Selective binding of chemically defined antigenic peptides to mouse lymphocytes

Immunologically active peptides containing either two different (N? 10? C) or two identical (N? 8? N) antigenic amino acid sequences, bridged by 10 and 8 glycine residues, respectively, were used to study antigen-binding lymphocyte populations in BALB/c mice. Spleen cells from mice immunized with either N? 10? C or N? 8? N were treated with either normal rabbit serum or brain associated anti-thymus (BA Θ) serum and subsequently examined by autoradiography for the numbers of cells binding ¹²⁵I-labeled N? 10? C, and ¹²⁵I-labeled conjugates of either the C antigenic determinant or the N antigenic determinant with poly? D? glutamic acid. In unimmunized mice there were more cells binding the N determinant than the C determinant. After anti-BA Θ treatment, the relative numbers of each population increased, implying that many of these cells were bone marrow-derived. In N? 10? C or N? 8? N immunized mice, the numbers of cells binding the N determinant remained virtually unchanged after anti-BA Θ treatment, indicating an equal distribution of these cells in both bone marrow or thymus-derived populations. However, the C determinant binding population in N? 10? C immunized animals appeared to be mainly of bone marrow origin. Since the C determinant exhibited mainly B cell reactivity, the role of thymus cells in immune responsiveness to a peptide (C? mal? 10? C) containing two C determinants was examined. Only those mice which were reconstituted with both bone marrow and thymus cells after thymectomy and irradiation were capable of responding normally to this antigen, demonstrating thymus involvement even with antigens that appear to be mainly reactive with B cells.     

Functional properties of subsets of T lymphocytes defined by specific antigens.

Heteroantisera raised to the acute lymphocytic T (ALL) cell line HSB2 and to Sézary cells react with distinct subpopulations of T lymphocytes. Each antiserum reacts with a different T cell antigen and defines a distinct subpopulation that represents approximately 50% of peripheral blood T lymphocytes. The anti-HSB2-positive subpopulation contained suppressor cells for pokeweed mitogen-dependent immunoglobin (Ig) synthesis whereas the anti-Sézary cell serum-positive population included helper cells for Ig synthesis and mixed lymphocyte responder cells.     

Cellular architecture of the mouse hippocampus: a quantitative aspect of chemically defined GABAergic neurons with stereology

The structural organization of the central nervous system is an infrastructure of the modern neuroscience. Especially, anatomical research at the cellular level can serve as a fundamental resource to understand various novel findings from the molecular level to the system level. Of the decade, the importance of rigorous quantitative neuroanatomy is gradually realized, but earlier anatomical reports are usually presented in qualitative terms or quantified with biased methods. This article quantitatively describes the spatial distributions of chemically defined GABAergic neurons in the mouse hippocampus by using unbiased stereological techniques. We focus on the expression of nine major neurochemical markers: glutamic acid decarboxylase 67, three calcium binding proteins (parvalbumin, calretinin, calbindin D28K), four neuropeptides (somatostatin, neuropeptide Y, cholecystokinin, vasoactive intestinal protein) and neuronal nitric oxide synthase. Here we deal with their laminar distributions, numerical densities and the relative ratio to the total GABAergic neurons, with special reference to their differentiation along the dorsoventral axis of the mouse hippocampus. Furthermore, we estimate the absolute numbers of GABAergic neurons contained in a 300-mum-thick hypothetical slice. The present data outline the quantitative aspects of the cellular architecture of hippocampal GABAergic system and also give complementary information to the recent multidisciplinary analyses at the single cell level.     

Xid mouse lymphocytes respond to TI-2 antigens when co-stimulated by TI-1 antigens or lymphokines

