A small percentage of normal mouse thymus and spleen lymphocytes form rosettes with autologous erythrocytes. The number of these autologous rosettes increases 15- to 20-fold after adult thymectomy and to a lesser degree with aging. Autologous rosette level is also abnormally high in nude (congenitally athymic) mice. The high level of autologous rosette-forming cells found after adult thymectomy is normalized by injecting ng amounts of purified circulating thymic factor. Autologous rosette-forming cells adhere to nylon, belong to the less dense spleen cells, are in majority steroid-resistant in the thymus. All these properties suggest that autologous rosette-forming cells might belong to immature T-cell (thymic-dependent cell) precursors.
Publisher Summary This chapter discusses the histocompatibility antigens of the guinea pig. Histocompatibility antigens in guinea pigs have been detected serologically either by the classical NIH eosin microlymphocytotoxicity technique or by 51Cr release lymphocytotoxicity. Four different loci linked on the same chromosome have been recognized up to now. The so-called first GPLA locus (B locus) has at present three well defined alleles, Bl, B2, and B3, and a fourth tentative allele, B4. The membrane antigens governed by Bl, B2, or B3 genes appear to be present on nonlymphoid cells. The GPLA locus B seems to be equivalent to the K (or D) locus of the H2 complex in mice.
This chapter presents a report of an examination of pure bone marrow derived colonies of mouse neutrophils, macrophages, and eosinophils grown in vitro during maturation for appearance and properties of receptors for C3 and immunoglobulin G (IgG). C3 receptor analysis of different colony types obtained from bone marrow cultures for this examination showed that colony macrophages and neutrophils but not colony eosinophils formed EAC rosettes. The chapter presents proportions of colony cells forming EAC rosettes harvested at different times of culture. In 3-day-old colonies, 70% macrophages were EAC positive, and in older colonies, 6–12 days, the value reached 90%. Neutrophilic colony cells also exhibited C3 receptors, but a significant proportion of EAC reactive cells was observed only after the fourth or fifth day of culture, and the EAC reactive cell population never exceeded 60%, even within morphologically well-differentiated colonies. The presence of receptors for IgG on colony cells was investigated by measuring the binding capacity of cells to complexes formed with 7S antibody-sensitized erythrocytes (EAzs). Neutrophil and eosinophil colony cells also bear a receptor for IgG but they only comprise half of the colony cells. The chapter presents the results of the examination that showed that macrophage and neutrophil colony cells grown in vitro from mouse bone marrow have receptors for both EAC1-3 mouse and EA7S, and that eosinophil colonies carry only receptors for EA7S. The characteristics and properties of these receptors on colony cells closely resemble those on macrophages and neutrophils obtained directly from peritoneal fluid and bone marrow. The chapter also presents further evidence of the examination that colony cells from bone marrow hematopoietic progenitor cells are capable of a high degree of differentiation and that cell membrane markers are of great value for monitoring the differentiation capacity of hematopoietic populations cloned in vitro.
This chapter discusses that lymphocyte-dependent antibody (LDA) cytotoxicity requires the presence of intact IgG molecule of anti-target antibody, which interacts with Fc receptor of the effector cells by its Fc portion. In the rabbit, the cell that mediates LDA cytotoxicity can be found in central lymphoid organs such as thymus and bone marrow as well as in peripheral lymphoid organs such as the spleen. The chapter discusses the characterization of a bridge or bridges between various receptors of effector and target cells in terms of lymphocyte-dependent antibody cytotoxicity. It describes the influence of heterologous anti-rabbit isotypic antibodies on LDA cytotoxicity of rabbit lymphocytes against ChRBC, coated with rabbit antitarget antibodies. The enhancement induced by antiallotype antibodies is an immunological phenomenon. The bridge formed by activated C3—between target cells coated with anti-Forssman antibody and the C3 receptor of effector cells—cannot induce hemolysis. Only the bridge established between IgG molecules of antibodies coating the target cells and the Fc or IgG receptors of effector cell could mediate the LDA cytotoxicity.
