Mitogenic activity of Neisseria gonorrhoeae surface antigens in mouse splenic lymphocyte culture.

The mitogenic effects of Neisseria gonorrhoeae endotoxin, fractionated envelope componenents, and intact cells were examined on unsensitized mouse splenic lymphocytes in vitro. The stimulatory effect of these substances was measured by increased [3H]thymidine incorporation in spleen cell cultures. Intact cells, purified lipopolysaccharide (LPS), and cell envelope preparations were highly stimulatory and the stimulation index was dose dependent. Fractionated components of the envelope demonstrated variable stimulation when tested at identical LPS concentrations, reflecting the mitogenic activity of the protein moieties. The stimulatory dose responses for purified N. gonorrhoeae and Escherichia coli LPS were compared and mitogenicity was higher with gonococcal LPS at all concentrations tested. Alkaline detoxification or succinylation of N. gonorrhoeae LPS results in loss of ability to induce blast transformation. The mitogenicity of cell-surface components of N. gonorrhoeae is discussed in terms of LPS and protein content.     

Epitope expression on primate lymphocyte surface antigens

The cross-reactivity of peripheral blood mononuclear cells from 28 nonhuman primates was investigated with ten kinds of Leu series of monoclonal antibodies specific to human T-, natural killer/killer-, and B-cells. The chimpanzees possessed all ten epitopes examined but the orangutan lacked Leu4 and Leu7 epitopes and the gibbons lacked Leu4, Leu7, and Leu12 epitopes. In addition to the above epitopes, the Old World monkeys lacked Leu1 and Leu10 epitopes. The Leu3a/Leu2a cell ratios varied from 0 to 1.56 among the 12 macaque species and this enabled classification of these species into three groups. In the New World monkeys, Leu2a epitope was absent, whereas Leu11a epitope was detected in several species and Leu3a epitope was found only in the owl monkeys. The prosimians expressed only HLA-DR epitope.     

Immunochemical identification of multiple cell surface antigens appearing during specific stages of mouse spermatogenesis

Cell surface antigens that appear in a defined temporal sequence during mouse spermatogenesis were previously detected serologically, but not identified biochemically, with four heterologous antibodies prepared against purified populations of pachytene spermatocytes (AP), round spermatids (ARS), vas deferens spermatozoa (AVDS), and mixed seminiferous cells (ASC) [Millette and Bellvé, J Cell Biol 74:86–97, 1977]. These antigens have now been identified immunochemically on nitrocellulose blots from SDS polyacrylamide gels. Three antisera (AP, ARS, and ASC) recognize a similar subset of determinants on one-dimensional immunoblots of germ cells and plasma membranes prepared from a mixed population of late spermatogenic cells. Comparisons of minor bands to reveal differences among these antisera. AVDS exhibits the least complex binding pattern. The results indicate that at least ten surface constituents appear during the pachytene stage of meiosis, coincident with a period of maximal RNA and protein synthesis [Monesi, Exp Cell Res 39:197–224, 1965]. Furthermore, two-dimensional immunoblot comparisons of plasma membranes isolated from pachytene spermatocytes and round spermatids reveal differences between surface determinants detectable at these two spermatogenic stages. For example, ASC recognizes two newly described proteins that are restricted to pachytene spermatocytes (? M_r 57,000, pI 6.45) and to round spermatids (? M_r 39,500, pI 4.85), respectively.     

Biosynthesis of lymphocyte surface IgD in the mouse

The synthesis of IgD was studied in mouse spleen cells by using [35S]methionine labeling followed by immunoprecipitation with monoclonal antibody and two-dimensional gel electrophoresis. After a 15-min pulse of [35S]methionine, a relatively basic form (IgD1) of apparent m.w. 59,000 was precipitated. Conversion into more acidic forms of m.w. 63 to 72,000 (IgD2) took place during a chase period of several hours. The acidic form was identical in mobility to that of IgD labeled by surface radioiodination, and was almost completely removed by treatment of intact cells with pronase. Neuraminidase treatment of the surface form (IgD2) produced a form resembling IgD1 in charge, but with no detectable change in m.w. Treatment of IgD1 with endoglycosidase H resulted in a form with an apparent m.w. of 50,000, whereas IgD2 was resistant to this enzyme. Both IgD1 and IgD2 bound to lentil lectin, whereas only IgD2 bound to Ricinus communis hemagglutinin, which binds to terminal galactose residues. These results indicate that IgD is synthesized as an incompletely glycosylated precursor possessing "high mannose" type oligosaccharide moieties, and passes relatively slowly through the cell. Shortly before surface appearance, galactose and sialic acid are added. No specific association with any other labeled protein was observed, and any IgD secretion was below the limits of detectability.     

Production of monoclonal antibodies to antigens of specific mouse T-suppressors

F1(MSU X WAG) rats were immunized with anti B6 BALB/c specific suppressor T cells (SSTC), purified by absorption/elution technique, with the following fusion of splenocytes to NS-I myeloma cell line. Hybrids were screened for their ability to affect SSTC, cytotoxic T lymphocytes (CTL) and producers of macrophage migration inhibition factor (MIF-producers) all triggered by in vivo priming with allogeneic cells. Two hybridoma cell lines--C1 and C4 inactivated SSTC by approximately 50%, leaving CTL and MIF-producers intact. C4 were also active in vivo, if injected as ascitic fluid from nu/nu mice, though to a lesser extent than in vitro.     

