Psychosine variants as antigens for natural killer T cells.

Natural killer T (NKT) cells play a central role in the interface between innate and adaptive immunity, and alpha-galactosylceramide was recently shown to be an endogenous antigen for these cells. The source of alpha-galactosylceramide has not yet been determined; however, in vivo degradation of alpha-galactosylceramide involves generation of alpha-psychosine (alpha-galactosylsphingosine). Alpha-psychosine stimulates cytokine release from NKT cells and constitutes an endogenous antigen for these cells. Alpha-psychosine contains a single lipid chain, while most antigens for NKT cells have two lipid chains, and we have investigated if other glycolipids with one lipid chain, derived from know antigens for NKT cells, stimulate cytokine release from NKT cells. Only psychosine variants derived from the most potent NKT cell antigens cause stimulation, and this stimulation occurs in vitro as well as in vivo. Truncated forms of weak antigens for NKT cells are not stimulatory.

A peptide-free, liposome-based oligosaccharide vaccine, adjuvanted with a natural killer T cell antigen, generates robust antibody responses in vivo.

Due to the prevalence of oligo- and polysaccharides on the surfaces of pathogenic organisms, carbohydrates are primary targets for recognition by antibodies generated by the immune systems of higher organisms. Consequently, substantial effort has been expended in efforts to develop vaccines based on carbohydrate epitopes. Typical approaches involve multivalent presentation of carbohydrate targets on antigenic peptides or proteins, which often involve substantial synthetic commitments and/or vaccines that are heterogeneous and difficult to characterize. We have developed a simple, liposome-based approach to generate multivalent carbohydrate vaccines, and in place of an antigenic peptide or protein, we have used a potent antigen for natural killer T cells. This vaccine, based on the Streptococcus pneumoniae serotype 14 polysaccharide, gave a response superior to that from a clinically used vaccine (Prevnar). The dependence of this response on liposome formation was demonstrated by comparison to a simple mixture of the oligosaccharide and the natural killer T cell adjuvant. The importance of the strength of the adjuvant was observed by use of a potent synthetic adjuvant and a weaker, bacterial derived glycolipid adjuvant. These results demonstrate the effectiveness of this novel and relatively simple means of generating carbohydrate-based vaccines.

The crystal structure of CD8 in complex with YTS156.7.7 Fab and interaction with other CD8 antibodies define the binding mode of CD8 alphabeta to MHC class I.

The CD8alphabeta heterodimer interacts with class I pMHC on antigen-presenting cells as a co-receptor for TCR-mediated activation of cytotoxic T cells. To characterize this immunologically important interaction, we used monoclonal antibodies (mAbs) specific to either CD8alpha or CD8beta to probe the mechanism of CD8alphabeta binding to pMHCI. The YTS156.7 mAb inhibits this interaction and blocks T cell activation. To elucidate the molecular basis for this inhibition, the crystal structure of the CD8alphabeta immunoglobulin-like ectodomains were determined in complex with mAb YTS156.7 Fab at 2.7 A resolution. The YTS156.7 epitope on CD8beta was identified and implies that residues in the CDR1 and CDR2-equivalent loops of CD8beta are occluded upon binding to class I pMHC. To further characterize the pMHCI/CD8alphabeta interaction, binding of class I tetramers to CD8alphabeta on the surface of T cells was assessed in the presence of anti-CD8 mAbs. In contrast to YTS156.7, mAb YTS105.18, which is specific for CD8alpha, does not inhibit binding of CD8alphabeta to class I tetramers, indicating the YTS105.18 epitope is not occluded in the pMHCI/CD8alphabeta complex. Together, these data indicate a model for the pMHCI/CD8alphabeta interaction similar to that observed for CD8alphaalpha in the CD8alphaalpha/pMHCI complex, but in which CD8alpha occupies the lower orientation (membrane proximal to the antigen presenting cell), and CD8beta occupies the upper position (membrane distal). The implication of this molecular assembly for the function of CD8alphabeta in T cell activation is discussed.

Crystal structure of the TCR co-receptor CD8alphaalpha in complex with monoclonal antibody YTS 105.18 Fab fragment at 2.88 A resolution.

