Edward J. Evans

The nature of molecular recognition by T cells.

Considerable progress has been made in characterizing four key sets of interactions controlling antigen responsiveness in T cells, involving the following: the T cell antigen receptor, its coreceptors CD4 and CD8, the costimulatory receptors CD28 and CTLA-4, and the accessory molecule CD2. Complementary work has defined the general biophysical properties of interactions between cell surface molecules. Among the major conclusions are that these interactions are structurally heterogeneous, often reflecting clear-cut functional constraints, and that, although they all interact relatively weakly, hierarchical differences in the stabilities of the signaling complexes formed by these molecules may influence the sequence of steps leading to T cell activation. Here we review these developments and highlight the major challenges remaining as the field moves toward formulating quantitative models of T cell recognition.

Crystal structure of a soluble CD28-Fab complex

Naive T cell activation requires signaling by the T cell receptor and by nonclonotypic cell surface receptors. The most important costimulatory protein is the monovalent homodimer CD28, which interacts with CD80 and CD86 expressed on antigen-presenting cells. Here we present the crystal structure of a soluble form of CD28 in complex with the Fab fragment of a mitogenic antibody. Structural comparisons redefine the evolutionary relationships of CD28-related proteins, antigen receptors and adhesion molecules and account for the distinct ligand-binding and stoichiometric properties of CD28 and the related, inhibitory homodimer CTLA-4. Cryo-electron microscopy–based comparisons of complexes of CD28 with mitogenic and nonmitogenic antibodies place new constraints on models of antibody-induced receptor triggering. This work completes the initial structural characterization of the CD28–CTLA-4–CD80–CD86 signaling system.

Structure and Interactions of the Human Programmed Cell Death 1 Receptor

Background: The inhibitory leukocyte receptor PD-1 binds two ligands, PD-L1 and PD-L2. Results: Nuclear magnetic resonance analysis and rigorous binding and thermodynamic measurements reveal the structure of, and the mode of ligand recognition by, PD-1. Conclusion: PD-L1 and PD-L2 bind differently to PD-1 and much more weakly than expected. Significance: Potent inhibitory signaling can be initiated by weakly interacting receptors. PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC′ sheet, which is flexible and completely lacks a C″ strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC′ sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1·ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3–4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.

An Early HIV Mutation within an HLA-B*57-Restricted T Cell Epitope Abrogates Binding to the Killer Inhibitory Receptor 3DL1

ABSTRACT Mutations within MHC class I-restricted epitopes have been studied in relation to T cell-mediated immune escape, but their impact on NK cells via interaction with killer Ig-like receptors (KIRs) during early HIV infection is poorly understood. In two patients acutely infected with HIV-1, we observed the appearance of a mutation within the B*57-restricted TW10 epitope (G9E) that did not facilitate strong escape from T cell recognition. The NK cell receptor KIR3DL1, carried by these patients, is known to recognize HLA-B*5703 and is associated with good control of HIV-1. Therefore, we tested whether the G9E mutation influenced the binding of HLA-B*5703 to soluble KIR3DL1 protein by surface plasmon resonance, and while the wild-type sequence and a second (T3N) variant were recognized, the G9E variant abrogated KIR3DL1 binding. We extended the study to determine the peptide sensitivity of KIR3DL1 interaction with epitopes carrying mutations near the C termini of TW10 and a second HLA-B*57-restricted epitope, IW9. Several amino acid changes interfered with KIR3DL1 binding, the most extreme of which included the G9E mutation commonly selected by HLA-B*57. Our results imply that during HIV-1 infection, some early-emerging variants could affect KIR-HLA interaction, with possible implications for immune recognition.

A human embryonic kidney 293T cell line mutated at the Golgi alpha-mannosidase II locus.

Disruption of Golgi α-mannosidase II activity can result in type II congenital dyserythropoietic anemia and induce lupus-like autoimmunity in mice. Here, we isolated a mutant human embryonic kidney (HEK) 293T cell line called Lec36, which displays sensitivity to ricin that lies between the parental HEK 293T cells, in which the secreted and membrane-expressed proteins are dominated by complex-type glycosylation, and 293S Lec1 cells, which produce only oligomannose-type N-linked glycans. Stem cell marker 19A was transiently expressed in the HEK 293T Lec36 cells and in parental HEK 293T cells with and without the potent Golgi α-mannosidase II inhibitor, swainsonine. Negative ion nano-electrospray ionization mass spectra of the 19A N-linked glycans from HEK 293T Lec36 and swainsonine-treated HEK 293T cells were qualitatively indistinguishable and, as shown by collision-induced dissociation spectra, were dominated by hybrid-type glycosylation. Nucleotide sequencing revealed mutations in each allele of MAN2A1, the gene encoding Golgi α-mannosidase II: a point mutation that mapped to the active site was found in one allele, and an in-frame deletion of 12 nucleotides was found in the other allele. Expression of the wild type but not the mutant MAN2A1 alleles in Lec36 cells restored processing of the 19A reporter glycoprotein to complex-type glycosylation. The Lec36 cell line will be useful for expressing therapeutic glycoproteins with hybrid-type glycans and as a sensitive host for detecting mutations in human MAN2A1 causing type II congenital dyserythropoietic anemia.

