptor for antigen as an example, we show that k-space image correlation spectroscopy of quantum dots blinking detects T cell receptor clusters on a scale of tens of nanometers and reports changes in clustering after T cell activation. Our results offer a general approach to the global analysis of lateral organization and receptor clustering in single cells, and can thus be applied when the cell type of interest is rare.     

Mechanobiology of antigen-induced T cell arrest

To mount an immune response, T cells must first find rare antigens present at the surface of antigen-presenting cells (APCs). They achieve this by migrating rapidly through the crowded space of tissues and constantly sampling the surface of APCs. Upon antigen recognition, T cells decelerate and polarise towards the APC, ultimately forming a specialised interface known as the immunological synapse. These conjugates form as the result of the interaction between pairs of receptors/ligands that are under mechanical stress due to the continuously reorganising cell cytoskeleton. In this review, we discuss the involvement of mechanical forces during antigen recognition by migrating T cells. We will explore this question from a conceptual and technical perspective, with the aim of providing new insights into the emerging field of mechanobiology.     

Generation of Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and Redifferentiation

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Highlights? Reprogramming of antigen-specific T cells to generate iPSCs (T-iPSCs) ? Redifferentiation of CD8⁺ T cells, with original antigen specificity, from T-iPSCs ? Newly differentiated T cells show high proliferation and elongated telomeres ? T cell antigen-specific cytotoxicity is maintained     

Modulation of MHC Binding by Lateral Association of TCR and Coreceptor

The structure of a T cell receptor (TCR) and its affinity for cognate antigen are fixed, but T cells regulate binding sensitivity through changes in lateral membrane organization. TCR microclusters formed upon antigen engagement participate in downstream signaling. Microclusters are also found 3–4 days after activation, leading to enhanced antigen binding upon rechallenge. However, others have found an almost complete loss of antigen binding four days after T cell activation, when TCR clusters are present. To resolve these contradictory results, we compared binding of soluble MHC-Ig dimers by transgenic T cells stimulated with a high (100 μM) or low (100 fM) dose of cognate antigen. Cells activated by a high dose of peptide bound sixfold lower amounts of CD8-dependent ligand K^b-SIY than cells activated by a low dose of MHC/peptide. In contrast, both cell populations bound a CD8-independent ligand L^d-QL9 equally well. Consistent with the differences between binding of CD8-dependent and CD8-independent peptide/MHC, Förster resonance energy transfer (FRET) measurements of molecular proximity reported little nanoscale association of TCR with CD8 (16 FRET units) compared to their association on cells stimulated by low antigen dose (62 FRET units). Loss of binding induced by changes in lateral organization of TCR and CD8 may serve as a regulatory mechanism to avoid excessive inflammation and immunopathology in response to aggressive infection.     

Liver-Primed Memory T Cells Generated under Noninflammatory Conditions Provide Anti-infectious Immunity

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Highlights? Memory-like CD8⁺ T cells are generated in the liver in the absence of inflammation ? An alternative pathway of T cell priming is facilitated by nonimmune cells ? Liver-primed T cells are rescued from deletion for anti-infectious immunity ? T cell priming in the liver complements conventional memory T cell generation     

Emergent Group Dynamics Governed by Regulatory Cells Produce a Robust Primary T Cell Response

The currently accepted paradigm for the primary T cell response is that effector T cells commit to autonomous developmental programs. This concept is based on several experiments that have demonstrated that the dynamics of a T cell response is largely determined shortly after antigen exposure and that T cell dynamics do not depend on the level and duration of antigen stimulation. Another experimental study has also shown that T cell responses are robust to variations in antigen-specific precursor frequency.     