Evolution of epitopes on human and nonhuman primate lymphocyte cell surface antigens

The T- and B-cell surface polypeptides detected by an international workshop panel of 100 mouse monoclonal antibodies (M.Ab) were biochemically defined by radioimmunoprecipitation. Eight T-cell-associated molecules and eight B-cell-associated molecules were identified by multiple antibodies in the panel. Clusters of antibodies specific for the same polypeptide were then compared for their reactivity against peripheral blood mononuclear cells (PBMC) from 11 nonhuman primate species. All the major T- and B-cell antigens present in humans were also expressed in some nonhuman primates. M.Ab to the same antigen were found to react with distinct epitope groups that differed in their phylogenetic distribution. Some epitopes were highly conserved, while other epitopes on the same molecule were only expressed in hominoids and were not detected in old world and new world monkeys. Our detailed analysis of the phylogeny of 37 T-cell antigen epitopes on ten different molecules revealed there was no clear correspondence between the number of epitopes shared and evolutionary distance. Rather the data suggest that parallelism with back mutation may be a common mechanism in the evolution of T-cell antigens. The data also show that the tissue distribution of T-cell antigens can differ between primate species; for example, M.Ab to human Tp45 Tla-Qa-like antigens that did not react with human PBMC did react with PBMC from new world monkeys.     

Antigen-specific mouse lymphocyte stimulation by DNP-conjugated T-independent antigens studied by photobleaching recovery

Fluorescence photobleaching recovery techniques have allowed us to measure the lateral mobility of T-independent antigens bound to antigen-specific mouse B cells. The in vitro immunogenicity or tolerogenicity of antigens we have examined, DNP-polymerized flagellin (DNP-POL), and DNP-linear dextran (DNP-DEX), depend upon the antigen dose and epitope density. These factors also determine the mobility of antigen bound to B cell surfaces. For DNP-POL bound to DNP-specific cells, the observed diffusion constants D decrease monotonically with increasing antigen dose and epitope density. Values of D range from 10.4 × 10^?11 cm² sec^?1 for DNP_0.4-POL at 0.15 μg/ml to 0.8 × 10^?11cm² sec^?1for DNP_3.5-POL at 30 μg/ml. For receptor-bound DNP-DEX, D depends strongly on antigen epitope density but not observably on antigen concentration. For epitope densities of 1.2 or less, D is close to the value of 21 × 10^?11cm²sec^?1 observed for single slg receptors. By an epitope density of 4.8, D has fallen to 2.1 × 10^?11cm²sec^?1. Peak immunogenicities for DNP-POL and DNP-DEX arc observed when antigen- receptor aggregates have mobilities 14-fold and 3-fold lower, respectively, than a single slg molecule.     

Changes in surface antigens on preimplantation mouse embryos.

Changes in the expression of specific cell surface antigens on preimplantation mouse embryos were examined by immunocytochemistry. Embryos were recovered at various times during the preimplantation phase of normal pregnancy, and from pregnancies with experimentally induced delayed implantation, and were probed with a panel of monoclonal antibodies against murine leukocyte antigens. Antibodies directed against certain macrophage surface glycoproteins (i.e., Mac-2 and Mac-3) and those against lysosome-associated membrane glycoproteins (i.e., LAMP-1 and LAMP-2) reacted specifically with cell surface determinants on the embryos. Differences in the spatiotemporal patterns of antibody binding during normal and delayed implantation indicate that expression of the antigenic determinants recognized by these antibodies is regulated individually in response to intrinsic as well as extrinsic signals at the time of implantation, and thus they may be important for the process of embryo implantation.     

Human T lymphocyte clones specific for malaria (Plasmodium falciparum) antigens.

Peripheral blood mononuclear cells (PBM) from a patient who had lived in a malarial-endemic area were cultured in the presence of malarial antigens (a lysate of Plasmodium-infected erythrocytes). Responding cells were grown in IL-2-containing medium and then cloned, and subsequently subcloned, in the presence of phytohemagglutinin and allogeneic irradiated PBM. Ten clones were specific for malarial antigens. They proliferated in response to P. falciparum extract, but not to a lysate of uninfected erythrocytes. The response was HLA-restricted. All the clones tested responded to lysates of cells infected with parasites of either African or Asian origin. Six clones had the T4+/T8- phenotype and four the T4-/T8+ phenotype. Two of the T4+ clones recognised a parasite antigen of apparent mol. wt. approximately 50 000. All of the clones tested produced gamma-interferon following antigen stimulation.     

Characterization of thymus-derived lymphocyte subsets in acute Epstein-Barr virus-induced infectious mononucleosis.

Changes in thymus-derived (T) lymphocyte subpopulation numbers were studied in patients with acute and convalescent Epstein-Barr virus (EBV)-induced infectious mononucleosis (LM). T cell subsets were characterized by the presence of Fc receptors for IgG (TG), for IgM (TM) or by the absence of either receptor (Tnon-M, non-G). We found that in acute IM, total numbers of T and B lymphocytes were elevated (p less than 0.01). Of the T lymphocyte subsets, the total number of Tnon-M, non-G lymphocytes was increased six fold compared to normal subjects (p less than 0.001) and included the majority of the atypical T lymphocytes. The number of total TG and TM lymphocytes was moderately increased (p less than 0.05). In convalescent IM patients, the number of total T cells remained slightly elevated (p less than 0.02) whereas proportions and absolute numbers of B lymphocytes and T cell subsets returned to near normal levels. Thus, acute Epstein-Barr virus-induced IM is associated with a T lymphocytosis which is composed predominantly of atypical T cells which lack detectable Fc receptors for IgG or IgM.