What Controls T Cell Receptor Phosphorylation

Document S1. Experimental Procedures and One FigurexDownload (.45 MB ) Document S1. Experimental Procedures and One Figure

The T cell surface--how well do we know it?

The overall degree of complexity of the T cell surface has been unclear, constraining our understanding of its biology. Using global gene expression analysis, we show that 111 of 374 genes encoding well-characterized leukocyte surface antigens are expressed by a resting cytotoxic T cell. Unexpectedly, of 97 stringently defined, T cell-specific transcripts with unknown functions that we identify, none encode proteins with the modular architecture characteristic of 80% of leukocyte surface antigens. Only two encode proteins with membrane topologies found exclusively in cell surface molecules. Our analysis indicates that the cell type-specific composition of the resting CD8+ T cell surface is now largely defined, providing an insight into the overall compositional complexity of the mammalian cell surface and a framework for formulating systematic models of T cell surface-dependent processes, such as T cell receptor triggering.

T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements.

Background: The T cell receptor (TCR) triggers signaling in T cells via an unknown mechanism. Results: The structure of the signaling subunit, CD3ϵ, is unchanged by signal-inducing antibodies, and mutations that would block intersubunit rearrangements do not affect signaling. Conclusion: Antibodies trigger TCR signaling without inducing large structural rearrangements. Significance: TCR triggering might generally occur in the absence of large structural rearrangements. Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3ϵ complexed with the mitogenic anti-CD3ϵ antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3ϵ from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3ϵ. Saturation mutagenesis of surface-exposed CD3ϵ residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3ϵ is highly exposed and reveals that no large new CD3ϵ interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.

Rigid-body ligand recognition drives cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor triggering

The inhibitory T-cell surface-expressed receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), which belongs to the class of cell surface proteins phosphorylated by extrinsic tyrosine kinases that also includes antigen receptors, binds the related ligands, B7-1 and B7-2, expressed on antigen-presenting cells. Conformational changes are commonly invoked to explain ligand-induced “triggering” of this class of receptors. Crystal structures of ligand-bound CTLA-4 have been reported, but not the apo form, precluding analysis of the structural changes accompanying ligand binding. The 1.8-Å resolution structure of an apo human CTLA-4 homodimer emphasizes the shared evolutionary history of the CTLA-4/CD28 subgroup of the immunoglobulin superfamily and the antigen receptors. The ligand-bound and unbound forms of both CTLA-4 and B7-1 are remarkably similar, in marked contrast to B7-2, whose binding to CTLA-4 has elements of induced fit. Isothermal titration calorimetry reveals that ligand binding by CTLA-4 is enthalpically driven and accompanied by unfavorable entropic changes. The similarity of the thermodynamic parameters determined for the interactions of CTLA-4 with B7-1 and B7-2 suggests that the binding is not highly specific, but the conformational changes observed for B7-2 binding suggest some level of selectivity. The new structure establishes that rigid-body ligand interactions are capable of triggering CTLA-4 phosphorylation by extrinsic kinase(s).

Molecular cloning and analysis of SSc5D, a new member of the scavenger receptor cysteine-rich superfamily

Glycoproteins of the scavenger receptor cysteine-rich (SRCR) superfamily contain one or more protein modules homologous to the membrane-distal domain of macrophage scavenger receptor I. These domains can be found in the extracellular regions of membrane proteins and in secreted glycoproteins, from the most primitive species to vertebrates. A systematic, bioinformatics-based search for putative human proteins related to the forty-seven known human group B SRCR domains identified a new family member that we have called Soluble Scavenger with 5 Domains (SSc5D). SSc5D is a new soluble protein whose expression is restricted to monocytes/macrophages and T-lymphocytes, and is particularly enriched in the placenta. The gene encoding SSc5D spans 30kb of genomic DNA, and contains fourteen exons producing a 4.8kb-long mRNA. The mature polypeptide is predicted to consist of 1573 amino acids comprising, towards the N-terminus, five very similar SRCR domains that are highly conserved among non-marsupial mammals, and a large (>250nm), very heavily glycosylated, mucin-like sequence towards the C-terminus. Each of the SRCR domains is encoded by a single exon, and contains eight cysteine residues, as observed for all other group B SRCR domains. A shorter isoform encoded by a weakly expressed, alternatively spliced transcript, which lacks the mucin-like C-terminal region, was also identified. It seems likely that SSc5D has a role at the interface between adaptive and innate immunity, or in placental function.