Spleen cells from male (CBA/N x DBA/2) F1 hybrid mice do not significantly respond to in vitro stimulation by trinitrophenyl-conjugated polyacrylamide beads (TNP-PAA), whereas the same antigen elicits high PFC responses in female F1 hybrid cells. Therefore, this antigen could be classified as a T-independent type 2 (TI-2) antigen. When male spleen cells were co-stimulated by TNP-PAA and TI type 1 antigen, either LPS or Brucella abortus, they produced vigorous anti-TNP responses. A similar increase of the in vitro responsiveness of male F1 hybrid spleen cells to TNP-PAA antigen was provoked by the addition of supernatants from P 388-D1 cells stimulated by muramyl-dipeptide (MDP) mainly containing interleukin-1 (IL-1) or supernatants from phorbol 12-myristate 13-acetate (PMA)-stimulated EL-4 cells that contained T-cell factors. The PFC response to another TI-2 antigen, TNP-Ficoll, was also significantly enhanced after co-stimulation by P 388-D1 supernatants. The response to TI-2 antigens being macrophage dependent, the influence of supernatants of peritoneal macrophages from male and female F1 hybrids incubated with TNP-Ficoll on the PFC response of normal DBA/2 mouse spleen cells to sheep erythrocytes was assessed. It was found that macrophage supernatants from female hybrids regularly increased by more than two times this anti-SRBC PFC response, whereas macrophage supernatants from male F1 hybrids did not. Moreover, in a specific proliferation test measuring IL-1 activity, when macrophage supernatants from female F1 produced a 13-fold increase of thymidine incorporation, supernatants from male F1 only produced a three-fold increase. It is concluded that, in addition to the known defects of B cells from Xid mice, their macrophages are also defective.     

Cellular co-operation in the production of human leucocyte inhibitory factor by lymphocyte subpopulations stimulated with antigens and allogeneic cells.

The ability of antigens and allogeneic cells to induce lymphokine synthesis and cellular co-operation in lymphokine production was investigated. Human peripheral blood lymphocytes were separated into T- and B-cell populations by sheep red blood cell rosette formation and centrifugation on Ficoll--Isopaque. The cells were then stimulated with PPD, SK-SD, candida and with allogeneic cells. The presence of leucocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose migration method. The results indicated that T lymphocytes produced LIF after stimulation with antigens and allogeneic cells. In addition, B cells responded to PPD. Except for PPD the stimulants did not induce significant T-cell LIF production in the absence of monocytes. Only autologous monocytes enhanced LIF synthesis after antigenic stimulation, whereas in mixed lymphocyte cultures allogeneic monocytes were as effective as autologous ones. The monocyte helper effect was mediated by soluble factors in mixed lymphocyte cultures and by soluble factors and direct cell--cell contact in antigen-stimulated cultures. No co-operation between T and B lymphocytes was found. B cells did not enhance LIF production by T cells, nor could T cells induce a B-cell response to antigens or allogeneic cells.     

Cellular and humoral immune responses in guinea pigs and rabbits to chemically defined synthetic peptides

Two peptides shown previously (Biochemistry 1971. 10: 1763) to constitute the two major antigenic determinants of performic acid-oxidized ferredoxin (O-Fd) from Clostridium pasteurianum were studied in terms of their immunological properties. These peptides, designated the “N” (the NH₂-terminal heptapeptide of O-Fd) and the “C” (the COOH-terminal pentapeptide of O-Fd) haptens, were synthesized and linked together in various ways to obtain peptides of a comparable length. The following peptides were prepared and used in this study: N-10-C, containing the N and C haptens linked by ten glycine residues, N-8-N, containing two N haptens linked by eight glycine residues and C-mal-10, constituting a symmetrical molecule with two C haptens each with a free COOH-terminal and bridged by a malonic acid and ten glycine residues. All of these peptides were immunogenic in that they elicited both immediate and delayed skin reactions in immunized guinea pigs and stimulated increased uptake of [³H]thymidine in lymphocytes from appropriately sensitized animals. The peptides N-10-C and N-8-N also elicited the production of circulating antibodies in immunized rabbits. No antibodies to C-mal-10-C were detected by the methods used here. Lymphocytes taken from guinea pigs sensitized to N-10-C were stimulated when they were cultured in the presence of either a conjugate of poly-D -glutamic acid and the C hapten (ratio of 1:22) or poly-D -glutamic and the N hapten (ratio of 1:18), indicating that the response was directed to the haptenic groups alone and not to the bridge structure. Lymphocytes from animals sensitized with C-mal-10-C were not stimulated by N-10-C and appeared to require the presence of at least two C haptens per molecule before cell transformation could be elicited. On the other hand, lymphocytes from animals sensitized to N-8-N were stimulated with N-10-C, but not with the N hapten alone, indicating that while only one haptenic group was essential for stimulation, it had to be presented to reactive cells in a form other than as the free hapten. The implications of the results are discussed.     