This chapter provides an overview of the interaction of lectins with human lymphocytes. The lymphocyte surface bears receptors that are related to the function and differentiation of these cells. The receptors for lectins consist of the carbohydrate chains of glycoproteins and possibly also glycolipids exposed on the external cell membrane. In an experiment described in the chapter, the receptors were studied by a surface-labeling technique. Lymphocytes contain several glycoproteins. Several of these glycoproteins act as receptors for the Ricinus communis agglutinin, concanavalin A, kidney bean leucoagglutinin, wheat germ, and soybean agglutinins. Of these lectins, concavalin A and leucoagglutinin are strongly lymphocyte-stimulating, while the Ricinus communis agglutinin inhibits DNA synthesis. The wheat germ and soybean agglutinins are neither toxic nor stimulating for normal lymphocytes. Several major membrane glycoproteins contain receptors for lymphocyte-stimulaing and nonstimulating lectins. In transforming lymphocytes, the external glycoprotein pattern changes and a protein that is strongly labeled without galactose oxidase treatment appears.
This chapter presents the genetic control of host versus graft (HvG) and graft versus host (GvH) reactions in dog bone marrow (BM) transplantation. Four loci are currently recognized in the major histocompatibility complex of the dog (DL-A), two coding for serologically defined (SD) and another two for lymphocyte defined (LD) structures. The chapter describes an experiment in which BM grafts were performed in this animal model to elucidate the genetic control of HvG and GvH reactions. In this experiment, the recipients received 750–800 rad total body irradiation (TBI) from two opposing X-ray machines and were subsequently treated with 4 x 108 allogeneic BM cells/kg body weight. The currently available methods of donor selection appeared to be insufficient for the avoidance of severe HvG and GvH reactions, thus, it is prevented by the addition of intravenous injection administration of silica particles to a conditioning regimen of TBI.
The human mixed lymphocyte reaction ( MLR ) has been shown to be controlled by the genes of the major histocompatibility complex distinct from but closely linked to the four locus of the HL-A region. This chapter describes an experiment that demonstrates that the two structures MLR-S inducing stimulation and MLR-R responsible for allogeneic recognition are expressed on the surface of different cells. In the first series of experiments, the effects of the two heterologous antisera, one specific for the human T cells (anti-HuTLA) and the other for the non-T lymphocytes, was investigated. They were found to react with monocytes in addition to B cells. Both antisera were found to block the MLR. These results were confirmed by culturing B- and T-enriched populations obtained by the formation of E rosettes and subsequent centrifugation on Ficoll isopaque. The results demonstrate that all the responding cells belonged to the T population (HuTLA and E rosette positive) and their activation was initiated only by the B lymphocytes or/and monocytes.
This chapter describes an experiment in which human lymphocytes, isolated from peripheral blood, were stimulated with phytohemagglutinin (PHA)-M prior to being seeded on a two-layer medium of soft agar. In the experiment, the number and size of colonies were not influenced by addition to the culture media of leukocyte feeder layers (106cells/ml) or of leukocyte-conditioned medium. A significant enhancing effect on the cloning potential of lymphocytes resulted from the addition of feeder layers of autologous, allogeneic, or mixed lymphocytes, prepared from peripheral blood. These supplements increased the number of colonies developing by 70%. Addition to the cultures of conditioned medium produced by 107 lymphocytes per ml (LCM) caused a similar enhancement. On the other hand, conditioned medium prepared from human spleen cells (SCM) (107cells/ml) inhibited the development of colonies from human peripheral lymphocytes by 66%. From these results, it can be inferred that the stimulation of the formation of lymphocyte colonies by feeder layer from normal lymphocytes and lymphocytes per ml (LCM) indicates the production of a colony stimulating factor. SCM appears to contain factors that promote the growth of macrophages and granulocytes and inhibits the development of lymphocyte colonies.
