Antigen-binding thymus-derived lymphocytes: II. Nature of the immunoglobulin determinants

Thymus-derived `rosette'-forming lymphocytes which have been separated from other SRBC-sensitive cells by means of cotton wool columns were examined for the presence of immunoglobulin. This was carried out by inhibition of rosette formation by anti-immunoglobulin sera. Inhibition was effected by a number of anti-IgM sera shown to contain antibodies with specificities directed towards the `hinge' region of the μ chain. No other heavy chain specific antisera were inhibitory.

The ratio of rosette inhibition by anti-κ and anti-λ light chain sera varied during the course of the response to SRBC, the latter inhibiting by 89 per cent 3 days post-immunization.

     

Cytophilic properties of surface immunoglobulin of thymus-derived lymphocytes

The cytophilic properties of released surface immunoglobulins of normal thymus lymphocytes and of activated thymus-derived lymphocytes (ATC) were analysed by lactoperoxidase-catalysed radioiodination in conjunction with immunological and autoradiographic techniques. Immunoglobulin from both normal T cells and ATC was cytophilic for macrophages (peritoneal exudate cells), but showed no detectable capacity to bind to either T lymphocytes or to bone-marrow-derived lymphocytes (B cells). Under the operative experimental conditions surface immunoglobulin of B cells did not show appreciable binding to macrophages. These results support the feasibility of models of collaboration between T cells and B cells which involve a soluble antigen-specific collaborative factor (T-cell Ig complexed with antigen) and show an obligatory requirement for macrophages.     

Characterization of thymus-derived and bone marrow-derived rosette-forming lymphocytes

An improved technique has been developed to accurately quantitate lymphoid rosette-forming cells (RFC) and to assess the morphology and antigen-binding ability of each RFC. Two essential features were involved. First, rosettes were fixed with glutaraldehyde (a concentration of 0.6 % minimized the formation of artefacts). This fixation prevented dissociation of rosettes; RFC of weak antigen-binding ability could therefore consistently be preserved. Second, rosette preparations were stained, thus permitting morphological classification of RFC and the elimination of false rosettes. The antigen-binding ability of each RFC was determined by the number of sheep red blood cells (SRBC) bound. By this technique, thymus-derived (T) RFC were found to bind fewer erythrocytes than bone marrow-derived (B) RFC. Pretreatment of the SRBC with neuraminidase increased the SRBC-binding ability of B RFC, thus making the latter readily distinguishable from T RFC. Functionally different subpopulations of T and B RFC were suggested.     

Rat transplantation antigens: II. Solubilization of multiple antigenic specificities

Lymphoid cells of the dark agouti (AgB 4 or Rt H1.1) rat were extracted with 3M KCl. This soluble extract was shown by inhibition of specific, cytotoxic alloantisera to contain the private antigen, AgB 4, and the public antigens, AgB 26, 30 and 29. The public antigens AgB 27 and 28 were not assayed. A species-specific antigen and a non-AgB antigen were also present. Equal concentrations of the various antigens were not obtained. Gel filtration of this extract over Sephadex G-200 yielded AgB 4,26, and 30 and the species antigen at the front of the inner bed volume with a minor AgB 4 peak at about 65,000 mol. wt. AgB 29 and the non-AgB antigen were not recovered. As AgB 29 was present in high concentration in the extract and shown not to be highly labile, it is suggested that it may be present on a molecule distinct from that carrying AgB 4,26 and 30.     

Selective in vitro response of thymus-derived lymphocytes from Treponema pallidum-infected rabbits.

The blastogenic response of nylon wool-separated peripheral-blood lymphocytes from Treponema pallidum-infected rabbits was tested in vitro with mitogens and T. pallidum antigens. The mitogenic response of the enriched T-cell population to concanavalin A and phytohemagglutinin was depressed during the first 3 to 4 weeks of infection, similar to the pattern observed with unfractionated cells. Shortly thereafter, levels of blastogenesis returned to values of uninfected cultures. Enhanced blast transformation was seen immediately when purified T-cells from infected rabbits were exposed in vitro to T. pallidum antigens. Although these relatively high levels of blastogenesis were maintained for the duration of the experiment, cultures of unfractionated lymphocytes from infected rabbits did not exhibit an increased blastogenic response to the same antigen preparation until 3 to 4 weeks after infection. Autologous serum from infected rabbits decreased the lymphocyte response to T. pallidum antigen. The stimulatory effects of anti-immunoglobulin G and lipopolysaccharide on nylon wool-fractionated or unfractionated lymphocytes from both infected and control rabbits were similar throughout the course of infection. During the first 6 weeks of experimental disease, there was a 25 to 31% increase in the number of lymphocytes circulating in the peripheral blood of T. pallidum-infected rabbits.     

Production of xenogeneic and allogeneic antisera specific for mouse thymus-derived lymphocytes.