Differentiation antigens defined by mouse monoclonal antibodies against human germ cell tumors

We have developed two mouse monoclonal antibodies, M912-2A2 and M912-2G10, against cell surface antigens of a human infantile embryonal carcinoma cell line, MTE. The distribution of these antigens (designated as 2A2 and 2G10) was almost identical in human germ cell tumors in which they hallmarked yolk sac components and some tubular endodermal structures. Immunoelectron-microscopically, the antigens were located on the microvilli of MTE tumor cells. These antigens were not found on other common childhood tumors. In normal and fetal tissues they exhibited quite different distributions. In the kidney, 2A2 and 2G10 were present on the collecting tubules and proximal/distal tubules, respectively. Expression of both antigens was already observed in fetal kidneys of 10 weeks gestational age. In hematopoietic cells 2G10 was present only on granulocytes and on erythrocytes regardless of ABO blood group, whereas 2A2 was not present on any peripheral blood cells. Both antigens were equally expressed in testis and epididymis. Biochemically, reactivity of both antibodies was abolished with periodate treatment, suggesting their carbohydrate nature. Further biochemical characterization revealed that antibody to 2G10 reacts with the nonreducing terminal structure of type 2 carbohydrate chain, Ga1 beta 1-4G1cNAc, common to nLc4 (paragloboside), nLc6 (neolactohexaose), and Y4 neutral glycolipids of O-type erythrocytes. These data illustrate the complexity of carbohydrate antigens on yolk sac components of human germ cell tumors and provide a basis for the study of primitive endodermal and yolk sac differentiation in these tumors.     

Subpopulations of mouse spleen lymphocytes. III. Cellular interactions in the response to concanavalin A.

The profile of response to concanavalin A (con A) of purified mouse T cells was found to differ appreciably from that of non-fractionated spleen cells, in agreement with results previously published by other investigators. Experiments designed to elucidate the reasons underlying these differences have revealed that the response of the spleen cells to con A is determined by a complex interplay between several cell types. (a) B cells contribute to the overall incorporation of thymidine in the presence of con A-stimulated T cells. However, the B cells participate in the response only if the T cells are dividing. (b) A population of 'adherent cells' is present in the spleen, which enhances the stimulation of the spleen cells by low doses of con A but suppresses the response to high doses of mitogen. These adherent cells include most likely the conventional macrophages, but probably also a population of 'suppressor T cells'. (c) Such 'suppressor T' cells can be readily detected among the peritoneal exudate cells. Addition of the exudate cells to cultures of purified T cells enhances the response to low doses of con A. This effect can be further increased by treating the peritoneal cells with a cell T-specific antiserum and complement, i.e. by eliminating the T cells.     

Protective anti-sporozoite antibodies induced by a chemically defined synthetic vaccine.

Chemically defined synthetic polymers, known as multiple antigen peptide systems (MAPs) represent an effective and novel approach for engineering peptide-based vaccines. Ten different mono and diepitope MAP models, containing different arrangements and stoichiometry of functional B and/or T helper epitopes from the circumsporozoite protein of Plasmodium berghei were used to immunize mice. High titers of antibody and protective immunity against sporozoite challenge were elicited by MAPs containing T and B epitopes arranged in tandem and in equimolar amounts. These results indicate that MAPs may serve as a basis for developing subunit vaccines to induce high levels of antibodies against sporozoites.     

Comparison of effects of Met-enkephalin on secretion of antibodies to various antigens by mouse lymphocytes

M. V. Lomonosov Moscow State University. M. N. Shemyakin Institute of Bioorganic Chemistry, Moscow. (Presented by Academician of the Russian Academy of Medical Sciences I. P. Ashmarin). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 12, pp. 629–631, December, 1992.     

Human tumor-associated antigens defined by monoclonal antibodies

Melanoma-associated antigens extracted from cultured human melanoma cells or isolated from their spent culture medium react specifically with monoclonal antibody and are represented by glycoproteins with molecular weights of 260,000 (260K), 250,000 (250K), and 100,000 (100K). The 260 and 250K antigens are present only in spent culture medium or detergent extracts of melanoma cells, respectively. The 100K antigen is an oncofetal marker present on tumor cells of different histological type. Indirect immunofluorescence data indicate that the 250K antigen is present on the surface of melanoma cells from excised melanoma tissues when the 100K antigen appears to be present in the cytosol. Subunit structure determination indicates that the 100K antigen is a single polypeptide chain whereas the 250K antigen extracted from melanoma cells is associated with a proteoglycan fragment which is important for its cell surface expression.     

Minor histocompatibility antigens, defined by graft-vs.-host disease-derived cytotoxic T lymphocytes, show variable expression on human skin cells.