Previous in vivo studies suggests that the expression of H-Y antigen on male tissues and the response by female hosts are at least in part determined by the H-2 complex This chapter discusses in vitro studies of H-2 restricted T-cell cytotoxic responses to H-Y antigen. From the studies, it can be concluded that the secondary in vitro cytotoxic responses to H-Y antigen are mediated by cytotoxic T-lymphocytes and the H-Y target cell antigen is specified by the H-2 complex. The results suggest that the H-Y target antigen maps at the D-end of H-2 in an H-2b system and at the K-end in an H-2d system. F1 female cells sensitized to one parental male cell lyse only targets homologous with that parental cell. The expression of H-Y antigen on male tissues and the response by female hosts are determined by the H-2 complex.
Several studies have revealed that within 1 day of injecting mice or rats with antigens, such as heterologous erythrocytes, lymphocytes reactive to these antigens are specifically withdrawn from the recirculating lymphocyte pool and become sequestered in organs, such as the spleen. In mice injected with H2-incompatible spleen cells, the capacity of thoracic lymphocytes (TDL) to evoke a graft-versus-host (GVH) reaction against the injected determinants is found to be specifically abolished at 1–2 days, near normal on day 3, and above normal on day 5. This chapter describes studies that were designed to determine whether the unresponsiveness towards the GVH determinants also applies to cells producing a mixed-lymphocyte reaction (MLR), allograft rejection, and cell-mediated lympholysis (CML). The results from the studies showed that injecting parental strain mice with irradiated H2-incompatible spleen cells virtually abolished the capacity of TDL, collected 1 day later, to respond specifically to determinants controlling the GVH reaction, allograft rejection, and CML. The near normal reactivity of cells taken from the spleen at this time suggests that the injected irradiated cells carrying these various H2 determinants induced antigen-specific selective recruitment of recirculating lymphocytes (ASRL) reactive to those determinants to organs such as the spleen.
Human hemopoietic cells can be characterized by the presence of an array of unique and shared receptors on their surface. These surface receptors can be used as differentiation markers because they have been shown to be present on cells only at specific stages of development. Thymocytes and peripheral T lymphocytes have receptors for sheep erythrocytes and thus form E rosettes. B lymphocytes, monocytes, and mature granulocytes have complement receptors and form EAC rosettes. B lymphocytes alone have receptors for mouse erythrocytes and form M rosettes. The expression of all of these markers has been evaluated on all cell fractions after incubation with or without inducers. This chapter reviews some experiments to discuss the induction of surface receptors on granulocyte, T, and B-cell precursors in human bone marrow by thymic and non-thymic agents. It provides an overview of the methods used in the experiments and the results of the experiments. These experiments suggest that the cell separation procedures provide sufficiently homogeneous cell populations to allow identification of the cells responsive to the polypeptide hormones and cyclic nucleotide studied. These agents induce the expression of the receptor for sheep erythrocytes by a direct action on lymphocytes, and the complement receptor by a direct action on immature granulocytes. Some induction of a B-cell marker was observed in the experiments, but it is not clear whether this is due to a direct effect of thymopoietin on B cells or an indirect effect via thymopoietin influence on T cells. The chapter further presents several examples of the hormonal induction of hematopoietic differentiation.
