Specific inhibition of macrophage migration inhibition factor by fucosylated glycolipid RM.

The effects of glycolipids on the interaction of the MIF (migration inhibition factor) with rat macrophages were examined using a migration inhibition assay system. MIF activity was specifically blocked by fucosylated Glycolipid RM [Gal alpha 1-3Gal(2-1 alpha Fuc) beta 1-3GalNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide, (1978) J. Biochem. 83, 85-90], but not by Cytolipin R, hematoside, or blood group B active glycolipid [Gal alpha 1-3Gal(2-1 alpha Fuc) beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide]. Inhibition of MIF activity was proportional to the concentration of Glycolipid RM. These findings suggest that Glycolipid RM acts as a receptor for MIF.     

T and B lymphocyte migration into syngeneic tumors.

The migration of splenic T and B lymphocytes into syngeneic tumors undergoing immunologic rejection was investigates. Spleen cells were obtained from normal BALC/c mice or BALB/c mice bearing tumors induced by murine sarcoma virus (MSV). Either whole spleen cells or immunoabsorbent purified T and B cells were radiolabeled with sodium chromate-51 and injected i.v. into normal or MSV inducted-tumor bearing syngeneic recipients. Twenty-four hours later the recipient mice were sacrificed and radioactivity was assessed for tumor, contralateral normal muscle, the lymph nodes draining the tumor and contralateral draining lymph nodes, peripheral lymph nodes, spleen, and liver. Both T and B lymphocytes from either normal or MSV tumor-bearing animals show greatly increased migration into the tumor when compared with normal muscle. Migration of T cells from both normal and MSV tumor bearers was 30 times that of migration to normal muscle. B cells from tumor-bearing mice, on the other hand, localized in the tumor itself only 50% as frequently as did B cells from normal animals. In addition, T cells from MSV tumor bearers were found in the highest proportion in the lymph node draining the tumor site. We conclude that T and B lymphocytes from either normal or tumor-bearing mice migrate to a syngeneic tumor undergoing immunologic rejection. In contrast, the migration of both T and B cells from tumor-bearing animals was decreased to the peripheral lymph nodes at the time of maximum tumor growth.     

Suppressor T cells regulate the cytolytic T lymphocyte response to syngeneic tumors induced by murine sarcoma virus (MSV) in the mouse.

Studies were designed to analyze the immune activities of spleen cells from mice previously injected with murine sarcoma virus (MSV) and undergoing the processes of MSV tumor growth and rejection. Fractionation of MSV-primed spleen cells according to cell size by velocity sedimentation at unit gravity showed that MSV-specific cytolytic T lymphocytes (CTL) generated in vivo underwent an apparent transition in size from large to small cells as the tumor regressed. The majority of CTL precursors, however, were invariably recovered among small to medium-sized MSV-immune cells, as revealed to CTL generation in vitro in secondary mixed leukocyte-tumor cell cultures (MLTC). Evidence was obtained for the existence in MSV-immune spleens of two suppressor cell populations capable of inhibiting CTL generation in vitro: one population probably consisted of macrophages and could be removed by treatment with carbonyl iron; the second population was comprised of T cells and inhibited the differentiation of tumor-immune CTL precursors in a selective manner. These results provide a preliminary overview of the mechanisms regulating the generation, differentiation, and activity of tumor-specific CTL in a syngeneic model system.     

Characterization of effector lymphocytes associated with immunity to murine sarcoma virus (MSV) induced tumors. I. Physical properties of cytolytic T lymphocytes generated in vitro and of their immediate progenitors.

Cytolytic T lymphocytes (CTL) were generated in secondary mixed leukocyte-tumor cell cultures (MLTC) with syngeneic RB1-5 tumor cells as stimulating cells and with responding spleen cells from regressor mice that had rejected a murine sarcoma virus (MSV)-induced tumor. CTL precursor cells were found to be exclusively of thymic origin and non-T cells were apparently not required for CTL generation. When the size variations of CTL from syngeneic MLTC were analyzed by velocity sedimentation it appeared that a transition from small precursor cells to larger effector cells occurred during the first 5 days in culture; this change in cell size was then followed by a shift toward small-sized cells. Furthermore, the CTL generated in syngeneic MLTC in the MSV tumor immune system were compared with those CTL obtained in allogeneic mixed leukocyte cultures (MLC) and were shown to exhibit fundamental similarities.     

