Studies on non-H-2-linked lymphocyte-activating determinants. IV. Ontogeny of the Mls product on murine B cells.

The minor lymphocyte stimulating (Mls) locus codes for lymphocyte activating determinants (LADs) on murine B lymphocytes, but not T lymphocytes. This observation was strengthened by a series of techniques which allow deletion and addition of T and B cells. These included the use of cytotoxic antisera such as anti-Thy 1.2, anti-MTLA, anti-MBLA, and complement, and the use of a goat anti-μ antisera, and finally the use of a fluorescence activated cell sorter (FACS).The studies in this report document the organ distribution and the ontogenetic appearance of the surface LADs on the surface of B lymphocytes from DBA/2N (H-2^d, Mls^a) and CBA/J (H-2^k, Mls^d) mice. Adult-like ability to stimulate H-2 identical BALB/c (H-2^d, Mls^b) and C3H/He (H-2^k, Mls^c) responder cells appeared at about 4–5 weeks of age. Inability of neonatal cells to induce an Mls-defined MLC was found not to be due to a low frequency of B lymphocytes or to the presence of suppressor cells, but due to the absence of the Mls-coded LADs on their surface. These data support the concept that the Mls-coded LADs are present on adult B lymphocytes and are specific markers of B-cell differentiation, which is preceded by membrane IgM and the δ homologue of human IgD, Ia, and the receptor for the third component of complement.     

Cytolytic T lymphocyte responses to metabolically inactivated stimulator cell. I. Metabolic inactivation impairs both CD and LD antigen signals

The effects of metabolic inactivation of spleen cells on antigen presentation to precursors of alloreactive cytolytic T lymphocytes (T_c) were examined. By serological methods, populations inactivated by ultraviolet irradiation, glutaraldehyde fixation or plasma membrane isolation were found to retain normal levels of H-2K/D and Ia antigens. However, comparison of the antigen doses required to stimulate secondary T_c responses in mixed leukocyte culture showed that the inactivated preparations were approximately 10-fold less immunogenic than X-irradiated spleen cells. Their total inability to stimulate primary cytolytic responses pointed to at least a 100-fold impairment of immunogenicity for unprimed T_c precursors in the case of uv-irradiated and glutaraldehyde-treated stimulator cells, and at least a 10-fold impairment for membrane fragments. Experiments showing that the capacity of cell monolayers to absorb precursor T_c from unprimed spleen populations was reduced following uv-irradiation or glutaraldehyde treatment provided direct evidence that this loss of immunogenicity was due in part to suboptimal antigen presentation to precursor T_c. It is concluded that, in addition to the traditional view that these treatments damage the “LD” signal to helper T lymphocytes, metabolic inactivation also impairs recognition of “CD” determinants by precursor T_c.     

Resistance to tolerance induction in B cells of autoimmune mice—Abnormal expansion of low affinity IgG-producing B-cell population

Several strains of mice are known to develop spontaneous autoimmune diseases like lupus erythematosus and they show various immunological abnormalities as well. Despite different genetic backgrounds, they manifest various immunological abnormalities in common, e.g., polyclonal B-cell activation (PBA) and resistance to tolerance induction. To elucidate mechanisms of the development of autoimmunity, tolerance inducibility was examined in autoimmune and normal mice using trinitrophenylated carboxymethyl cellulose (TNP-CMC) as tolerogen which is known to induce TNP-specific B-cell tolerance without the participation of T cells. NZB and MRL/Mp-lpr/lpr mice were used as autoimmune mice and C57BL/6, BALB/c, and MRL/Mp-+/+ mice as nonautoimmune mice. When TNP-CMC-injected mice were challenged with T-independent antigens, all of the mice tested were shown to be tolerant. In contrast, when TNP-CMC-injected mice were challenged with T-dependent antigen and secondary IgG responses were assessed, autoimmune mice showed rather hyperreactivity, while nonautoimmune mice showed hyporesponsiveness. Cyclophosphamide improved this defective tolerance inducibility. By the solid-phase radioimmunoassay it was revealed that average affinity of serum anti-TNP antibodies produced in TNP-CMC-injected mice was low. Such low affinity antibodies were produced in large amount in autoimmune mice. Hence, it was suggested that B-cell clones destined to produce low affinity IgG antibodies were responsible for the resistance to tolerance induction and such clones were expanding in autoimmune mice.     

Evidence for the occurrence of two forms of β-lipotropin in rat pituitary

A peptide isolated from porcine gut according to its glucagon-like activity in liver (bioactive enteroglucagon) has been characterized immunologically, biologically and chemically: its potency relative to pancreatic glucagon in interacting with an antiglucagon antibody, hepatic glucagon-binding sites and hepatic adenylate cyclase was ～100%, 20% and 10%, respectively. In contrast, it is ～20-times more potent than glucagon in oxyntic glands, justifying the term ‘oxyntomodulin’. Chemically, it consists in the 29 amino acid-peptide glucagon elongated at its C-terminal end by the octapeptide Lys—Arg—Asn—Lys—Asn—Asn—Ile &;—Ala; accordingly, it is called ‘glucagon-37’     

Identification of a calmodulin-dependent glycogen synthase kinase in rabbit skeletal muscle, distinct from phosphorylase kinase

A glycogen synthase kinase that is completely dependent on Ca2+ and calmodulin has been identified in mammalian skeletal muscle, and purified approximately 3000-fold by chromatography on phosphocellulose and calmodulin--Sepharose. The presence of 50 mM NaCl in the homogenisation buffer was critical for extraction of the enzyme. The calmodulin-dependent glycogen synthase kinase (app. Mr 850 000) is distinct from myosin light-chain kinase and phosphorylase kinase, but phosphorylates the same serine residue on glycogen synthase as phosphorylase kinase. The physiological role of the enzyme is discussed.     

