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Biomedical subjects

H R MacDonald

Publications and source records attributed to H R MacDonald.

At least 235 records · Page 13Linked to original sources
PubMed1986

Dissociation of interleukin 2-dependent and -independent B cell proliferation with monoclonal anti-interleukin 2 receptor antibody.

In a recent report from our laboratory (J. Exp. Med. 1984. 160: 1070), it has been shown that B cells which have been activated with lipopolysaccharide (LPS) plus anti-Ig antibodies express interleukin 2 (IL2) receptors and proliferate in response to pure IL2. In the present study, we have investigated the role of IL2 during proliferative B cell responses supported by various T cell supernatants (SN) as well as by LPS itself. The following results were obtained: (a) B cells activated with LPS plus anti-Ig were found to proliferate in response to either recombinant IL2, T cell SN or fresh LPS; (b) a monoclonal anti-IL2 receptor antibody (PC61) could completely inhibit the effects of recombinant IL2 or various T cell SN (cloned T helper cell SN, EL4 SN, concanavalin A-spleen cell SN or mixed leukocyte culture SN), but did not interfere with the effect of LPS; (c) B cells activated with LPS or LPS plus anti-Ig were not found to secrete detectable amounts of IL2 by themselves. Thus, the data demonstrate that LPS-stimulated B cell proliferation requires neither B cell autocrine nor T cell-derived IL2. On the other hand, with regard to LPS plus anti-Ig-activated B cells, IL2 appeared to be the only growth-promoting activity that could be detected in the supernatants obtained from total normal spleen cell populations.

Animals↗
PubMed1986

Intracellular pathway of interleukin 2 following receptor-mediated endocytosis.

Electron microscope autoradiography was used to examine the intracellular pathway of radioiodinated interleukin 2 [( 125I]IL2) following its receptor-mediated endocytosis in CTLL cells. Direct measurement of the kinetics of endocytosis showed that 60-70% of surface-bound [125I]IL2 was internalized after 1 h at 37 degrees C. [125I]IL2 was observed to enter cells through invagination of coated pits at the cell surface and to concentrate into lysosomal multivesicular bodies as early as 5-10 min after endocytosis. These results provide ultrastructural information on the intracellular pathway of IL2 and on its probable site of degradation within the cell.

Biological Transport↗
PubMed1985

Differential requirements for the induction of interleukin 2 responsiveness in L3T4+ and Lyt-2+ T cell subsets.

Minimal requirements for the induction of interleukin 2 (IL-2) responsiveness in purified subsets of murine T lymphocytes have been investigated. Whereas Lyt-2+ cells could be induced to IL-2-dependent growth by lectin, phorbol ester, or calcium ionophore, none of these stimuli was by itself sufficient for L3T4+ cells. The latter cells could, however, be induced to respond to IL-2 by combinations of lectin plus phorbol ester or ionophore plus phorbol ester (but not lectin plus ionophore). Under optimal conditions, growth of L3T4+ cells (like Lyt-2+ cells) was independent of accessory cells and cell-cell contact.

Animals↗
PubMed1985

Production and characterization of monoclonal anti-Thy-1 antibodies that stimulate lymphokine production by cytolytic T cell clones.

In an effort to derive monoclonal antibodies (mAb) which can induce production of macrophage-activating factor (MAF) by cloned murine cytolytic T lymphocyte (CTL) lines, we have fused spleen cells from a rat immunized with a CTL clone with the nonsecreting mouse myeloma X63-Ag8.653. Three mAb (designated I-22, III-5 and V-8) were found to stimulate MAF production by the immunizing CTL clone and (with a single exception) two other unrelated CTL clones. However, none of these mAb inhibited the cytolytic activity of the clones. Immunoprecipitation studies indicated that the three mAb reacted primarily with a 25-30-kDa protein which could not be distinguished from that precipitated by either a reference anti-Thy-1.2 mAb or a polyclonal rabbit anti-Thy-1 antiserum. Moreover, competition binding experiments demonstrated that the three mAb competed with each other and with the reference anti-Thy-1.2 mAb. Flow cytofluorometric analysis of the strain distribution of the molecules defined by the mAb revealed that two of the antibodies (I-22 and III-5) were directed against nonpolymorphic determinants of Thy-1, whereas V-8 mAb reacted only with Thy-1.2+ lymphocytes. One of the mAb (III-5) was also able to stimulate proliferation and interleukin 2 secretion by normal splenic T cells. Since mAb directed against a number of other surface structures on CTL clones did not stimulate MAF production, it thus appears that Thy-1 (or molecules associated with Thy-1) may play a functional role in T lymphocyte triggering.