Murine lymphoid cells sensitized against allografts express two types of cell-mediated cytotoxicity (CMC) measured in vitro in short-term assay of 2–3 hr by 51Cr release. One type is specific against target cells carrying the sensitizing alloantigens, and the other type is nonspecific against syngeneic, allogeneic, and xenogeneic target cells and is dependent on the presence of phytohaemagglutinin (PHA) or concanavalin A (Con A). This chapter presents several lines of evidence demonstrating that lectin-dependent cell-mediated cytotoxicity (LDCC) and specific CMC are mediated by the same effector T-cell subpopulation: (a) treatment of immune lymphocytes with anti-θ serum and complement abrogates both types of cytotoxicity; (b) absorption of immune cells on corresponding target cell monolayers depletes the non-adherent fraction of both specific CMC and LDCC; (c) kinetics of in vivo development of cytotoxic lymphocytes parallel the kinetics observed in LDCC; (d) LDCC due to either polyclonal mitogenic activation of cytotoxic cells or antibody dependent cellular cytotoxicity were ruled out; and (e) specific CMC is not augmented by lectin. In humans, normal peripheral blood leukocytes (PBL) are also found to be cytotoxic to target cells in the presence of PHA or ConA. The chapter further discusses the results in murine systems, which demonstrate that a single clone of alloimmune T lymphocytes can perform specific CMC through antigen-binding receptors and nonspecific CMC through PHA and Con A receptors. By analogy to the murine system, LDCC in human PBL represents a subpopulation of activated T cells and can therefore be utilized as a marker for circulating sensitized T cells.
This chapter presents studies on the mechanism by which one subcellular structure, cytoplasmic microtubule (MT) may interact with membranes to regulate surface topography. The degree of MT assembly and MT–membrane interaction in intact cells varies in a dynamic manner, depending on binding events occurring at the surface. The degree of assembly of MT and extent of MT–membrane interactions appears to be enhanced after ligand or particle binding with surface receptors. This enhancement may be mediated in part via the stimulation of cyclic GMP generation, which seems to increase MT stability. The mobility of proteins in cell membranes is regulated at least in part by MT. An intermediate step, the generation of cyclic GMP may follow ligand or particle binding and determine the stability of MT and/or of MT–membrane interaction. The immobilization of leukocyte membranes by phenylglyoxal is unrelated to changes in lipid fluidity. An arginine-rich intramembranous protein (or proteins) is involved in determining the mobility of proteins in leukocyte membranes and in controlling the deformability of the whole membrane. Although proteins are mobile in cell membranes, it is clear that they do not always diffuse at random in the lipid matrix of the membrane but rather are subject to considerable restraints on mobility.
This chapter discusses a study in which rabbit mesenteric lymph node cells (LNC) were rosetted with goat anti-rabbit Ig antibody-coated erythrocytes (Ab-E). The rosette Ig-bearing cells were separated from unrosetted (Ig-) cells by centrifugation on Ficoll–Hypaque. Ig- cells behaved like T cells; they responded to T-cell mitogens and none of them secreted Ig when tested with rabbit anti-Ig allotype Ab-E for reverse hemolytic plaque formation. The Ig+ cells were further separated into two approximately equal subpopulations when treated with autologous serum and centrifuged on Ficoll–Hypaque: (1) a stable population (Ig+S) and (2) a labile population (Ig+L). The study showed that Ig+L cells differentiated to Ig+S cells in the presence of Ig- cells, but they did not differentiate when Ig+S were already present in the culture. Two subpopulations of Ig+ cells were separated, and one of these, the Ig+L cells, is probably a developmental precursor to the other subpopulation, the Ig+S cells.
Several studies on the mechanism of human lymphocytes has revealed that under optimal experimental conditions, equilibrium interactions between 125I-PHA (phytohemagglutinin) and its specific lymphocyte membrane receptors result in a curvilinear Scatchard plot, suggestive of the heterogeneity of receptor sites with different binding affinity or the existence of site–site interactions. The chapter describes an experiment to demonstrate that PHA-containing sites lower the affinity of adjacent sites for PHA, a phenomenon known as negative cooperativity. The chapter illustrates the Scatchard plot of the binding data obtained under steady state conditions in the experiment conducted. The slope of the line, which reflects the equilibrium constant of the receptor–ligand interactions, shows a progressive change with increasing ligand concentrations. The demonstration of negative cooperativity rests on the displacement of receptor-bound 125I-PHA induced by an excess of PHA, a phenomenon that can easily be measured from the resulting acceleration of the complex dissociation.