The CD8 glycoprotein functions as an essential element in the control of T-cell selection, maturation and the TCR-mediated response to peptide antigen. CD8 is expressed as both heterodimeric CD8alphabeta and homodimeric CD8alphaalpha isoforms, which have distinct physiological roles and exhibit tissue-specific expression patterns. CD8alphaalpha has previously been crystallized in complex with class I pMHC and, more recently, with the mouse class Ib thymic leukemia antigen (TL). Here, we present the crystal structure of a soluble form of mouse CD8alphaalpha in complex with rat monoclonal antibody YTS 105.18 Fab fragment at 2.88 A resolution. YTS 105.18, which is commonly used in the blockade of CD8+ T-cell activation in response to peptide antigen, is specific for mouse CD8alpha. The YTS 105.18 Fab is one of only five rat IgG Fab structures to have been reported to date. Analysis of the YTS 105.18 Fab epitope on CD8alpha reveals that this antibody blocks CD8 activity by hydrogen bonding to residues that are critical for interaction with both class I pMHC and TL. Structural comparison of the liganded and unliganded forms of soluble CD8alphaalpha indicates that the mouse CD8alphaalpha immunoglobulin-domain dimer does not undergo significant structural alteration upon interaction either with class I pMHC or TL.

Crystal structure of an isolated V(alpha) domain of the 2C T-cell receptor.

The T-cell receptor (TCR) is a heterodimeric cell-surface protein consisting of two chains, alpha and beta, each of which is composed of a variable (V) and a constant (C) domain. Crystals of the isolated V(alpha) domain of the murine TCR 2C were grown by serendipity from a solution containing the extracellular domains of the intact TCR 2C and CD3 gamma epsilon-chains. The V(alpha) crystal structure shows how crystal packing can substitute for another V(alpha) domain in a different fashion from that observed in V(alpha)/V(alpha) homodimer and V(alpha)/V(beta) heterodimer structures. Significant conformational changes occur in the CDR3 and beta(3)beta(4) loops that normally form part of the dimer interface. The monomeric V(alpha) domain provides the unique opportunity to study the effect of dimerization on the conformation of the unliganded complementarity-determining regions (CDR) of a TCR. This structure of an individual V(alpha) module has implications for stability and bioengineering of isolated antibody and immunoglobulin domains.

Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory.

Emerging evidence indicates that CD8+ and CD4+ T-cell immunity is differentially regulated. Here we have delineated differences and commonalities among antiviral T-cell responses by enumeration and functional profiling of eight specific CD8+ and CD4+ T-cell populations during primary, memory and recall responses. A high degree of coordinate regulation among all specific T-cell populations stood out against an approximately 20-fold lower peak expansion and prolonged contraction phase of specific CD4+ T-cell populations. Surprisingly, although CD8+ T-cell memory was stably maintained for life, levels of specific CD4+ memory T cells gradually declined. However, this decay, which seemed to result from less efficient rescue from apoptosis, did not affect functionality of surviving virus-specific CD4+ T cells. Our results indicate that CD4+ T-cell memory might become limiting under physiological conditions and that conditions precipitating CD4+ T-cell loss might compromise protective immunity even in the presence of unimpaired CD8+ T-cell responses.

The crystal structures of K(bm1) and K(bm8) reveal that subtle changes in the peptide environment impact thermostability and alloreactivity.

The K(bm1) and K(bm8) natural mutants of the murine MHC class I molecule H-2K(b) were originally identified by allograft rejection. They also bind viral peptides VSV8 and SEV9 with high affinity, but their peptide complexes have substantially decreased thermostability, and the K(bm1) complexes do not elicit alloreactive T cell responses. Crystal structures of the four mutant complexes at 1.7-1.9 A resolution are similar to the corresponding wild-type K(b) structures, except in the vicinity of the mutated residues, which alter the electrostatic potential, topology, hydrogen bonding, and local water structure of the peptide binding groove. Thus, these natural K(b) mutations define the minimal perturbations in the peptide environment that alter antigen presentation to T cells and abolish alloreactivity.

