Keith G. Gould

T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand

The binding of a T-cell antigen receptor (TCR) to peptide antigen presented by major histocompatibility antigens (pMHC) on antigen-presenting cells (APCs) is a central event in adaptive immune responses. The mechanism by which TCR–pMHC ligation initiates signalling, a process termed TCR triggering, remains controversial. It has been proposed that TCR triggering is promoted by segregation at the T cell–APC interface of cell-surface molecules with small ectodomains (such as TCR–pMHC and accessory receptors) from molecules with large ectodomains (such as the receptor protein tyrosine phosphatases CD45 and CD148). Here we show that increasing the dimensions of the TCR–pMHC interaction by elongating the pMHC ectodomain greatly reduces TCR triggering without affecting TCR–pMHC ligation. A similar dependence on receptor–ligand complex dimensions was observed with artificial TCR–ligand systems that span the same dimensions as the TCR–pMHC complex. Interfaces between T cells and APCs expressing elongated pMHC showed an increased intermembrane separation distance and less depletion of CD45. These results show the importance of the small size of the TCR–pMHC complex and support a role for size-based segregation of cell-surface molecules in TCR triggering.

Lipoprotein Access to MHC Class I Presentation During Infection of Murine Macrophages with Live Mycobacteria

Following uptake by macrophages, live mycobacteria initially reside within an immature phagosome that resists acidification and retains access to recycling endosomes. Glycolipids are exported from the mycobacterial phagosome and become available for immune recognition by CD1-restricted T cells. The aim of this study was to explore the possibility that lipoproteins might similarly escape from the phagosome and act as immune targets in cells infected with live mycobacteria. We have focused on a 19-kDa lipoprotein from Mycobacterium tuberculosis that was previously shown to be recognized by CD8+ T cells. The 19-kDa Ag was found to traffic separately from live mycobacteria within infected macrophages by a pathway that was dependent on acylation of the protein. When expressed as a recombinant protein in rapid-growing mycobacteria, the 19-kDa Ag was able to deliver peptides for recognition by MHC class I-restricted T cells by a TAP-independent mechanism. Entry into the class I pathway was rapid, dependent on acylation, and could be blocked by killing the mycobacteria by heating before infection. Although the pattern of 19-kDa trafficking was similar with different mycobacterial species, preliminary experiments suggest that class I presentation is more efficient during infection with rapid-growing mycobacteria than with the slow-growing bacillus Calmette-Guérin vaccine strain.

Controlled intracellular release of peptides from microcapsules enhances antigen presentation on MHC class I molecules.

To understand the time course of action of any small molecule inside a single cell, one would deposit a defined amount inside the cell and initiate its activity at a defined moment. An elegant way to achieve this is to encapsulate the molecule in a micrometer-sized reservoir, introduce it into a cell, remotely open its wall by a laser pulse, and then follow the biological response by microscopy. The validity of this approach is validated here using microcapsules with defined walls that are doped with metallic nanoparticles so as to enable them to be opened with an infrared laser. The capsules are loaded with a fluorescent antigenic peptide and introduced into mammalian cultured cells where, upon laser-induced release, the peptide binds to major histocompatibility complex (MHC) class I proteins and elicits their cell surface transport. The concept of releasing a drug inside a cell and following its action is applicable to many problems in cell biology and medicine.

Human Cell Lymphotropic Virus (HTLV) Type-l-Specific CD8+ Cells: Frequency and Immunodominance Hierarchy

Human T cell lymphotropic virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We used interferon- gamma enzyme-linked immunospot assays with overlapping peptides spanning the entire HTLV-1 proteome to test whether the HTLV-1-specific CD8(+) T cells differed significantly in frequency or immunodominance hierarchy between patients with HAM/TSP and asymptomatic carriers and whether the frequency correlated with provirus load. Tax was the immunodominant target antigen. There was no significant qualitative or quantitative difference in the HTLV-1-specific CD8(+) T cell response between the 2 groups. Virus-specific CD8(+) T cell frequency alone does not indicate the effectiveness of the cytotoxic T lymphocyte response in controlling provirus load at equilibrium.

High Circulating Frequencies of Tumor Necrosis Factor Alpha- and Interleukin-2-Secreting Human T-Lymphotropic Virus Type 1 (HTLV-1)-Specific CD4+ T Cells in Patients with HTLV-1-Associated Neurological Disease

ABSTRACT Significantly higher frequencies of tumor necrosis factor alpha- and interleukin-2-secreting human T-lymphotropic virus type 1 (HTLV-1)-specific CD4+ T cells were present in the peripheral blood mononuclear cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients than in those of asymptomatic carriers with similar provirus loads. The data suggest that HTLV-1-specific CD4+ T cells play a role in the pathogenesis of HAM/TSP.

Matched sizes of activating and inhibitory receptor/ligand pairs are required for optimal signal integration by human natural killer cells.

It has been suggested that receptor-ligand complexes segregate or co-localise within immune synapses according to their size, and this is important for receptor signaling. Here, we set out to test the importance of receptor-ligand complex dimensions for immune surveillance of target cells by human Natural Killer (NK) cells. NK cell activation is regulated by integrating signals from activating receptors, such as NKG2D, and inhibitory receptors, such as KIR2DL1. Elongating the NKG2D ligand MICA reduced its ability to trigger NK cell activation. Conversely, elongation of KIR2DL1 ligand HLA-C reduced its ability to inhibit NK cells. Whereas normal-sized HLA-C was most effective at inhibiting activation by normal-length MICA, only elongated HLA-C could inhibit activation by elongated MICA. Moreover, HLA-C and MICA that were matched in size co-localised, whereas HLA-C and MICA that were different in size were segregated. These results demonstrate that receptor-ligand dimensions are important in NK cell recognition, and suggest that optimal integration of activating and inhibitory receptor signals requires the receptor-ligand complexes to have similar dimensions.