Animals↗
PubMed1985

Secretion of IgG1 induction factor by T cell clones and hybridomas.

IgG1 induction factor elevates the IgG1 response induced by lipopolysaccharide and suppresses the lipopolysaccharide-induced IgG3 and IgG2b responses in cultures of mouse spleen cells. We have developed new T cell lines secreting this factor by cloning mixed lymphocyte culture populations. Using supernatants of one of these T cell lines it was found that the assay is quantitative, reproducible and accurate, both when induction of IgG1 as well as reduction of IgG3 and IgG2b were measured. Using this analysis, different conditions to induce maximal production of the factor were tested. The cell line was thereafter used as fusion partner with a T cell lymphoma. The hybrids were selected in the presence of T cell growth factor and all of them secreted IgG1 induction factor.

Animals↗
PubMed1985

Antigen stimulation of cytolytic T lymphocyte precursors: minimal requirements for growth and acquisition of cytolytic activity.

Minimal requirements for growth and acquisition of cytolytic activity by alloantigen-stimulated cytolytic T lymphocyte precursors (CTL-P) have been investigated. CTL-P from C57BL/6 spleen were purified by staining with monoclonal anti-Lyt-2 antibodies followed by positive selection on a fluorescence-activated cell sorter. Microcultures containing 2000 purified Lyt-2+ cells were set up with irradiated P815 (DBA/2 mastocytoma) stimulating cells and pure interleukin 2 obtained by recombinant DNA technology (rIL2). Proliferation and generation of antigen-specific cytolytic activity were observed to be both antigen and IL2-dependent in such microcultures. Furthermore, P815 stimulating cells could be replaced by 5 other H-2d cell lines of differing tissue origin. In limiting dilution studies, 1-2% of Lyt-2+ CTL-P (contaminated by less than 0.2% macrophages) could be induced to grow and express cytolytic activity in the presence of irradiated P815 and rIL2. Taken together, these data indicate that neither accessory cells nor differentiation factors (other than IL2) are absolutely required for alloantigen-induced growth and differentiation of CTL-P.

Animals↗
PubMed1985

Stimulator requirements for primed alloreactive T cells: macrophages and dendritic cells activate T cells across all genetic disparities.

The cellular requirements for stimulating primed alloreactive T cells have been investigated. In vitro-primed secondary alloreactive cells, long-term lines, and Ly 1+2- noncytolytic clones which reacted with allo-H-2K, D, or Mls (M locus) antigens were tested. The data indicated that a specialized antigen-presenting cell such as a macrophage or a dendritic cell was required for stimulating primed alloreactive cells across all the genetic disparities tested. B and T lymphocytes were ineffective stimulators. The stimulator requirement for secondary and Ly 1+2- clone responses was heterogeneous, since both macrophages and dendritic cells were effective stimulators. Thus, the allostimulator requirement for inducing proliferation and mediator secretion by the primed T-cell populations closely paralleled the requirement for stimulating unprimed populations. The only exception found was the peritoneal washout population, which did not stimulate a primary response but did stimulate secondary responses. The failure of peritoneal macrophages to stimulate a primary response was shown to be due to an inhibitory pathway which did not occur when the responding population was alloantigen primed.

Animals↗
PubMed1985

Clonal analysis of cytolytic T lymphocyte-mediated lysis of target cells with inducible antigen expression: correlation between antigen density and requirement for Lyt-2/3 function.

