Stanley G. Nathenson

Identification of the β cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes

Type 1 diabetes is an autoimmune disease in which autoreactive T cells attack and destroy the insulin-producing pancreatic β cells. CD8+ T cells are essential for this β cell destruction, yet their specific antigenic targets are largely unknown. Here, we reveal that the autoantigen targeted by a prevalent population of pathogenic CD8+ T cells in nonobese diabetic mice is islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Through tetramer technology, IGRP-reactive T cells are readily detected in islets and peripheral blood directly ex vivo. The human IGRP gene maps to a diabetes susceptibility locus, suggesting that IGRP also may be an antigen for pathogenic T cells in human type 1 diabetes and, thus, a new, potential target for diagnostic and therapeutic approaches.

Efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex

Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide–major histo-compatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen. These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.

Structural basis for co-stimulation by the human CTLA-4/B7-2 complex

Regulation of T-cell activity is dependent on antigen-independent co-stimulatory signals provided by the disulphide-linked homodimeric T-cell surface receptors, CD28 and CTLA-4 (ref. 1). Engagement of CD28 with B7-1 and B7-2 ligands on antigen-presenting cells (APCs) provides a stimulatory signal for T-cell activation, whereas subsequent engagement of CTLA-4 with these same ligands results in attenuation of the response. Given their central function in immune modulation, CTLA-4- and CD28-associated signalling pathways are primary therapeutic targets for preventing autoimmune disease, graft versus host disease, graft rejection and promoting tumour immunity. However, little is known about the cell-surface organization of these receptor/ligand complexes and the structural basis for signal transduction. Here we report the 3.2-Å resolution structure of the complex between the disulphide-linked homodimer of human CTLA-4 and the receptor-binding domain of human B7-2. The unusual dimerization properties of both CTLA-4 and B7-2 place their respective ligand-binding sites distal to the dimer interface in each molecule and promote the formation of an alternating arrangement of bivalent CTLA-4 and B7-2 dimers that extends throughout the crystal. Direct observation of this CTLA-4/B7-2 network provides a model for the periodic organization of these molecules within the immunological synapse and suggests a distinct mechanism for signalling by dimeric cell-surface receptors.

The three-dimensional structure of H-2Db at 2.4 A resolution: implications for antigen-determinant selection

Solution at 2.4 A resolution of the structure of H-2Db with the influenza virus peptide NP366-374 (ASNEN-METM) and comparison with the H-2Kb-VSV (RGY-VYQGL) structure allow description of the molecular details of MHC class I peptide binding interactions for mice of the H-2b haplotype, revealing a strategy that maximizes the repertoire of peptides than can be presented. The H-2Db cleft has a mouse-specific hydrophobic ridge that causes a compensatory arch in the backbone of the peptide, exposing the arch residues to TCR contact and requiring the peptide to be at least 9 residues. This ridge occurs in about 40% of the known murine D and L allelic molecules, classifying them as a structural subgroup.

Three cDNA clones encoding mouse transplantation antigens: Homology to immunoglobulin genes

We constructed cDNA libraries from poly(A)+ RNA isolated from cell lines of two different inbred strains of mice, and screened the libraries with a cDNA clone encoding a human transplantation antigen. Three cDNA clones were identified, sequenced and found to encode amino acid sequences highly homologous to portions of a known mouse transplantation antigen. Comparison of the cDNA sequences of mouse transplantation antigens with the constant region domains of the mouse immunoglobulin mu gene reveals a striking homology, which suggests that the two genes share a common ancestor. Antibody genes undergo DNA rearrangement during B cell differentiation that are correlated with their expression. In contrast, DNA blots with these cDNA probes suggest that the genes for the transplantation antigens are not rearranged in the genomes of liver or embryo cells, which express these antigens, as compared with sperm cells, which do not express these antigens. In Bam Hl-digested liver DNAs from different inbred strains of mice, 10-15 bands of hybridization were found. Accordingly, the genes encoding the transplantation antigens appear to constitute a multigene family with similar gene numbers in different mice.

Structural and functional analysis of the costimulatory receptor programmed death-1.