Specific inhibition of human lymphocyte responses by primed autologous lymphocytes. I. Evaluation of MLR inhibition as a model for suppression.

Human lymphocytes from person A, primed for 10 to 14 days in MLC against lymphocytes from person B, inhibit specifically the proliferative response to B by fresh (i.e., unprimed) lymphocytes of A. Gamma-irradiated (2000 R) primed lymphocytes likewise inhibit specifically, although less strongly. Cells of A, primed with cells of B and then irradiated, usually can inhibit the response of A to cells of any individual sharing HLA-D antigens with B, and the effect tends to be independent of the number of stimulating cells. We also often see inhibition of responses to cells sharing HLA-A and -B antigens with person B, but this effect tends to be lost when the number of stimulating cells is increased. Similarly, at low doses, cells primed for HLA-D antigen a appear not to inhibit the response to an irrelevant HLA-D antigen b on the same stimulating cell. At higher doses of primed cells, even the response to the irrelevant antigen is inhibited. These data suggest to us that at least two mechnaisms may be involved: one directed at the stimulating cell (most likely cell-mediated cytolysis), and predominant at high ratios of primed cells to stimulating cells; the other directed at specific clones of responding cells, and predominant at low ratios.     

Specific binding of T lymphocytes to macrophages. I. Kinetics of binding.

Peritoneal exudate lymphocytes obtained from immune guinea pigs and cultured for 1 week on antigen-pulsed autologous macrophages were tested for their ability to bind to fresh antigen-pulsed autologous macrophages or to macrophages pulsed with an irrelevant antigen. Up to 30% of the lymphocytes bound to macrophages bearing the relevant antigen whereas only 2 to 5% remained nonspecifically bound to macrophages after vigorous washing. Specific binding was observed in cultures as early as 1 hr. Analysis of the kinetics of binding suggests that the observed nonspecific binding is not a step in specific binding. The possibility that weaker antigen-independent association between lymphocytes and macrophages precedes specific binding cannot be excluded. No evidence was obtained that serum antibody adsorbed to the macrophage or T cell plays a role in this cell interaction or that the T cell can bind antigen directly. We suggest that the observed specific binding represents the initial event in stimulation of T lymphocytes by antigen.     

Radiation leukemia virus-transformed immunocompetent T cells. II. Antigen-induced macrophage migration inhibition factor and leukocyte migration inhibition factor production

OVA-specific T cells were immortalized by infection with radiation leukemia virus (RadLV). Some clones derived from such population were shown to exhibit helper activity. We then tested clones without such function and found among them some that secreted macrophage migration inhibition factor (MIF) and leukocyte migration inhibition factor (LIF) upon exposure to the antigen in vitro. The lymphokine-producing clones, which were Thy-1+, Ly-1+ and Ly-2-, did not secrete MIF and LIF constitutively. Like other antigen-specific T cells, the immortalized clones could not be stimulated by free soluble antigen but required macrophages for presentation and for triggering the lymphokine production. The antigen-activated clones exclusively produced MIF and LIF, but not interleukin 2 or colony-stimulating factor. They neither provided helper activity nor induced delayed-type hypersensitivity. The data suggest that the T-cell clones carry the antigen receptors and that their antigen-inducible biological function is restricted to the migration inhibitory factor production.     