Xeongeneic and allogeneic antisera were raised in rabbits and mice using purified mouse T cell-specific antigens. These antisera were shown to be reactive in complement and antibody-dependent cell-mediated cytotoxicity tests as well as in immunofluorescence, with 30% of mouse spleen cells, 80% of lymph node cells and 100% of thymocytes or various T lymphoblastoid cell lines labeled. No reaction could be detected with bone marrow cells or non-T cell lines. Both types of reagents bound to the same spleen lymphocyte subpopulations and competed for the same antigenic sites as anti-Thy-1.2 alloantiserum. These antisera raised with small amounts of immunogens did not require any absorption to be rendered specific for mouse T cells.     

The discovery of thymus function and of thymus-derived lymphocytes

Summary: For centuries the thymus remained an enigmatic organ with unknown functions. The first demonstration of its crucial role in establishing the development of a normal immune system was provided in 1961, when it was shown that mice thymectomized immediately after birth had poorly developed lymphoid tissues, impaired immune responses and inordinate susceptibility to intercurrent infections. Although thymus lymphocytes were believed to be immunoincompetent, it was shown in 1967 that they could respond to antigen by proliferating and giving rise to a progeny of cells that could not produce antibody, but enabled other lymphocytes, derived from bone marrow, to differentiate to antibody-forming cells. This was the first unequivocal demonstration, in mammalian species, of the existence of two major interacting subsets of lymphocytes, T and B cells. It required a re-evaluation of many immunological phenomena, such as tolerance, memory and autoimmunity, and it was followed by an avalanche of work elucidating many of the mysteries of the immune system.     

Specific immune lysis of paramyxovirus-infected cells by H-2-compatible thymus-derived lymphocytes.

Mice exposed to paramyxovirus (Sendai) generate specifically sensitized thymus-derived lymphocytes (T cells) which, in an in vitro 51Cr release assay, interact only with virus-infected target cells sharing strong transplantation antigens. Reciprocal exclusion of cytotoxic T-cell activity is found for Sendai virus, lymphocytic choriomeningitis virus and ectromelia virus. Immune T cells are detected as early as 3 days after intraperitoneal inoculation with a large dose of Sendai virus, and cytotoxicity is generally maximal on days 5-7. Lysis is restricted to interactions where sensitized lymphocytes and virus-infected target cells (fibroblasts, tumour cells or macrophages) are compatible at the K or the D locus of one H-2 haplotype. Identity of immune response (Ir) genes is neither sufficient nor necessary. Levels of T-cell responsiveness show some variation with H-2 type. Cytotoxic T-cell activity associated with H-2b is less than that recognized for H-2k or H-2d. These differences are, however, not obviously related to Ir gene control.     

The T3 complex on human thymus-derived lymphocytes contains two different subunits of 20 kDa

The human cell surface antigen T3 is involved in several T lymphocyte specific functions, as determined by the effect of monoclonal antibodies (OKT3, anti-Leu-4, UCHT1) directed at this molecular structure. The main target antigen of these monoclonal antibodies is a glycoprotein of 20 kDa. It is associated with four, less predominant, structurally distinct glycoproteins of 25-28 kDa, 37 kDa and 44 kDa. Of these molecules only the 20-kDa T3 antigen could be labeled with the hydrophobic reagent 5-iodonaphthyl-1-azide (INA). Here we present evidence that the main 20-kDa T3 antigen is comprised of, in fact, two structurally different molecules. One of these is a glycoprotein with a protein backbone of 14 kDa, the other is an unglycosylated protein of 20 kDa. This unglycosylated protein is labeled specifically with INA. Additional evidence for the existence of two different 20-kDa T3 antigens is provided by studies using the enzymes endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F and the drug tunicamycin. We hypothesize that the specific susceptibility to labeling with INA of the unglycosylated 20-kDa T3 form reflects a positioning in the lipid bilayer different from that of the glycosylated 20-kDa T3 form.     

Lack of IFN-gamma production in response to antigenic stimulation in human IFN-tau-treated lymphocytes.

Interferon-tau (IFN-tau) is a type I IFN responsible for maternal recognition of the fetus in ruminants. In addition to its physiologic role, IFN-tau also inhibits HIV replication in human lymphocytes and macrophages and displays immunomodulatory effects but lacks the toxicity associated with other type I IFNs. Human IFN-alpha promotes a Th1 response, whereas IFN-tau has anti-inflammatory properties, inducing the production of Th2 cytokines in murine models of experimental autoimmune encephalitis (EAE) or fetal loss. We compared the effects of ovine IFN-tau (OvIFN-tau) and human IFN-alpha (HuIFN-alpha) on cytokine mRNA and protein production in human peripheral blood mononuclear cells (PBMCs) activated with a recall antigen, such as purified protein derivative (PPD) of tuberculin or with a proinflammatory stimulus, such as lipopolysaccharide (LPS). In both cases, IFN-alpha increased IFN-gamma production, whereas IFN-tau did not and thereby promoted Th2 cytokine production. This original property renders IFN-tau a potential candidate for therapeutic applications in immune disorders, such as multiple sclerosis (MS), but its therapeutic use in the treatment of HIV infection should be considered with caution.     