Bystander-Activated Memory CD8 T Cells Control Early Pathogen Load in an Innate-like,NKG2D-Dependent Manner

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Highlights? Memory CD8 T cells become cytotoxic when activated by inflammation (BA-CTLs) ? Bystander activation of memory CD8 T cells occurs with minimal TCR signaling ? BA-CTLs eliminate target cells in an innate-like, NKG2D-dependent manner ? BA-CTLs are necessary to limit pathogen replication early after an infection     

Phosphorylation-dependent regulation of PSF by GSK3 controls CD45 alternative splicing

Signal-induced alternative splicing of the CD45 gene in human T?cells is essential for proper immune function. Skipping of the CD45 variable exons is controlled, in large part, by the recruitment of PSF to the pre-mRNA substrate upon T?cell activation; however, the signaling cascade leading to exon exclusion has remained elusive. Here we demonstrate that in resting T?cells PSF is directly phosphorylated by GSK3, thus promoting interaction of PSF with TRAP150, which prevents PSF from binding CD45 pre-mRNA. Upon T?cell activation, reduced GSK3 activity leads to reduced PSF phosphorylation, releasing PSF from TRAP150 and allowing it to bind CD45 splicing regulatory elements and repress exon inclusion. Our data place two players, GSK3 and TRAP150, in the complex network that regulates CD45 alternative splicing and demonstrate a paradigm for signal transduction from the cell surface to the RNA processing machinery through the multifunctional protein PSF.     

ALK1 regulates the internalization of endoglin and the type III TGF-β receptor

Complex formation and endocytosis of transforming growth factor-β (TGF-β) receptors play important roles in signaling. However, their interdependence remained unexplored. Here, we demonstrate that ALK1, a TGF-β type I receptor prevalent in endothelial cells, forms stable complexes at the cell surface with endoglin and with type III TGF-β receptors (TβRIII). We show that ALK1 undergoes clathrin-mediated endocytosis (CME) faster than ALK5, type II TGF-β receptor (TβRII), endoglin, or TβRIII. These complexes regulate the endocytosis of the TGF-β receptors, with a major effect mediated by ALK1. Thus, ALK1 enhances the endocytosis of TβRIII and endoglin, while ALK5 and TβRII mildly enhance endoglin, but not TβRIII, internalization. Conversely, the slowly endocytosed endoglin has no effect on the endocytosis of either ALK1, ALK5, or TβRII, while TβRIII has a differential effect, slowing the internalization of ALK5 and TβRII, but not ALK1. Such effects may be relevant to signaling, as BMP9-mediated Smad1/5/8 phosphorylation is inhibited by CME blockade in endothelial cells. We propose a model that links TGF-β receptor oligomerization and endocytosis, based on which endocytosis signals are exposed/functional in specific receptor complexes. This has broad implications for signaling, implying that complex formation among various receptors regulates their surface levels and signaling intensities.     

CD45 is required for CD40-induced inhibition of DNA synthesis and regulation of c-Jun NH2-terminal kinase and p38 in BAL-17 B cells

Stimulation of B cell antigen receptor (BCR) may induce proliferation, differentiation, or apoptosis, depending upon the maturational stage of the cell and the presence or absence of signals transmitted via coreceptors. One such signal is delivered via CD40; for instance, ligation of CD40 rescues B cells from BCR-induced apoptosis. Here we show that, in contrast to WEHI-231 cells, CD40 ligation did not reverse BCR-induced growth inhibition in the BAL-17 mature B cell line and CD40 ligation itself inhibited proliferation. This inhibitory signaling was not observed in CD45-deficient cells. Further analyses demonstrate that transfection of dominant-negative form of SEK1 or treatment with SB203580 strongly reduced CD40-induced inhibition of BAL-17 proliferation, suggesting a requirement for c-Jun NH2-terminal kinase and p38 in CD40-induced inhibition of proliferation. Interestingly, CD40-initiated activation of c-Jun NH2-terminal kinase and p38 was enhanced and sustained in CD45-deficient cells, and these phenotypes were reversed by transfecting CD45 gene. However, CD40-mediated induction of cell surface molecules was not affected in CD45-deficient cells. Taken collectively, these results suggest that CD45 exerts a decisive effect on selective sets of CD40-mediated signaling pathways, dictating B cell fate.     

Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8+ T Cells

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Highlights? iPSCs generated from T cells specific for the MART-1 melanoma epitope ? Differentiation of iPSCs into T cells with a MART-1 specific T cell receptor ? MART-1-based stimulation of T cells demonstrates retained antigen specificity     

T cell microvilli simulations show operation near packing limit and impact on antigen recognition

T cells are immune cells that continuously scan for foreign-derived antigens on the surfaces of nearly all cells, termed antigen-presenting cells (APCs). They do this by dynamically extending numerous protrusions called microvilli (MVs) that contain T cell receptors toward the APC surface in order to scan for antigens. The number, size, and dynamics of these MVs, and the complex multiscale topography that results, play a yet unknown role in antigen recognition. We develop an anatomically informed model that confines antigen recognition to small areas representing MVs that can dynamically form and dissolve and use the model to study how MV dynamics impact antigen sensitivity and discrimination. We find that MV surveillance reduces antigen sensitivity compared with a completely flat interface, unless MV are stabilized in an antigen-dependent manner, and observe that MVs have only a modest impact on antigen discrimination. The model highlights that MV contacts optimize the competing demands of fast scanning speeds of the APC surface with antigen sensitivity. Our model predicts an interface packing fraction that corresponds closely to those observed experimentally, indicating that T cells operate their MVs near the limits imposed by anatomical and geometric constraints. Finally, we find that observed MV contact lifetimes can be largely influenced by conditions in the T cell/APC interface, with these lifetimes often being longer than the simulation or experimental observation period. This work highlights the role of MVs in antigen recognition.     

A Theory of Immunodominance and Adaptive Regulation

Immunodominance refers to the phenomenon in which simultaneous T cell responses against multiple target epitopes organize themselves into distinct and reproducible hierarchies. In many cases, eliminating the response to the most dominant epitope allows responses to subdominant epitopes to expand more fully. The mechanism that drives immunodominance is still not well understood, although various hypotheses have been proposed. One of the more prevalent views is that immunodominance is driven by passive T cell competition for space on antigen presenting cells (APCs) or for access to specific MHC:epitope complexes on the surface of APCs. However, several experimental studies suggest that passive competition alone may not fully explain the robustness of immunodominance under physiological conditions or varying proportions of antigen-specific precursor T cells and APCs. These studies propose that a mechanism of active suppression among T cells gives rise to immunodominance.     

Soft Polydimethylsiloxane-Supported Lipid Bilayers for Studying T Cell Interactions

Much of what we know about the early stages of T cell activation has been obtained from studies of T cells interacting with glass-supported lipid bilayers that favor imaging but are orders of magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing “soft bilayers” with physiological levels of mechanical resistance (Young’s modulus of 4 kPa). Comparisons of T cell behavior on soft and glass-supported bilayers revealed that whereas late stages of T cell activation are thought to be substrate-stiffness dependent, early calcium signaling was unaffected by substrate rigidity, implying that early steps in T cell receptor triggering are not mechanosensitive. The exclusion of large receptor-type phosphatases was observed on the soft bilayers, however, even though it is yet to be demonstrated at authentic cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.     

The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation.

Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not known. Site-directed mutagenesis of the individual catalytic residues of the two CD45 phosphatase domains indicates that the catalytic activity of the membrane-proximal domain is both necessary and sufficient for restoration of TCR signal transduction in a CD45-deficient cell. The putative catalytic activity of the distal phosphatase domain is not required for proximal TCR-mediated signaling events. Moreover, in the context of a chimeric PTPase receptor, the putative catalytic activity of the distal phosphatase domain is not required for ligand-induced negative regulation of PTPase function. We also demonstrate that the phosphorylation of the C-terminal tyrosine of Lck, a site of negative regulation, is reduced only when CD45 mutants with demonstrable in vitro phosphatase activity are introduced into the CD45-deficient cells. These results demonstrate that the phosphatase activity of CD45 is critical for TCR signaling, and for regulating the levels of C-terminal phosphorylated Lck molecules.     