The mouse CD1d-restricted repertoire is dominated by a few autoreactive T cell receptor families.

To define the phenotype and T cell receptor (TCR) repertoire of CD1d-dependent T cells, we compared the populations of T cells that persisted in major histocompatibility complex (MHC)-deficient mice, which lack mainstream T cells, with those from MHC/CD1d doubly deficient mice, which lack both mainstream and CD1d-dependent T cells. Surprisingly, up to 80% of the CD1d-dependent T cells were stained by tetramers of CD1d/alpha-galactosylceramide, which specifically identify the previously described CD1d autoreactive Valpha14-Jalpha18/Vbeta8 natural killer (NK) T cells. Furthermore, zooming in on the CD1d-dependent non-Valpha14 T cells, we found that, like Valpha14 NK T cells, they mainly expressed recurrent, CD1d autoreactive TCR families and had a natural memory phenotype. Thus, CD1d-restricted T cells differ profoundly from MHC-peptide-specific T cells by their predominant use of autoreactive and semiinvariant, rather than naive and diverse, TCRs. They more closely resemble other lineages of innate lymphocytes such as B-1 B cells, gammadelta T cells, and NK cells, which express invariant or semiinvariant autoreactive receptors. Finally, we demonstrate that the MHC-restricted TCR repertoire is essentially non-cross-reactive to CD1d. Altogether, these findings imply that lipid recognition by CD1d-restricted T cells may have largely evolved as an innate rather than an adaptive arm of the mouse immune system.

Crystal structure of the murine NK cell-activating receptor NKG2D at 1.95 A.

NKG2D, a homodimeric lectin-like receptor, is a unique stimulatory molecule that is found on natural killer cells,T cells and activated macrophages. The natural ligands for murine NKG2D are distant major histocompatibility complex homologs, retinoic acid early transcript (Rae1) and H-60 minor histocompatibility antigen. The crystal structure of the extracellular region of murine NKG2D reveals close homology with other C-type lectin receptors such as CD94, Ly49A, rat MBP-A and CD69. However, the precise mode of dimeric assembly varies among these natural killer receptors, as well as their surface topography and electrostatic properties. The NKG2D structure provides the first structural insights into the role and ligand specificity of this stimulatory receptor in the innate and adaptive immune system.

The I-Ag7 MHC class II molecule linked to murine diabetes is a promiscuous peptide binder.

Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-Ag7, shares a common alpha-chain with I-Ad but has a peculiar beta-chain. As with most beta-chain alleles linked to diabetes susceptibility, I-Ag7 contains a nonaspartic residue at position beta57. We have produced large amounts of empty I-Ag7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phage-displayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-Ag7. Structural information was critical in evaluating the binding studies. I-Ag7, like I-Ad, appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-Ag7 over I-Ad as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-Ad and I-Ag7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the beta-chain. These characteristics link I-Ag7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.

In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers.

The CD1 family of major histocompatibility complex (MHC)-like molecules specializes in presenting lipid and glycolipid antigens to alpha/beta T lymphocytes, but little is known about the size of the CD1-restricted T cell population or the frequency of T lymphocytes specific for a given glycolipid antigen. Here, we report the generation and use of mouse CD1d1-glycolipid tetramers to visualize CD1d-restricted T cells. In contrast with previous BIAcore-based estimates of very short half-lives for CD1d-glycolipid complexes, we found that the dissociation rate of several different CD1d-glycolipid complexes was very slow. Fluorescent tetramers of mouse CD1d1 complexed with alpha-galactosylceramide (alphaGalCer), the antigen recognized by mouse Valpha14-Jalpha281/Vbeta8 and human Valpha24-JalphaQ/Vbeta11 natural killer T (NKT) cell T cell receptors (TCRs), allowed us for the first time to accurately describe, based on TCR specificity, the entire population of NKT cells in vivo and to identify a previously unrecognized population of NK1.1-negative "NKT" cells, which expressed a different pattern of integrins. In contrast, natural killer (NK) cells failed to bind the tetramers either empty or loaded with alphaGalCer, suggesting the absence of a CD1d-specific, antigen-nonspecific NK receptor. Mouse CD1d1-alphaGalCer tetramers also stained human NKT cells, indicating that they will be useful for probing a range of mouse and human conditions such as insulin-dependent diabetes mellitus, tumor rejection, and infectious diseases where NKT cells play an important role.