Surface properties of LDL-binding lymphocytes in human peripheral blood.

Surface properties of low density lipoprotein (LDL)-binding lymphocytes were evaluated to determine whether LDL binds with a subpopulation of human peripheral blood lymphocytes (PBL). B- and T-cell rich fractions were prepared from PBL using E-rosette formation or nylon reticulum columns. Binding of FITC-labelled LDL with these cell fractions was determined with a fluorescent microscope and a fluorescence-activated cell sorter (FACS II). The specificity of the binding was evaluated by a dose-dependent inhibition of LDL binding with the addition of unlabelled lipoproteins. In parallel studies, surface properties including E-rosette formation, surface immunoglobulins, and receptors for IgG-Fc, as well as human and mouse C3 were examined. LDL binding lymphocytes were enriched in the B-cell rich fraction, and depleted in the T-cell rich fraction. In addition, FITC-LDL binding lymphocytes were selectively collected by the FACS II. These LDL binding cells restored surface immunoglobulins after incubation in serum-free medium following trypsinization. The majority of lymphocytes stimulated by PHA and PWM in vitro bound with LDL. It is concluded that LDL binds with B cells in fresh human PBL, while it binds with B and T cells in mitogen-stimulated lymphocytes. It is suggested that the selective collection of LDL binding lymphocytes by the FACS II can be applied to the evaluation of cellular interaction of these cells in various immunological reactions.     

Lysis of enucleated tumor cells with allogeneic and syngeneic cytotoxic thymus-derived lymphocytes.

These studies were initiated to understand the minimal requirements for lysis of target cells by cytotoxic thymus-derived lymphocytes (CTL). In particular, the significance of the nucleus to the susceptibility of the target cell to be lysed by CTL have been studied. P815 mastocytoma cells and EL4 lymphoma cells were enucleated by cen- trifugation through a discontinuous Ficoll gradient containing 10 μg/ml of cytochalasin B. The resultant vesicles were then analyzed for susceptibility to lysis by different CTL specific for the H-2K and H-2D gene products, minor histocompatibility antigens, trinitrophenyl groups, and Sendai virus-specific antigens. The results indicate that enucleated vesicles obtained from both EL 4 and P 815 cells are susceptible to lysis by these CTL. The lysis of EL 4-enucleated vesicles (unlike P 815-enucleated vesicles), however, required 2–4 times more effector cells than were required for equivalent lysis of intact EL 4 cells. This reduced susceptibility to lysis of enucleated EL 4 vesicles was not the result of an inability of the CTL to recognize and bind the vesicles. This conclusion was suggested by the fact that both enucleated EL 4 vesicles and intact cells inhibited equally well the lysis of ⁵¹Cr-labeled concanavalin A-stimulated BALB. B spleen cells by anti-H-2^b CTL. Although enucleated vesicles obtained from both P 815 and EL 4 cells were susceptible to lysis by CTL, they exhibited a reduced capacity to “cap” their serologically defined H-2 antigens. Lysis of these enucleated vesicles by CTL has the same requirements as lysis of intact cells. This conclusion was based upon the requirement for Ca⁺⁺ and Mg⁺⁺ and also because lysis of the modified vesicles (trinitrophenyl or viral antigens) occurred in an H-2-restricted fashion. Moreover, the enucleated vesicles were susceptible to antibody and complement-mediated lysis and also lectin-dependent cell-mediated cytotoxicity.     

Antigen-binding thymus-derived lymphocytes: I. Rapid method for isolation of theta-positive antigen-stimulated cells

A rapid method for separating SRBC-stimulated populations of theta (θ)-positive thymus-derived lymphocytes from bone marrow-derived lymphocytes and plaque-forming cells has been developed using cotton wool columns. The column effluent was monitored for plaque-forming and `rosette'-forming cells. Stimulated θ-negative cells were found to adhere preferentially to cotton wool. The total recovery of small lymphocytes was 12±3 per cent and the yield of rosette-forming cells was 5.5±4 per cent of which 85±5 per cent were θ-positive.     

Mechanism of formation of non-immune rosettes between guinea-pig thymus-derived lymphocytes and rabbit erythrocytes.

Several observations reported here suggest that spontaneous rosette formation between rabbit erythrocytes and guinea-pig T lymphocytes is mediated by natural anti-guinea-pig T-cell antibodies bound to the surface of the rabbit erythrocyte. First, normal rabbit sera frequently contain antibodies specifically cytotoxic for guinea-pig T lymphocytes. Second, the activity of rabbit erythrocytes in spontaneous rosette formation is reduced after incubation for 5 days at pH 6-1, but can be restored to levels seen with fresh erythrocytes by a brief incubation in normal rabbit serum containing natural anti-guinea-pig antibodies; normal serum absorbed with thymocytes does not restore activity to the erythrocytes. Third, the activity of rabbit erythrocytes in forming spontaneous rosettes can be specifically blocked by treatment with anti-allotype and heterologous anti-Ig sera.