Leukocyte adherence inhibition response to murine sarcoma virus-induced tumors. II. Requirement for T-cell subpopulations and macrophages

Murine sarcoma virus (MSV)-immune T cells from C57BL/6 mice respond to intact RBL-5 tumor cells with the production of leukocyte adherence inhibition factor (LAIF), which mediates an adherence inhibition response of macrophages. LAIF is elaborated by isolated Lyt-2+ cells incubated with RBL-5 cells, whereas Lyt-1+ cells elaborate a substance that enhances macrophage adherence. Spleen macrophages or peritoneal exudate macrophages from MSV-immune mice when present at concentrations of 0.1% changed the response of Lyt-1+ cells from the formation of an adherence enhancing factor to the formation of an adherence inhibiting factor. Migration inhibition factor (MIF) was formed by Lyt-1+ cells, but not by Lyt-2+ cells under identical culture conditions. Addition of either spleen macrophages from mice with progressively growing tumors or tumor-infiltrating macrophages suppressed LAIF formation by both Lyt-1+ and Lyt-2+ cells. Tumor-infiltrating macrophages elicited an adherence enhancing factor from Lyt-2+ cells when present at high concentrations. The results suggest that the extent of macrophage adherence in vitro is the outcome of an interaction of macrophages with mediators that have opposing effects.     

T cell-mediated immunity to oncornavirus-induced tumors. I. Ly phenotype of precursor and effector cytolytic T lymphocytes

Regression of tumor induced by murine sarcoma virus (MSV) is accompanied by the formation of specific cytolytic T lymphocytes (CTL). Selection of T-cells sets by Ly phenotype determination allows separation of T sets involved in the cytolytic reaction. After MSV inoculation we demonstrate that a) Ly123+ cells contain precursors of CTL, b) direct cytolysis is mainly mediated by Ly23 cells, 3) cytolytic memory is divisible into "early" memory, carried by Ly23 cells, and "late" memory, which reverts to an Ly123 precursor population, and d) Ly1 cells are required to induce anti-MSV antibody formation.     

Specific absorption of T lymphocytes committed to soluble protein antigens by incubation on antigen-pulsed macrophage monolayers.

Monolayers of macrophages (Mphi) pulsed with antigen were used as immunosorbents for T lymphocytes from guinea pigs primed to soluble protein antigens. T lymphocytes were cultured on the Mphi monolayers for 4 hr, then aspirated and reincubated on a fresh monolayer pulsed with the same antigen for a second and a third step. T lymphocytes so treated were selectively deprived of cells responding in assay for antigen-dependent proliferation against the antigen used for pulsing the absorbing monolayer, but maintained their response to other antigens. The lymphocytes adhering to the Mphi of the absorbing monolayer were capable of giving a full response to the antigen used for pulsing the Mphi of the monolyers. The proliferative response of F1 T lymphocytes to antigen in association with Mphi of either parental strain could be absorbed leaving the response to antigen in association with Mphi of the other parental strain. The absorption of the proliferative response was not inhibited by addition of excess soluble antigen to the medium of the absorption culture. Our results indicate that specific guinea pig T lymphocytes responding by proliferation to soluble protein antigens recognize and bind specifically to a complex of Ia antigen and protein antigen at the surface of the Mphi.     

Specific inhibition of in vitro lymphocyte transformation by an anti-pan T cell (gp67) ricin A chain immunotoxin

The toxin A chain of ricin has been conjugated by a disulfide bond to a murine monoclonal antibody that recognizes the gp67kD antigen present on 95% of peripheral T lymphocytes. The immunotoxin retains both functions of its component parts: it binds to human peripheral blood lymphocytes, and it inhibits protein synthesis in a cellfree reticulocyte system. The immunotoxin has been evaluated for its ability to inhibit in vitro T lymphocyte transformation. In the presence of 20 mM NH4Cl, the immunotoxin decreases lymphocyte proliferation in response to phytohemagglutinin to less than 8% of untreated controls. The proliferative response in mixed lymphocyte culture and the development of allocytotoxic T cells is also dramatically inhibited by this immunotoxin. Monoclonal antibody alone does not inhibit these responses. Specificity of the immunotoxin has been established: the effect of the immunotoxin can be blocked by unconjugated monoclonal antibody, but not by a control monoclonal antibody that recognizes another T lymphocyte differentiation antigen or by a control monoclonal antibody that does not recognize human peripheral blood leukocytes. Treatment of human bone marrow cells with the immunotoxin preserves hematopoietic progenitor cells, as measured by granulocyte-macrophage, erythroid, and multipotential hematopoietic progenitor cell assays. These results indicate that an anti-pan T lymphocyte-ricin A chain immunotoxin is an effective agent against immunocompetent T lymphocytes in vitro, and may be an effective agent for use in clinical bone marrow transplantation.     

