Michael G. Mage

Mouse lymphocytes with and without surface immunoglobulin: preparative scale separation in polystyrene tissue culture dishes coated with specifically purified anti-immunoglobulin.

Mouse spleen cells could be preparatively separated into immunoglobulin positive (Ig+) and immunoglobulin-netative (Ig-)populations by incubating as many as 2 X 10(8) cells per 100 mm diameter petri plate coated with specifically purified goat anti-mouse immunoglobulin. The non-adherent population was 95% or more Ig-, and possessed graft versus host and cytotoxic effector activities, as would be expected for T cells. They could also give a mixed lymphocyte reaction and generate cytotoxic effector activity on culture in vitro. The adherent cells could not be released undamaged from plates coated with undiluted anti-Ig, but they could be released from plates coated with a 1/4 or 1/10 dilution of anti-Ig in an irrelevant antibody. The released cells were over 90% viable by trypan-blue staining, and 94% or more of the viable cells were Ig+.

Guinea pig heterophil and eosinophil peroxidase

Abstract Guinea pig heterophil and eosinophil peroxidases were purified from bone marrow leukocytes in order to compare certain physical properties. Purified heterophil peroxidase (homogeneous in the ultracentrifuge, by immunologic test, and by gel electrophoresis) had a Soret maximum at 437 nm, an A 437 A 280 ratio of 0.79, and a mol wt of 137,000. The molecule consisted of two subunits of similar size, as assessed by its appearance in the electron microscope and by electrophoresis in SDS polyacrylamide gels. Eosinophil peroxidase required the cationic detergent cetyltrimethylammonium bromide to prevent binding to the materials used in its purification. The purified esoinophil enzyme, homogeneous by immunologic criteria, was resolved by gel filtration into two peroxidase components having mol wts of 75,000 and 150,000, respectively. Each component had the same specific activity, absorption spectrum (Soret maximum at 425 nm), and A 415 A 280 ratio (0.83). Treatment of each with SDS and mercaptoethanol produced a single component with a mol wt of about 65,000. Examination of crude bone marrow extracts resolved only one eosinophil peroxidase component with a mol wt of about 80,000, indicating that aggregation occurred during purification. The leukocyte peroxidases, therefore, have substantially different properties despite their location in analogous organelles of closely related cells.

Automatic digital data acquisition and computer calculation in amino acid analysis

Abstract Amino acid analysis data acquisition and computation have been automated by (1) measuring the ninhydrin color with a spectrophotometer which is linear with respect to optical density, (2) digitizing the data directly from the photometer circuit by means of a digital voltmeter, and (3) formating and punching the digital data in paper tape so that each data word is identified as to channel of information (i.e., cuvet position and wavelength), and processing the punched data on a computer using a FORTRAN program. The program edits the data with regard to noise, compensates for baseline changes of various types, splits unresolved peaks, and calculates peak areas over a range of optical densities from 0 to 3.000. This system, in daily use in our laboratory, provides increased accuracy and saves approximately 2 man hours per analysis.

The Peptide-Receptive Transition State of MHC Class I Molecules: Insight from Structure and Molecular Dynamics

MHC class I (MHC-I) proteins of the adaptive immune system require antigenic peptides for maintenance of mature conformation and immune function via specific recognition by MHC-I–restricted CD8+ T lymphocytes. New MHC-I molecules in the endoplasmic reticulum are held by chaperones in a peptide-receptive (PR) transition state pending release by tightly binding peptides. In this study, we show, by crystallographic, docking, and molecular dynamics methods, dramatic movement of a hinged unit containing a conserved 310 helix that flips from an exposed “open” position in the PR transition state to a “closed” position with buried hydrophobic side chains in the peptide-loaded mature molecule. Crystallography of hinged unit residues 46–53 of murine H-2Ld MHC-I H chain, complexed with mAb 64-3-7, demonstrates solvent exposure of these residues in the PR conformation. Docking and molecular dynamics predict how this segment moves to help form the A and B pockets crucial for the tight peptide binding needed for stability of the mature peptide-loaded conformation, chaperone dissociation, and Ag presentation.

Isolation and characterization of sheep γ1- and γ2-immunoglobulins and their polypeptide chains

Abstract The two electrophoretically distinct ‘7-S’ γ1- and γ2-sheep immunoglobulins were isolated from serum, and each was separated into its component heavy and light polypeptide chains. The heavy chains as well as the intact immunoglobulins differed antigenically, electrophoretically, and in amino acid composition while no such differences were found between their light chains.

Crystal structure of a TAPBPR-MHC I complex reveals the mechanism of peptide editing in antigen presentation.

Two snapshots of the TAPBPR-MHC I complex Cytotoxic CD8+ T cells recognize infected and cancerous cells by scrutinizing the antigenic peptides presented by the major histocompatibility complex class I (MHC I). Peptide binding and exchange occurs in the endoplasmic reticulum in a sequence of events mediated by the chaperones tapasin and TAPBPR (see the Perspective by Cresswell). Thomas and Tampé resolved the crystal structure of the TAPBPR-MHC I editing complex by using a photocleavable high-affinity peptide to stabilize the MHC molecule. Jiang et al. crystalized MHC I molecules inhabited by truncated disulfide-linked peptides that still permit TAPBPR to bind. These complimentary snapshots elucidate the dynamic process by which chaperones stabilize the groove of peptide-free MHC I molecules. This helps MHC I sample peptide candidates and facilitates the generation of peptide repertoires enriched with high-affinity antigenic peptides. Science, this issue p. 1060, p. 1064; see also p. 992 Two different approaches yield complimentary structures of TAPBR in complex with MHC I. Central to CD8+ T cell–mediated immunity is the recognition of peptide–major histocompatibility complex class I (p–MHC I) proteins displayed by antigen-presenting cells. Chaperone-mediated loading of high-affinity peptides onto MHC I is a key step in the MHC I antigen presentation pathway. However, the structure of MHC I with a chaperone that facilitates peptide loading has not been determined. We report the crystal structure of MHC I in complex with the peptide editor TAPBPR (TAP-binding protein–related), a tapasin homolog. TAPBPR remodels the peptide-binding groove of MHC I, resulting in the release of low-affinity peptide. Changes include groove relaxation, modifications of key binding pockets, and domain adjustments. This structure captures a peptide-receptive state of MHC I and provides insights into the mechanism of peptide editing by TAPBPR and, by analogy, tapasin.