Function and dysfunction of T cell receptor: structural studies.

The engagement of the T cell receptor (TCR) to its ligand, the major histocompatibility complex (MHC)-peptide complex, leads to T cell activation. The molecular mechanisms leading to this activation are still unknown. Dimerization or substantial conformational changes following TCR ligation have not been observed by classical biochemical methods or by X-ray crystallography of the TCR/MHC complex. However, most of these experiments have used reductionist approaches in which only MHC and TCR molecules were taken into account. In fact, the TCR is only one of many molecules forming the TCR complex (TCRC), and the interplay among the components of this larger complex have not been studied in depth. The reconstitution of a complete TCRC using recombinant molecules is our goal and will be the first step to new structural and functional studies.

A structural framework for deciphering the link between I-Ag7 and autoimmune diabetes.

Susceptibility to murine and human insulin-dependent diabetes mellitus correlates strongly with major histocompatibility complex (MHC) class II I-A or HLA-DQ alleles that lack an aspartic acid at position beta57. I-Ag7 lacks this aspartate and is the only class II allele expressed by the nonobese diabetic mouse. The crystal structure of I-Ag7 was determined at 2.6 angstrom resolution as a complex with a high-affinity peptide from the autoantigen glutamic acid decarboxylase (GAD) 65. I-Ag7 has a substantially wider peptide-binding groove around beta57, which accounts for distinct peptide preferences compared with other MHC class II alleles. Loss of Asp(beta57) leads to an oxyanion hole in I-Ag7 that can be filled by peptide carboxyl residues or, perhaps, through interaction with the T cell receptor.

A population of murine gammadelta T cells that recognize an inducible MHC class Ib molecule.

Although gammadelta T cells are implicated in regulating immune responses, gammadelta T cell-ligand pairs that could mediate such regulatory functions have not been identified. Here, the expression of the major histocompatibility complex (MHC) class Ib T22 and the closely related T10 molecules is shown to be activation-induced, and they confer specificity to about 0.4% of the gammadelta T cells in normal mice. Thus, the increased expression of T22 and/or T10 might trigger immunoregulatory gammadelta T cells during immune responses. Furthermore, the fast on-rates and slow off-rates that characterize this receptor/ligand interaction would compensate for the low ligand stability and suggest a high threshold for gammadelta T cell activation.

A functional hot spot for antigen recognition in a superagonist TCR/MHC complex.

A longstanding question in T cell receptor signaling is how structurally similar ligands, with similar affinities, can have substantially different biological activity. The crystal structure of the 2C TCR complex of H-2Kb with superagonist peptide SIYR at 2.8 A elucidates a structural basis for TCR discrimination of altered peptide ligands. The difference in antigen potency is modulated by two cavities in the TCR combining site, formed mainly by CDRs 3alpha, 3beta, and 1beta, that complement centrally located peptide residues. This "functional hot spot" allows the TCR to finely discriminate amongst energetically similar interactions within different ligands for those in which the peptide appropriately stabilizes the TCR/pMHC complex and provides a new structural perspective for understanding differential signaling resulting from T cell cross-reactivity.

Structural basis of T cell recognition.

Exciting breakthroughs in the last two years have begun to elucidate the structural basis of cellular immune recognition. Crystal structures have been determined for full-length and truncated forms of alpha beta T cell receptor (TCR) heterodimers, both alone and in complex with their peptide-MHC (pMHC) ligands or with anti-TCR antibodies. In addition, a truncated CD8 coreceptor has been visualized with a pMHC. Aided in large part by the substantial body of knowledge accumulated over the last 25 years on antibody structure, a number of general conclusions about TCR structure and its recognition of antigen can already be derived from the relatively few TCR structures that have been determined. Small, but important, variations between TCR and antibody structures bear on their functional differences as well as on their specific antigen recognition requirements. As observed in antibodies, canonical CDR loop structures are already emerging for some of the TCR CDR loops. Highly similar docking orientations of the TCR V alpha domains in the TCR/pMHC complex appear to play a primary role in dictating orientation, but the V beta positions diverge widely. Similar TCR contact positions, but whose exact amino acid content can vary, coupled with relatively poor interface shape complementarity, may explain the flexibility and short half-lives of many TCR interactions with pMHC. Here we summarize the current state of this field, and suggest that the knowledge gap between the three-dimensional structure and the signaling function of the TCR can be bridged through a synthesis of molecular biological and biophysical techniques.