Peptide-Major Histocompatibility Complex Dimensions Control Proximal Kinase-Phosphatase Balance during T Cell Activation

T cell antigen recognition requires binding of the T cell receptor (TCR) to a complex between peptide antigen and major histocompatibility complex molecules (pMHC), and this recognition occurs at the interface between the T cell and the antigen-presenting cell. The TCR and pMHC molecules are small compared with other abundant cell surface molecules, and it has been suggested that small size is functionally important. We show here that elongation of both mouse and human MHC class I molecules abrogates T cell antigen recognition as measured by cytokine production and target cell killing. This elongation disrupted tyrosine phosphorylation and Zap70 recruitment at the contact region without affecting TCR or coreceptor binding. Contact areas with elongated forms of pMHC showed an increase in intermembrane distance and less efficient segregation of CD3 from the large tyrosine phosphatase CD45. These findings demonstrate that T cell antigen recognition is strongly dependent on pMHC size and are consistent with models of TCR triggering requiring segregation or mechanical pulling of the TCR.

Hepatitis B virus core antigen epitopes presented by HLA-A2 single-chain trimers induce functional epitope-specific CD8+ T-cell responses in HLA-A2·1/Kb transgenic mice

The potency of CD8+ cytotoxic T lymphocyte (CTL) responses toward core antigen has been shown to affect the outcomes of hepatitis B virus (HBV) infection. Since single‐chain trimers (SCT) composed of peptide epitope β2‐microglobulin (β2m) and major histocompatiblity complex (MHC) class I heavy chain covalently linked together in a single molecule have been shown to stimulate efficient CTL responses, we investigated the properties of human leucocyte antigen (HLA)‐A2 SCTs encoding the HBV core antigen (HBcAg) epitopes C18−27 and C107−115. Transfection of NIH‐3T3 cells with pcDNA3.0‐SCT‐C18−27 and SCT‐C107−115 leads to stable presentation of HBcAg epitopes at the cell surface. HLA‐A2.1/Kb transgenic mice vaccinated with the SCT constructs, either as a DNA vaccine alone or followed by a boost with recombinant vaccinia virus, were shown to generate HBcAg‐specific CTL responses by enzyme‐linked immunospot assay (ELISPOT) and in vitro interferon‐γ release experiments. HBcAg‐specific CTLs from vaccinated HLA‐A2.1/Kb transgenic mice were able to inhibit HBV surface and e antigen expression as indicated by HepG2.2.15 cells. Our data indicate that a DNA vaccine encoding a human HLA‐A2 SCT with HBV epitopes can lead to stable, enhanced HBV core antigen presentation, and may be useful for the control of HBV infection in HLA‐A2‐positive HBV carriers.

Competition Between MHC Class I Alleles for Cell Surface Expression Alters CTL Responses to Influenza A Virus

Mammalian cells express up to six different MHC class I alleles, many of which differ in terms of their interaction with components of the Ag presentation pathway and level of cell surface expression. However, it is often assumed in Ag presentation studies that class I alleles function independently of each other. We have compared cell surface expression levels and function of MHC class I molecules in F1 hybrid mice with those in the homozygous parental strains. The level of cell surface expression of certain alleles in F1 mice differed significantly from 50% of that found on the same cell type in the corresponding parental strain, suggesting allele-specific competition for cell surface expression, and not expression solely according to gene dosage. The strongest effect was observed in H-2b × H-2k F1 mice, in which the H-2b class I molecules dominated over the H-2k class I molecules. The magnitude of H-2k-restricted CTL responses to influenza A virus infection was similar in the F1 hybrid and parental H-2k mice. However, in H-2k mice expressing a Kb transgene, cell surface levels of the endogenous class I molecules were down-regulated to a greater degree than in F1 hybrid mice, and H-2k-restricted CTL responses against influenza A virus were greatly reduced, although the CTL repertoire was apparently present. Therefore, certain MHC class I molecules compete with each other for cell surface expression, and the resulting low cell surface expression of specific alleles can lead to a severe reduction in the ability to generate a CTL response.

Novel detection of in vivo HLA–B27 conformations correlates with ankylosing spondylitis association

OBJECTIVE The class I major histocompatibility complex (MHC) molecule HLA-B27 exhibits a strong association with the autoimmune inflammatory arthritis disorder ankylosing spondylitis (AS) and with other related spondylarthropathies. In the absence of both a defined autoimmune response and a target autoantigen(s), the propensity of HLA-B27 to misfold has been hypothesized to be a major parameter in disease pathogenesis. We undertook this study to test the hypothesis that HLA-B27 misfolding is due to exposure of cysteine residues within the heavy chain to the oxidizing environment of the endoplasmic reticulum. METHODS A rapid acidification and alkylation modification method was used to examine cysteine residue exposure and accessibility within AS-associated and non-AS-associated HLA-B27 subtypes. RESULTS This novel approach to probing in vivo class I MHC structure revealed that the HLA-B27 heavy chain adopts conformations not previously described. Furthermore, amino acid residues specific to subtypes HLA-B*2706, B*2709, and B*2704 can have an impact on these novel conformations and on cysteine residue exposure. CONCLUSION HLA-B27 can adopt novel conformations, resulting in differential accessibility of cysteine residues, which can explain the propensity to misfold. Cysteine exposure in the HLA-B27 heavy chain is also affected by residues within the 114 and 116 regions, thereby providing a potential biochemical basis for the association of HLA-B27 subtypes with AS.