EWI-2 negatively regulates TGF-β signaling leading to altered melanoma growth and metastasis

In normal melanocytes, TGF-β signaling has a cytostatic effect. However, in primary melanoma cells, TGF-β-induced cytostasis is diminished, thus allowing melanoma growth. Later, a second phase of TGF-β signaling supports melanoma EMT-like changes, invasion and metastasis. In parallel with these “present-absent-present” TGF-β signaling phases, cell surface protein EWI motif-containing protein 2 (EWI-2 or IgSF8) is “absent-present-absent” in melanocytes, primary melanoma, and metastatic melanoma, respectively, suggesting that EWI-2 may serve as a negative regulator of TGF-β signaling. Using melanoma cell lines and melanoma short-term cultures, we performed RNAi and overexpression experiments and found that EWI-2 negatively regulates TGF-β signaling and its downstream events including cytostasis (in vitro and in vivo), EMT-like changes, cell migration, CD271-dependent invasion, and lung metastasis (in vivo). When EWI-2 is present, it associates with cell surface tetraspanin proteins CD9 and CD81 — molecules not previously linked to TGF-β signaling. Indeed, when associated with EWI-2, CD9 and CD81 are sequestered and have no impact on TβR2-TβR1 association or TGF-β signaling. However, when EWI-2 is knocked down, CD9 and CD81 become available to provide critical support for TβR2-TβR1 association, thus markedly elevating TGF-β signaling. Consequently, all of those TGF-β-dependent functions specifically arising due to EWI-2 depletion are reversed by blocking or depleting cell surface tetraspanin proteins CD9 or CD81. These results provide new insights into regulation of TGF-β signaling in melanoma, uncover new roles for tetraspanins CD9 and CD81, and strongly suggest that EWI-2 could serve as a favorable prognosis indicator for melanoma patients.     

Exclusion of CD45 from the T-cell receptor signaling area in antigen-stimulated T lymphocytes

T lymphocytes are activated by the engagement of their antigen receptors (TCRs) with complexes of peptide and major histocompatibility complex (MHC) molecules displayed on the cell surface of antigen-presenting cells (APCs) [1]. An unresolved question of antigen recognition by T cells is how TCR triggering actually occurs at the cell-cell contact area. We visualized T-cell-APC contact sites using confocal microscopy and three-dimensional reconstruction of z-sections. We show the rapid formation of a specialized signaling domain at the T-cell-APC contact site that is characterized by a broad and sustained area of tyrosine phosphorylation. The T-lymphocyte cell-surface molecule CD2 is rapidly recruited into this signaling domain, whereas TCRs progressively percolate from the entire T-cell surface into the phosphorylation area. Remarkably, the highly expressed phosphatase CD45 is excluded from the signaling domain. Our results indicate that physiological TCR triggering at the T-cell-APC contact site is the result of a localized alteration in the balance between cellular kinases and phosphatases. We therefore provide experimental evidence to support current models of T-cell activation based on CD45 exclusion from the TCR signaling area [2] [3] [4].     

Homooligomerization of the cytoplasmic domain of the T cell receptor zeta chain and of other proteins containing the immunoreceptor tyrosine-based activation motif

Antigen receptors on T cells, B cells, mast cells, and basophils all have cytoplasmic domains containing one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor engagement in an early and obligatory event in the signaling cascade. How clustering of receptor extracellular domains leads to phosphorylation of cytoplasmic domain ITAMs is not known, and little structural or biochemical information is available for the ITAM-containing cytoplasmic domains. Here we investigate the conformation and oligomeric state of several immune receptor cytoplasmic domains, using purified recombinant proteins and a variety of biophysical and biochemical techniques. We show that all of the cytoplasmic domains of ITAM-containing signaling subunits studied are oligomeric in solution, namely, T cell antigen receptor zeta, CD3epsilon, CD3delta, and CD3gamma, B cell antigen receptor Igalpha and Igbeta, and Fc receptor FcepsilonRIgamma. For zeta(cyt), the oligomerization behavior is best described by a two-step monomer-dimer-tetramer fast dynamic equilibrium with dissociation constants in the order of approximately 10 microM (monomer-dimer) and approximately 1 mM (dimer-tetramer). In contrast to the other ITAM-containing proteins, Igalpha(cyt) forms stable dimers and tetramers even below 10 microM. Circular dichroic analysis reveals the lack of stable ordered structure of the cytoplasmic domains studied, and oligomerization does not change the random-coil-like conformation observed. The random-coil nature of zeta(cyt) was also confirmed by heteronuclear NMR. Phosphorylation of zeta(cyt) and FcepsilonRIgamma(cyt) does not significantly alter their oligomerization behavior. The implications of these results for transmembrane signaling and cellular activation by immune receptors are discussed.