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

Significance This report explores the biochemical and structural basis of the interactions of TAP binding protein, related (TAPBPR), a tapasin homolog, with MHC-I molecules. TAPBPR associates with MHC-I molecules early in their biosynthesis and folding but is not part of the peptide-loading complex (PLC). Here, by examining the interactions of recombinant TAPBPR with peptide-free and peptide-complexed MHC-I molecules, we show that TAPBPR serves as a peptide editor. Structural comparison of TAPBPR with tapasin indicates the similarities of the two molecules and provides a basis for evaluating the steps of peptide loading. Understanding the molecular underpinnings of peptide loading of MHC-I by TAPBPR and tapasin has wide-ranging influence on our ability to modulate peptide loading for vaccine design and T-cell recognition. Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8+ T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.

Cell-mediated immunity to chemically xenogenized tumors: II. Evidence for accessory function and self-antigen presentation by a highly immunogenic tumor variant

To determine whether antigen-presenting ability might be involved in the superior immunogenicity of chemically xenogenized tumors over that of parental cells, we tested a murine lymphoma line xenogenized by a triazene derivative for expression of Ia antigens, ability to present soluble antigen in vitro, and production of factor(s) active in a mouse thymocyte assay. Results showed that Ia antigens, absent on nonimmunogenic parental L5178Y cells, were expressed on a xenogenized, highly immunogenic tumor variant (clone D), as detected by immunofluorescence. While the ability of parental cells to stimulate lymphocyte proliferation in vitro was lost on removal of Ia+ cells from the responder population, considerable augmentation of reactivity was observed upon depletion of Ia+ cells from the population of splenocytes responding to the xenogenized cells. Under these conditions, stimulation was blocked by anti-Ia antibodies, or an anti-L3T4 reagent or antibodies to the novel antigenic determinants induced by xenogenization. In addition, no stimulating activity was observed following exposure of clone D cells to glutaraldehyde or lysosomotropic agents such as chloroquine and ammonia. When the ability of clone D cells to present ovalbumin in vitro was assayed, it was found that the xenogenized cells could present the soluble antigen to specifically primed lymphocytes. Moreover, clone D cells could substitute for splenic adherent cells in the proliferative reaction of splenocytes to concanavalin A. Finally, when the supernate from clone D-cell culture pulsed with phorbol myristic acetate was tested in a mouse thymocyte assay, considerable IL-1-like activity was disclosed.

Cell-mediated immunity to chemically xenogenized tumors I. Inhibition by specific antisera and H-2 association of the novel antigens

SummaryT cell-mediated proliferative and cytotoxic responses occur in vitro to syngeneic tumor cells antigenically altered by mutagen treatment. One such xenogenized variant of the murine L5178Y lymphoma elicits IgG antibodies reactive with determinants on variant cells that are not expressed at detectable levels on parental or normal cells of the same H-2d haplotype and are also unrelated to public specificites of H-2b or H-2k histocompatibility antigens. In the present study we investigated the effect of those antibodies on development of cell-mediated responses in vitro to the xenogenized cells used for induction of the humoral response. The proliferative reaction, generation of cytolytic activity and target cell lysis were all inhibited by the anti-xenogenized tumor immune serum, whereas the corresponding reactions to the parental cells by syngeneic or allogeneic effector lymphocytes were not. In order to investigate the possible H-2 association of T cell-mediated responses to xenogenized cells, we also examined the effect on those reactions of antibodies specific for Class I or Class II products of the H-2d complex. The results obtained suggested a role for I-Ad molecules in the T cell proliferative response to the xenogenized cells, and also indicated a preferential association of the cytotoxic response with H-2Kd determinants.

Purification of alkaline phosphatase from guinea pig bone marrow

Abstract A new method of purifying alkaline phosphatase (AP) from specific granules of guinea pig bone marrow granulocytes is described. The method devised resulted in a 500-fold purification of AP with a specific activity of 81,000 μmoles of p -nitrophenyl phosphate hydrolyzed min −1 mg −1 and a 39% yield. The purified enzyme was free of protein contaminants as judged by immunoprecipitation and gel electrophoresis. Its molecular size was estimated by gel filtration to be 4 × 10 5 . The purified enzyme behaved like a lipoprotein on ion-exchange chromatography. Lipid extracted from purified enzyme contained 2–4 μg P/mg of protein. Like other alkaline phosphatases, purified AP appeared to be a zinc metalloenzyme, that is, it was inhibited by various chelating agents, including o -phenanthroline, and contained a stoichiometrically significant amount of zinc, 3–5 g-atoms of zinc/400,000 g of protein. Magnesium activated the purified enzyme 4- to 7-fold.