Structure and function of a membrane-bound murine MHC class I molecule.

MHC molecules are expressed at the surface of nucleated cells to present peptides to T cells. Structural information on MHC molecules has been gathered by x-ray crystallography techniques by using soluble proteins. Although relationships between MHC molecules and cell membranes have not been studied in detail, they are of critical importance for T cell recognition. Using a chemically modified lipid, we have been able to capture and orient histidine-tagged MHC molecules on lipid membranes. Surface plasmon resonance experiments show that the protein binds to the nickel lipid in a specific manner and in an oriented fashion, which allows T cell receptor binding. Similar lipid surfaces have been used to grow two-dimensional crystals and to determine the structure of a membrane-anchored murine H-2Kb MHC class I molecule. The docking of the crystallographic structure into the three-dimensional reconstructed structure derived from the two-dimensional crystals allows us to determine that the histidine tag is near the membrane surface and that the MHC molecule is in an upright position, exposing the peptide/alpha1-alpha2 domains toward the T cell.

Selection of functional T cell receptor mutants from a yeast surface-display library.

The heterodimeric alphabeta T cell receptor (TCR) for antigen is the key determinant of T cell specificity. The structure of the TCR is very similar to that of antibodies, but the engineering of TCRs by directed evolution with combinatorial display libraries has not been accomplished to date. Here, we report that yeast surface display of a TCR was achieved only after the mutation of specific variable region residues. These residues are located in two regions of the TCR, at the interface of the alpha- and beta-chains and in the beta-chain framework region that is thought to be in proximity to the CD3 signal-transduction complex. The mutations are encoded naturally in many antibody variable regions, indicating specific functional differences that have not been appreciated between TCRs and antibodies. The identification of these residues provides an explanation for the inherent difficulties in the display of wild-type TCRs compared with antibodies. Yeast-displayed mutant TCRs bind specifically to the peptide/MHC antigen, enabling engineering of soluble T cell receptors as specific T cell antagonists. This strategy of random mutagenesis followed by selection for surface expression may be of general use in the directed evolution of other eukaryotic proteins that are refractory to display.

Effects of complementarity determining region mutations on the affinity of an alpha/beta T cell receptor: measuring the energy associated with CD4/CD8 repertoire skewing.

It has been proposed that the generally low affinities of T cell receptors (TCRs) for their peptide-major histocompatibility complex (pMHC) ligands (Kd approximately 10(-4) to 10(-7) M) are the result of biological selection rather than an intrinsic affinity limitation imposed by the TCR framework. Using a soluble version of the 2C TCR, we have used complementarity determining region (CDR)-directed mutagenesis to investigate whether the affinity of this receptor for its allogeneic pMHC ligand can be improved upon. We report that several mutants at positions lying within CDR3alpha and CDR2beta showed increased affinities for pMHC compared with the wild-type receptor. Additionally, we have investigated whether Valpha mutations that have been implicated in the phenomenon of CD8(+) repertoire skewing achieve this skewing by means of generalized increases in affinity for MHC-I molecules. Two mutants (S27F and S51P), which each promote skewing toward a CD8(+) phenotype, exhibited significantly reduced affinity for pMHC-I, consistent with a quantitative-instructional model of CD4/CD8 lineage commitment. This model predicts that CD8 is downregulated on thymocytes that have TCR-ligand interactions above a minimal energy threshold. Together, the results (a) demonstrate that engineering higher affinity TCRs is feasible, and (b) provide TCR-pMHC energy values associated with CD4/CD8 repertoire skewing.

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells.

Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC). At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR), whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal 1 to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal 1 alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-1 can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and ICAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.