PD-1, a member of the CD28/CTLA-4/ICOS costimulatory receptor family, delivers negative signals that have profound effects on T and B cell immunity. The 2.0 A crystal structure of the extracellular domain of murine PD-1 reveals an Ig V-type topology with overall similarity to the CTLA-4 monomer; however, there are notable differences in regions relevant to function. Our structural and biophysical data show that PD-1 is monomeric both in solution as well as on cell surface, in contrast to CTLA-4 and other family members that are all disulfide-linked homodimers. Furthermore, our structure-based mutagenesis studies identify the ligand binding surface of PD-1, which displays significant differences compared to those present in the other members of the family.

Atomic structure of an alphabeta T cell receptor (TCR) heterodimer in complex with an anti-TCR fab fragment derived from a mitogenic antibody.

Each T cell receptor (TCR) recognizes a peptide antigen bound to a major histocompatibility complex (MHC) molecule via a clonotypic αβ heterodimeric structure (Ti) non‐covalently associated with the monomorphic CD3 signaling components. A crystal structure of an αβ TCR‐anti‐TCR Fab complex shows an Fab fragment derived from the H57 monoclonal antibody (mAb), interacting with the elongated FG loop of the Cβ domain, situated beneath the Vβ domain. This loop, along with the partially exposed ABED β sheet of Cβ, and glycans attached to both Cβ and Cα domains, forms a cavity of sufficient size to accommodate a single non‐glycosylated Ig domain such as the CD3ϵ ectodomain. That this asymmetrically localized site is embedded within the rigid constant domain module has implications for the mechanism of signal transduction in both TCR and pre‐TCR complexes. Furthermore, quaternary structures of TCRs vary significantly even when they bind the same MHC molecule, as manifested by a unique twisting of the V module relative to the C module.

Programmed death-1 (PD-1)–deficient mice are extraordinarily sensitive to tuberculosis

The programmed death-1 (PD-1) costimulatory receptor inhibits T and B cell responses and plays a crucial role in peripheral tolerance. PD-1 has recently been shown to inhibit T cell responses during chronic viral infections such as HIV. In this study, we examined the role of PD-1 in infection with Mycobacterium tuberculosis, a common co-infection with HIV. PD-1–deficient mice showed dramatically reduced survival compared with wild-type mice. The lungs of the PD-1−/− mice showed uncontrolled bacterial proliferation and focal necrotic areas with predominantly neutrophilic infiltrates, but a lower number of infiltrating T and B cells. Proinflammatory cytokines, such as TNF-α, IL-1, and especially IL-6 and IL-17 were significantly increased in the lung and sera of infected PD-1−/− mice, consistent with an aberrant inflammation. Microarray analysis of the lungs infected with M. tuberculosis showed dramatic differences between PD-1−/− and control mice. Using high-stringency analysis criteria (changes of twofold or greater), 367 transcripts of genes were differentially expressed between infected PD-1−/− and wild-type mice, resulting in profoundly altered inflammatory responses with implications for both innate and adaptive immunity. Overall, our studies show that the PD-1 pathway is required to control excessive inflammatory responses after M. tuberculosis infection in the lungs.

Identification of a CD8 T Cell That Can Independently Mediate Autoimmune Diabetes Development in the Complete Absence of CD4 T Cell Helper Functions

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic β cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic β cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4αβ Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted β cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.

Evidence that multiple residues on both the α-helices of the class I MHC molecule are simultaneously recognized by the T cell receptor

Single amino acid substitutions at nine different positions on the H-2Kb molecules from in vitro-mutagenized, immunologically altered, somatic cell variants were correlated with their patterns of recognition by monoclonal antibodies (MAbs) and allogeneic cytotoxic T lymphocyte (CTL) clones. While MAbs were found to detect spatially discrete, domain-specific sites, CTLs interacted simultaneously with multiple residues on the alpha 1 and alpha 2 domains of the Kb molecule. The computer graphic three-dimensional Kb model structure showed that, of the seven CTL-specific residues analyzed, six residues were located on the alpha-helical regions of the two domains. Every CTL clone was found to interact with a distinct pattern of residues composed of a specific subset of the CTL-specific residues.