The lysis by allospecific cytolytic T lymphocytes (CTL) of the BALB/c lymphoma ST-4.5, a cell line that can be induced by interferon-gamma (IFN-gamma) to express increased amounts of major histocompatibility complex (MHC) class I antigens, was investigated. Culture of ST-4.5 in IFN-gamma increased the surface expression of Kd molecules from originally low levels and Dd from undetectable amounts by approximately fivefold as determined by fluorescence-activated cell sorter (FACS) analysis, whereas the levels of several other antigens (Ld, I-Ad, Thy-1, Lyt-2, L3T4, and LFA-1) were not affected. The lysis of ST-4.5 by Dd- and Ld-specific CTL clones correlated with the expression of those antigens on target cells as determined by both FACS and biochemical analysis. Lysis of ST-4.5 by CTL clones specific for Kd antigen fell into two distinct groups: those that could lyse targets cultured either normally or in IFN-gamma, and those that could only lyse targets that had been precultured in IFN-gamma. The apparent sensitivity to antigen exhibited by the Kd-specific CTL clones predicted their sensitivity to inhibition of target lysis by anti-Lyt-2/3 antibody. Those CTL clones that were only active against ST-4.5 expressing higher amounts of surface antigen (resulting from IFN-gamma preculture) were readily inhibited by anti-Lyt-2/3 antibody, whereas those CTL capable of lysing normally cultured targets having lower amounts of surface antigen were heterogeneous in their sensitivity to anti-Lyt-2/3; some were inhibitable, whereas others were resistant. In addition, another CTL clone that was resistant to inhibition by anti-Lyt-2/3 alone was readily inhibited by a synergistic combination of anti-Lyt-2/3 plus anti-Kd (but not anti-Dd or Ld) antibodies. These results indicate that CTL antigen receptor sensitivity to (or affinity for) antigen and the level of specific antigen expression by the target cell may both be important criteria in assessing Lyt-2/3 molecule function in CTL-mediated cytolysis. The function of recognition-associated molecules such as Lyt-2/3 may be to strengthen and increase the number of receptor-ligand binding events that facilitate CTL-target membrane interactions that lead to the lysis of the target cell.

Animals↗
PubMed1985

Cell surface glycoproteins involved in the stimulation of interleukin 1-dependent interleukin 2 production by a subline of EL4 thymoma cells. I. Functional characterization by monoclonal antibodies.

Three rat monoclonal antibodies (MAb) capable of stimulating interleukin 2 (IL 2) production by a variant subline of EL4 thymoma cells (EL4-6.1) have been produced. The stimulatory capacity of these MAb (designated RL73, RL119, and RL388) was originally found to be dependent on the presence of irradiated peritoneal exudate cells; however, this requirement could be replaced by the cellfree supernatant of the "macrophage-like" cell line P388D1 or by biochemically purified human interleukin 1 (IL 1). A number of other rat MAb directed against cell surface structures did not stimulate IL 1-dependent IL 2 production by EL4-6.1 cells; however, certain MAb directed against Thy-1 as well as the lectin phytohemagglutin did have this capacity. Furthermore, the stimulatory activity of MAb RL73, RL119, and RL388 appeared to be restricted to the EL4-6.1 variant line, because neither the parental EL4 line from which it was derived nor a series of ovalbumin-specific T-T hybrids responded to these MAb. The cell surface antigens recognized by MAb RL73, RL119, and RL388 were present on a wide variety of T cell lines and T-T hybrids, as well as on lines of B cell, macrophage, and fibroblast origin. Interestingly, the MAb reacted with the majority (approximately 85%) of thymocytes but not (or only to a very small extent) with resting T lymphocytes. After stimulation by concanavalin A, however, the three MAb reacted strongly with activated T lymphoblasts. The latter data suggest that MAb RL73, RL119, and RL388 may react with cell surface structures that are normally expressed as a consequence of lymphocyte activation.

Animals↗
PubMed1985

Antigenic stimulation regulates the expression of IL 2 receptors in a cytolytic T lymphocyte clone.

We have isolated and characterized an H-2Kb-specific CTL clone, B1.8, whose growth is strictly dependent on IL 2 and periodic exposure to stimulator cells. A single, brief encounter with antigen induces on resting B1.8 cells the expression of high-affinity IL 2 receptors, which is maintained for several days, thus allowing cells to undergo at least six rounds of division in the presence of affinity-purified IL 2. We show with the use of monoclonal anti-Lyt-2 or -H-2Kb antibodies, which block specific antigen recognition by B1.8 cells, that continuous antigenic stimulation is not required during this period of growth. After this time, B1.8 cells lose their ability to respond to IL 2 and revert to a resting state, a process that is reversed upon addition of fresh stimulator cells. The decline and subsequent arrest of cell growth correlates with a loss of IL 2 receptors; growth-arrested cells express a 10-fold lower number of receptors than actively growing cells.

Animals↗
PubMed1985

Interleukin 2 is both necessary and sufficient for the growth and differentiation of lectin-stimulated cytolytic T lymphocyte precursors.

