David H. Margulies

A simple method for polyethylene glycol-promoted hybridization of mouse myeloma cells.

A simple method is described for promoting the fusion of mouse myeloma cells in suspension with polyethylene glycol (PEG 1000). By carefully controlling the concentration of PEG and the time of exposure of the cells, it was possible to obtain hybridization frequencies several-hundred-fold higher than those obtained with Sendai virus.

T cell receptor-MHC class I peptide interactions: affinity, kinetics, and specificity

The critical discriminatory event in the activation of T lymphocytes bearing alpha beta T cell receptors (TCRs) is their interaction with a molecular complex consisting of a peptide bound to a major histocompatibility complex (MHC)-encoded class I or class II molecule on the surface of an antigen-presenting cell. The kinetics of binding were measured of a purified TCR to molecular complexes of a purified soluble analog of the murine MHC class I molecule H-2Ld (sH-2Ld) and a synthetic octamer peptide p2CL in a direct, real-time assay based on surface plasmon resonance. The kinetic dissociation rate of the MHC-peptide complex from the TCR was rapid (2.6 x 10(-2) second-1, corresponding to a half-time for dissociation of approximately 27 seconds), and the kinetic association rate was 2.1 x 10(5) M-1 second-1. The equilibrium constant for dissociation was approximately 10(-7) M. These values indicate that TCRs must interact with a multivalent array of MHC-peptide complexes to trigger T cell signaling.

Enhanced Antigen-Specific Antitumor Immunity with Altered Peptide Ligands that Stabilize the MHC-Peptide-TCR Complex

T cell responsiveness to an epitope is affected both by its affinity for the presenting MHC molecule and the affinity of the MHC-peptide complex for TCR. One limitation of cancer immunotherapy is that natural tumor antigens elicit relatively weak T cell responses, in part because high-affinity T cells are rendered tolerant to these antigens. We report here that amino acid substitutions in a natural MHC class I-restricted tumor antigen that increase the stability of the MHC-peptide-TCR complex are significantly more potent as tumor vaccines. The improved immunity results from enhanced in vivo expansion of T cells specific for the natural tumor epitope. These results indicate peptides that stabilize the MHC-peptide-TCR complex may provide superior antitumor immunity through enhanced stimulation of specific T cells.

Crystal structure of a lectin-like natural killer cell receptor bound to its MHC class I ligand.

Natural killer (NK) cell function is regulated by NK receptors that interact with MHC class I (MHC-I) molecules on target cells. The murine NK receptor Ly49A inhibits NK cell activity by interacting with H-2Dd through its C-type-lectin-like NK receptor domain. Here we report the crystal structure of the complex between the Ly49A NK receptor domain and unglycosylated H-2Dd. The Ly49A dimer interacts extensively with two H-2Dd molecules at distinct sites. At one interface, a single Ly49A subunit contacts one side of the MHC-I peptide-binding platform, presenting an open cavity towards the conserved glycosylation site on the H-2Dd α2 domain. At a second, larger interface, the Ly49A dimer binds in a region overlapping the CD8-binding site. The smaller interface probably represents the interaction between Ly49A on the NK cell and MHC-I on the target cell, whereas the larger one suggests an interaction between Ly49A and MHC-I on the NK cell itself. Both Ly49A binding sites on MHC-I are spatially distinct from that of the T-cell receptor.

A targeted glucocorticoid receptor antisense transgene increases thymocyte apoptosis and alters thymocyte development

The exquisite sensitivity of thymocytes to steroid-induced apoptosis, the steroidogenic potential of thymic epithelial cells, and the ability of steroid synthesis inhibitors to enhance antigen-specific deletion of thymocytes in fetal thymic organ cultures suggest a role for glucocorticoids in thymocyte development. To address this further, transgenic mice that express antisense transcripts to the glucocorticoid receptor (GR) specifically in immature thymocytes were generated. The consequent hyporesponsiveness of thymocytes to glucocorticoids was accompanied by a reduction in thymic size, primarily owing to a decrease in the number of CD4+CD8+ cells. While an enhanced susceptibility to T cell receptor (TCR)-mediated apoptosis appeared to be partially responsible for this reduction, thymocyte loss could also be detected before thymocytes progressed to the CD4+CD8+ TCR alpha beta-expressing stage. These results suggest that glucocorticoids are necessary for survival and maturation of thymocytes, and are consistent with a role for steroids in both the transition from CD4-CD8- to CD4+CD8+ cells and the survival of CD4+CD8+ cells stimulated via the TCR.

The TLR3 signaling complex forms by cooperative receptor dimerization

Toll-like receptors (TLRs) initiate immune responses by recognizing pathogen-associated molecules, but the molecular basis for recognition is poorly understood. In particular, it is unclear how receptor-ligand interactions lead to the initiation of downstream signaling. Here, we describe the mechanism by which TLR3 recognizes its ligand, double-stranded RNA (dsRNA), and forms an active signaling complex. We show that dsRNA binds saturably, specifically, and reversibly to a defined ligand-binding site (or sites) on the TLR3 ectodomain (TLR3ecd). Binding affinities increase with both buffer acidity and ligand size. Purified TLR3ecd protein is exclusively monomeric in solution, but through a highly cooperative process, it forms dimers when bound to dsRNA, and multiple TLR3ecd dimers bind to long dsRNA strands. The smallest dsRNA oligonucleotides that form stable complexes with TLR3ecd (40–50 bp) each bind one TLR3ecd dimer, and these are also the smallest oligonucleotides that efficiently activate TLR3 in cells. We conclude that TLR3 assembles on dsRNA as stable dimers and that the minimal signaling unit is one TLR3 dimer.