Little is known on the effector mechanisms inducing the cutaneous lesions observed during acute graft-vs.-host disease (aGvHD) after allogeneic bone marrow transplantation (BMT). Histological findings have indicated that infiltrating CD8+ lymphocytes probably play a role. We addressed the question of whether host minor histocompatibility (mH) antigen-reactive cytotoxic T lymphocytes (CTL) could account for this phenomenon via direct lysis of the epidermal cell layer. Six CTL clones, obtained from peripheral blood lymphocytes of patients suffering from aGvHD, each recognizing a well-characterized MHC class I-restricted mH antigen epitope, were tested on cultured keratinocytes of nine MHC and mH antigen-typed donors. Four of six mH antigen-specific CTL clones lysed unstimulated MHC class I-expressing, as well as recombinant interferon-gamma (rIFN-gamma)-activated, ICAM-1, MHC class I- and II-expressing keratinocytes. Two strongly cytolytic CTL clones showed no recognition of keratinocytes of donors whose phytohemagglutinin-activated T cell lines were readily lysed. With respect to a GvHD, the results imply that some class I-restricted CTL obtained from peripheral blood lymphocytes of a GvHD patients have the in vitro potential to destroy resting as well as IFN-gamma-activated epidermal cells, whereas others do not. In other words, CTL-defined human mH antigens vary with respect to their expression in the skin. It is intriguing that those minor H antigens which cannot be detected on human keratinocytes in vitro are those known to be associated with the occurrence of GvHD.     

Plasmodium falciparum: characterization of defined antigens by monoclonal antibodies.

Monoclonal antibodies directed against Plasmodium falciparum detect stage-specific, species-specific and common antigenic determinants of Plasmodia. These antibodies provide new tools for purification and characterization of Plasmodium falciparum antigens in relation to future procedures for immunoprophylaxis.     

Production of exotoxin A by Pseudomonas aeruginosa in a chemically defined medium.

A defined medium was developed in which easily measured quantities of exotoxin A (PE) were produced by Pseudomonas aeruginosa PA-103. The medium contained three L-amino acids (arginine, aspartic acid, and alanine), basal and trace salts including 14 mM K2HPO4, 14 mM glucose, and 140 mM glycerol. The concentrations of amino acids which yielded most satisfactory results were 6 mM alanine, 13 mM aspartic acid, and 16 mM arginine. The identity of PE in the culture supernatant fluid was demonstrated by adenosine diphosphate-ribosyl transferase activity and by immunodiffusion with sheep antitoxin elicited with purified PE and with PE produced in Trypticase soy broth dialysate and pure PE as controls. PE production was also demonstrated by mouse lethality and passive hemagglutination. As compared to Trypticase soy broth dialysate, P. aeruginosa produced 25 to 50% PE in the defined medium. Different strains of P. aeruginosa produced PE in the defined medium in proportions relative to those in Trypticase soy broth dialysate.     

Education of mouse lymphocytes against human lymphoblastoid cell lines: specific recognition of determinants independent of serologically defined HLA allotypes and Epstein-Barr virus-coded antigens.

In this report, a model of xenogeneic education is described, whereby cytotoxic mouse spleen cells are generated against human target cell antigens. To generate a significant response, a primary in vivo immunization followed by an in vitro boosting 4 to 8 weeks later is required. Lysis is totally abrogated after treatment of the effector cells by anti-Thy-1.2 antiserum plus complement confirming the T origin of these effectors. Such cytotoxic T lymphocytes (CTL) do not discriminate between two lymphoblastoid cell lines carrying different HLA-A and HLA-B specificities, either by direct assay or by competition assay with unlabeled target cells. Moreover, Daudi cells which lack the serologically defined (SD) HLA antigens, are susceptible to CTL specifically raised against Daudi. However, CTL raised against another B line do not lyse Daudi, and, reciprocally, anti-Daudi CTL do not lyse other B lines thus suggesting a polymorphic distribution of xenogeneic determinants on human lymphoid lines. A third specificity is revealed when mouse spleen cells are educated against human T lines. The CTL generated under these conditions lyse not only the line used for sensitization but the three other human T lines of the panel. On the other hand, they affect none of the B cell lines (including Daudi) nor phytohemagglutinin or concanavalin A-induced T blasts. In conclusion, the mouse T cell repertoire is capable of recognizing a polymorphic system of antigens expressed on human lymphoblastoid cell lines that is not related to the expression of HLA-SD antigens nor to that of the Epstein-Barr virus genome.