The development of cell-mediated lympholysis in the mouse depends on H-2 incompatibility and the MHS controls both the antigens, which serve as targets for destruction and the determinants involved in the generation of effectors. The genes that control the generation of effectors for chronic myelogenous leukemia (CML) are different from the genes that make the targets for destruction in CML testing. Genes controlling the generation of effector capacity are found in the I region of the H-2 gene complex close to but separate from the strong leukocyte adhesion deficiency (LAD) genes. The chapter describes the mapping of the area concerned, called the effector cells stimulating (ECS), carried out using 20 congenic and recombinant mouse strains. This is now been extended to 39 strain combinations. ECS amplification acts on the target cells and is the major genetic factor in the control of antigenic strength as applied to cell-mediated lympholysis.
This chapter presents the results of studies using two types of mouse T-cell populations that are free from contamination by B cells, plasma cells, or mouse serum proteins. The first type of population comprises Thy-1-antigen-bearing T lymphoma cells that have been cloned and grown in continuous cell culture. The second population of cells comprises thymocytes obtained from fetal thymuses taken early in gestation, before the appearance of small lymphocytes, and grown in vitro as organ cultures for one or two weeks. Thymuses cultured for these periods contain newly formed Thy-1-positive lymphocytes but no detectable plasma cells or B cells. Two types of cell populations have been used as models to demonstrate that Ig is exhibited on the surface of Thy-l-positive cells and that this Ig is synthesized by these cells. The chapter also describes certain physico-chemical properties of the surface immunoglobulin (Ig) isolated from these cells.
Although immunoglobulin of the D class is found in only minute amounts in serum, it is a major membrane-bound immunoglobulin of human B-lymphocytes. When cultured in vitro, human B-lymphocytes differentiate into cells, synthesizing and secreting each of the three major serum immunoglobulins—IgG, IgM, and IgA. This chapter presents the effect of antibody to IgD on immunoglobulin synthesis in vitro. Anti-IgD suppresses the synthesis of IgG, IgM, and IgA. The degree of suppression is found to be dependent on the amount of antibody added to the cultures. Suppressive activity is greatly reduced by prior absorption of antibody to IgD on IgD–sepharose but remains unaffected following prior absorption on IgG–sepharose columns. IgD-specific regions of the antibody molecule are responsible for the suppression of immunoglobulin synthesis and the Fc portion of the molecule is not required. The purified antibody to IgD suppresses IgM, IgG, and IgA synthesis by human blood lymphocytes cultured in vitro in the presence of fetal bovine serum.
A peptide consisting of 10 amino acids derived from the CH3 region of human IgG was shown to bind to monocytes and to inhibit rosette formation of antibody-coated erythrocytes with human monocytes. Two myeloma proteins of the IgG1 and IgG3 subclass, both with known deletions in the CH2 region of the gamma chain, showed unimpaired ability to bind to monocytes. These experiments suggest that the isolated peptide represents the primary site of attachment of IgG to monocytes.
This chapter discusses the expression of Ia antigens on different tissues and the possible involvement of Ia antigens in immune reactions. The central part of the mouse H-2 complex, the I-region, is involved in several different immunological phenomena such as genetic control of the immune response, cellular cooperation, mixed lymphocyte reaction, and graft versus host reaction. As many of these reactions are mediated by surface structures of lympoid cells, the recently discovered Ia antigens that are coded for by the I-region are of interest. Recent studies showed that the Ia antigen system is a complex polymorphic system with at least 3 different I-subregions controlling more than 15 different Ia specificities, some of which occur in allelic forms. Knowledge of the involvement of the I-region in immune reactions leads to the assumption that the I-region controlled la antigens participate in these reactions. This hypothesis is examined by attempts to block Ia determinants of lymphocytes participating in in vitro secondary immune responses to sheep red blood cells (SRBC) by inclusion of alloantisera into the cultures in the absence of complement.