Minimal requirements for the growth and differentiation of lectin-stimulated cytolytic T lymphocyte precursors (CTL-P) have been investigated. CTL-P from murine spleen were purified by staining with monoclonal anti-Lyt-2 antibodies, followed by positive selection on a fluorescence-activated cell sorter. Microcultures containing 2000 purified Lyt-2+ cells were set up with optimal concentrations of lectin (leukoagglutinin or concanavalin A) and pure interleukin 2 (IL 2) obtained either by immunoaffinity chromatography or recombinant DNA technology. Proliferation and generation of CTL activity in lectin-stimulated Lyt-2+ cells were IL 2 dose-dependent. Furthermore, no increased response was observed when crude supernatants containing other putative CTL differentiation factors were added, even when IL 2 concentrations were suboptimal. When highly purified Lyt-2+ cells contaminated by 1% Lyt-2- cells and less than 0.1% macrophages were cultured at limiting dilution, as few as 20 responding cells proliferated and differentiated into CTL in the presence of leukoagglutinin and pure IL 2; in fact, CTL activity and proliferation were strongly correlated in individual microcultures. In addition, 10 to 30% of individual CTL-P were growth-inducible under these experimental conditions. Together, these data show that the proliferation and differentiation of lectin-stimulated CTL-P can occur in pure IL 2 without any demonstrable requirement for other differentiation factors or accessory cells.

Animals↗
PubMed1985

Biosynthesis and maturation of the Lyt-2/3 molecular complex in mouse thymocytes.

The biosynthesis and maturation of the three subunits alpha (Mr = 37,000), beta (Mr = 32,000), and gamma (Mr = 27,000) of the mouse Lyt-2/3 antigenic complex have been studied by using two monoclonal antibodies directed against a monomorphic determinant of the Lyt-2 antigen. Short time-pulse labeling of thymocytes reveals three different high mannose intermediates that give rise upon endo-beta-N-acetyl-glucosaminidase H digestion to three distinct precursor polypeptides of Mr = 22,000 (alpha P), Mr = 18,000 (beta P), and Mr = 19,500 (gamma P). Pulse-chase analysis indicates rapid posttranslational processing, because mature forms already appear after 10 min of chase. The half-life of the endo-H-sensitive early forms are in the range of 20 to 30 min. Both the alpha and beta subunits are suggested to contain three N-asparagine-linked oligosaccharides, one of which is of the high mannose type. In contrast, the gamma-chain contains only one such glycan unit of the complex type. Moreover, the results presented show that all three chains undergo additional posttranslational modifications. Finally, the data suggest that the cytoplasmic domains of these chains are of different size.

Animals↗
PubMed1985

Cell surface glycoproteins involved in the stimulation of interleukin 1-dependent interleukin 2 production by a subline of EL4 thymoma cells. II. Structure, biosynthesis, and maturation.

In the present study, we examined the biosynthesis and the maturation of two distinct membrane glycoproteins detected by two monoclonal antibodies (RL388 and RL119), which were selected on the basis of their ability to stimulate the production of interleukin 2 by a subline of the murine EL4 thymoma. RL388 detected a disulfide-linked heterodimer complex (Mr = 130,000) composed of a glycosylated heavy (Mr = 86,000) and a nonglycosylated light (Mr = 39,000) subunit. The unglycosylated precursor of the heavy chain was a polypeptide of Mr = 57,500, which was converted upon maturation into a Mr = 73,000 core-glycosylated intermediate, and then into the Mr = 86,000 surface-expressed molecule. Partial endo-H digestion of the core-glycosylated form suggested the presence of four N-linked glycan units. The antibody reacted with a protein determinant expressed on the mature form as well as the unglycosylated precursor of the heavy chain. Moreover, both subunits assembled rapidly during biosynthesis, and the glycosylation of the heavy chain was not required for this association. Taken together, these data suggest that the antigen detected by RL388 may be the murine homologue of the human 4F2 antigen. The antigen identified by RL119 was a surface glycoprotein of Mr = 55,000 with three to five N-linked glycan units. The unglycosylated precursor polypeptide was of Mr = 29,000. The fully core-glycosylated form of Mr = 41,000, which was detected after inhibition of glucosidase I with 1-deoxynojirimycin, was converted into a Mr = 39,000 intermediate, and upon further trimming, into a Mr = 36,000 endo-H-sensitive form. The latter could be detected for chase periods of over several hours, thus suggesting a low rate of intracellular processing. The wide cellular distribution of the molecules identified by RL388 and RL119 and their preferential expression on the surface of growing cells suggests that they may be associated with cell activation events.

Animals↗