Lyt phenotype analysis of the effector cells responsible for evoking lymphocyte-induced angiogenesis (LIA)

Lymphocyte-induced angiogenesis (LIA) is a vascular response observed when allogeneic or semiallogeneic immunocompetent lymphocytes are inoculated intradermally into immunosuppressed or irradiated host mice. The reported experiments were carried out to characterize the effector cell population(s) responsible for causing LIA. Lyt 1.2, Lyt 2.2, and monoclonal Thy 1.2 antisera were used for negative selection with complement (C′) to investigate the ability of selected subsets of lymphocytes to evoke angiogenesis. Treatment of C57BL/6 spleen cells with either anti-Lyt 1.2 or anti-Thy 1.2 and C′ resulted in an almost complete abrogation of the LIA reaction. In contrast, depletion of Lyt 2+ cells, under conditions which fully abrogated their ability to generate cell-mediated cytotoxicity in allogeneic mixed leukocyte cultures, resulted only in a partial (45%) reduction in the induced vascular response. Synergistic interaction between cell preparations treated separately with either anti-Lyt 1.2 or anti-Lyt 2.2 serum was not observed. We conclude that (i) Lyt 1 + 2?T lymphocytes can induce a significant LIA reaction; (ii) lymphocytes resistant to negative selection with anti-Lyt-1.2 serum are incapable of inducing such a reaction; and (iii) Lyt 1 + 2+ cells directly or indirectly play an additional role in generating a maximal LIA response.     

Activation and mechanism of action of suppressor macrophages

Intravenous administration of Corynebacterium parvum to alloimmunized mice activates splenic suppressor macrophages that effectively curtail primary and secondary generation of cytotoxic T lymphocytes (CTLs) in vitro. CTL generation was significantly inhibited in suppressed primary cultures by Day 3, the earliest time point that activity is first detected in control cultures. Suppressor macrophages had to be present during the first 24–48 hr of culture to effectively curtail the generation of CTLs. However, if suppressor macrophages were reactivated by 48-hr in vitro culture and then added to primary sensitizations that had been initiated 48 hr previously, they were capable of significant suppression. Suppressor cells produced a soluble factor that mediated the inhibition of CTL generation. The production or action of this factor could not be counteracted by indomethacin.     

The effect of interferon on lymphocyte-mediated effector cell functions: selective enhancement of natural killer cells.

The effect of human interferons on different types of lymphocyte-mediated killer assays was explored. Killing by T cells generated through mixed lymphocyte cultures as well as antibody-dependent lymphocyte-mediated cytotoxicity was not influenced by the addition of interferon. Enhancement of cytolysis produced by natural killer cells was observed when interferon was added during the assay, but enhancement could also be induced if the effector cells were pretreated with interferon for 2 hr prior to the lytic reaction. Killing of a cell line susceptible to natural killing was increased and a cell line which is normally relatively resistant to this type of killing became a susceptible target.     

Inhibition of modulation of thymus-leukemia antigens by concanavalin A.

When incubated at 37 °C in medium containing antibodies specific for thymus-leukemia (TL) antigens, viable cells bearing these antigens become resistant to the cytolytic effects of guinea pig complement, a process termed antigenic modulation. Antibody-induced membrane redistribution of the TL antigens, detected by indirect immunofluorescence, occurs with a similar pace. When high concentrations of concanavalin A (Con A) were included with antibodies in the incubation medium, TL antigenic modulation as well as antigen patching and capping were markedly inhibited, similar to effects of Con A on membrane immunoglobulin redistribution with murine spleen cells. Colchicine antagonized the inhibition by Con A suggesting the involvement of microtubules. In parallel experiments high concentrations of Con A failed to alter the quantity of TL antigen expression or its rate of change with time during incubation in cognate antisera. These results support the hypotheses that (a) generalized alterations in membrane receptor mobility may be induced by ligand binding to the cell membrane, and (b) under certain conditions stable interactions occur between normally independent cell surface antigens.     

Immunological properties of Fc receptor on lymphocytes. 1. Functional differences between Fc receptor-positive and negative lymphocytes in humoral immune responses.

Using an EA rosetting system, it was observed that Fc receptors (FcR) were present on the surface of T cells as well as B cells, and that functional differences existed between FcR-positive (FcR⁺) and FcR-negative (FcR^?) cells in both T and B cells in in vivo humoral immune responses. Approximately 15% of splenic T cells obtained by nylon wool passage are FcR⁺. The number of surface immunoglobulinbearing cells as detected by immunofluorescent staining accounted for less than 10% of these FcR⁺ cells. FcR⁺ and FcR^? T+B-cell populations obtained from spleens contain 60 and 20% of surface immunoglobulin-positive cells, respectively. In the adoptive primary response in which horse RBC and dinitrophenyl-conjugated dextran (DNP-DE) were used as T-dependent and T-independent antigens, respectively, the majority of precursor B cells were FcR^?. In the secondary response using hapten-primed B cells and carrier-primed T cells, the majority of memory B cells for a haptenic determinant were also FcR^?. Furthermore, the majority of functional cells exerting helper activity in the same hapten-carrier system are FcR^? cells, and FcR⁺ T cells collaborate much less effectively with either memory B cells or helper FcR^? T cells.