Post‐thymectomy autoimmune gastritis: fine specificity and pathogenicity of anti‐H/K ATPase‐ reactive T cells

Thymectomy at day 3 of life (d3Tx) results in the development of organ‐specific autoimmunity. We have recently shown that d3Tx BALB/c mice which develop autoimmune gastritis contain CD4+ T cells specific for the gastric parietal cell proton pump, H/K ATPase. Here, we demonstrate that freshly explanted gastric lymph node (LN) cells from d3Tx mice react significantly to the H/K ATPase α chain, but only marginally to the β chain. Two H/K ATPase‐reactive T cell lines were derived from the gastric LN of d3Tx mice. Both are CD4+ , TCR α / β+ , and I‐Ad restricted, and recognize distinct peptides from the H/K ATPase α chain. One cell line secretes Th1 and the other Th2 cytokines, but both are equally potent in inducing gastritis with distinct profiles of cellular infiltration in nu/nu recipient animals. Neither of the cell lines induced disease in normal BALB/c recipients and transfer of disease to nu/nu recipients was blocked by co‐transfer of normal BALB/c spleen cells containing CD4+ CD25+ cells. Although CD4+ CD25+ T cells are thought to emigrate from the thymus after day 3 of life, they could be identified in LN of 2‐day‐old animals. The capacity of CD4+ CD25+ T cells to abrogate the pathogenic activity in vivo of both activated Th1/Th2 lines strongly suggests that this suppressor T cell population may have a therapeutic role in other models of established autoimmunity. The availability of well‐characterized lines of autoantigen‐specific T cells should greatly facilitate the analysis of the mechanism of action and target of the CD4+ CD25+ immunoregulatory cells.

Somatic cell hybridization of mouse myeloma cells

Somatic cell hybrides between different mouse myeloma cell lines have been readili isolated using modifications of existing techniques. The hybrid nature of these cells was established by HAT or HAT-ouabain selective procedures, their chromosome number, and, in one case, H-2 surface antifen expression. Three hybrid cell lines are described here in detail: an IgG2B, K X LgG2a, k; an IgG1, k X IgG2b, k; and an IgG1, k X IgM, Lambda. In all cases, both parental types of H and L chains are expressed in the hybrid cells and no new chains are observed. However, molecules possessing disulfide-bonded mixtures of parental H and/or L chains are seen. Analysis of subclones of these hybrids indicates considerable stability in the expression of the immunoglobulins for up to 13 months. However, segregant clones no longer synthesizing one or more of the parental H or L chains arise frequently.

Inhibition of an allospecific T cell hybridoma by soluble class I proteins and peptides: Estimation of the affinity of a T cell receptor for MHC

To investigate the molecular basis of the interaction between the T cell receptor and the MHC class I antigen in an allogeneic response, a soluble counterpart of the murine class I molecule, H-2Kb, was genetically engineered. Cells secreting this soluble molecule, H-2Kb/Q10b, inhibited stimulation of an H-2Kb-reactive T cell hybridoma by cells transfected with H-2Kbm10, a weak stimulus, but not by H-2Kb- or H-2Kbm6-transfected cells. Soluble purified H-2Kb/Q10b protein also blocked T cell stimulation. In addition, a peptide from the wild-type H-2Kb molecule spanning the region of the bm10 mutation specifically inhibited activation of the T cell hybridoma by H-2Kbm10 cells, thus suggesting that amino acid residues 163-174 of H-2Kb define a region important for T cell receptor binding. An estimate for the Kd of the T cell receptor for soluble H-2Kb/Q10b was 10(-7) M, while the Kd for soluble peptide 163-174 was 10(-4) M.

Abacavir induces loading of novel self-peptides into HLA-B*57: 01: an autoimmune model for HLA-associated drug hypersensitivity.

Background:Abacavir drug hypersensitivity in HIV-treated patients is associated with HLA-B*57:01 expression. To understand the immunochemistry of abacavir drug reactions, we investigated the effects of abacavir on HLA-B*57:01 epitope-binding in vitro and the quality and quantity of self-peptides presented by HLA-B*57:01 from abacavir-treated cells. Design and methods:An HLA-B*57:01-specific epitope-binding assay was developed to test for effects of abacavir, didanosine or flucloxacillin on self-peptide binding. To examine whether abacavir alters the peptide repertoire in HLA-B*57:01, a B-cell line secreting soluble human leucocyte antigen (sHLA) was cultured in the presence or absence of abacavir, peptides were eluted from purified human leucocyte antigen (HLA), and the peptide epitopes comparatively mapped by mass spectroscopy to identify drug-unique peptides. Results:Abacavir, but not didansosine or flucloxacillin, enhanced binding of the FITC-labeled self-peptide LF9 to HLA-B*57:01 in a dose-dependent manner. Endogenous peptides isolated from abacavir-treated HLA-B*57:01 B cells showed amino acid sequence differences compared with peptides from untreated cells. Novel drug-induced peptides lacked typical carboxyl (C) terminal amino acids characteristic of the HLA-B*57:01 peptide motif and instead contained predominantly isoleucine or leucine residues. Drug-induced peptides bind to soluble HLA-B*57:01 with high affinity that was not altered by abacavir addition. Conclusion:Our results support a model of drug-induced autoimmunity in which abacavir alters the quantity and quality of self-peptide loading into HLA-B*57:01. Drug-induced loading of novel self-peptides into HLA, possibly by abacavir either altering the binding cleft or modifying the peptide-loading complex, generates an array of neo-antigen peptides that drive polyclonal T-cell autoimmune responses and multiorgan systemic toxicity.