Human macrophage-lymphocyte interaction in proliferation to soluble antigen: I. Specific deletion of lymphocyte proliferative activity on macrophage monolayers

The interaction between human T lymphocytes and autologous macrophages (Mφ) in the proliferative response to soluble antigen was investigated. The presence of Mφ was a requirement for maximal lymphocyte proliferation to tetanus toxoid antigen. Brief exposure of Mφ to TT resulted in “antigen-pulsed” Mφ which could stimulate proliferation by lymphocytes in the absence of free antigen. Taking advantage of the adherent nature of Mφ, monolayers of antigen-pulsed Mφ were used to specifically adsorb antigen-reactive lymphocytes. This immunoadsorption was manifested by a specific deletion of proliferative activity in the fraction of lymphocytes failing to bind to the monolayers.     

T lymphocyte-enriched murine peritoneal exudate cells. I. A reliable assay for antigen-induced T lymphocyte proliferation.

The in vitro activation of murine thymus-derived (T) lymphocytes by soluble protein and synthetic antigens has been difficult to assess because of the lack of a specific and reliable proliferation assay. The present report describes the development of an assay system which overcomes these problems by making use of a population of nylon wool column-purified T lymphocytes obtained from thioglycollate-induced peritoneal exudates of immunized mice. PETLES (peritoneal exudate, T lymphocyte-enriched cells) were composed mainly of T lymphocytes, eosinophils and small numbers of macrophages. Contamination with bone marrow-derived (B) lymphocytes averaged only 2%. When PETLES from immunized mice were stimulated in microtiter cultures with the immunizing antigen, large degrees of proliferation ensued as measured by incorporation of 3H-methyl-thymidine 5 days after initiation. As few as 1.25 x 10(4) cells and as little as 50 ng/ml of antigen gave significant stimulation. Maximum responses were obtained witn a series of 10 experiments under these optimal conditions, gave a mean incorporation of 70,900 cpm while the controls cultured without antigen showed only 3,600 cpm. PETLES from nonimmunized mice or from mice immunized to other antigens did not respond to DNP5OVA although they did respond to mitogens. The antigen-induced proliferation was shown to require the presence of immune T lymphocytes by two criteria: elimination of the response by treatment with anti-Thy 1.2 serum plus complement and failure to reconstitute the response when the few remaining immune B lymphocytes left after anti-Thy 1.2 treatment were added to nonimmune T lymphocytes. In addition, the system exhibited carrier specificity. Because of the paucity of B lymphocytes in the population, their contribution to the overall magnitude of the proliferative response was negligible as demonstrated by the small response to B cell mitogens. Thus, the assay appears to be a quantitative as well as a qualitative assay for one aspect of T lymphocyte function. This technique should prove useful for the study of murine T lymphocytes in vitro.     

Adherent cell function in murine T lymphocyte antigen recognition. II. Definition of genetically restricted and nonrestricted macrophage functions in T cell proliferation.

The mechanisms by which adherent cells, presumably of mononuclear phagocytic lineage, influence in vitro antigen-specific activation of murine T lymphocytes was examined. Two distinct functions for macrophages could be discerned. One macrophage function is dependent on a soluble factor produced by cultured adherent cells and is most easily studied with complex multideterminant antigens. This factor is neither antigen-specific nor MHC-restricted in its action in that PEC, regardless of haplotype, produce factor in the absence of antigen. A second function, antigen-specific T cell activation, is seen when antigens of more restricted heterogeneity are used, such as those under the control of Ir genes. This latter activity demands identity or partial identity between the antigen-presenting cell and the primed T cell, thus suggesting an additional specific, genetically restricted function for macrophages in in vitro antigen recognition. Whether these adherent cell functions are mediated by all or distinct subsets of cells was not established.