Generation of thymus-derived helper cells by macrophage-associated antigen

A double cell transfer system was used to investigate the efficacy of macrophage bound antigen in generating carrier-reactive helper T cells. Macrophage bound antigen was found to be three orders of magnitude more potent than soluble antigen in T cell priming. The results are discussed with reference to the involvement of macrophages or related cells in the normal processes of immune induction.     

Nonspecific cytotoxicity of spleen cells and the specific cytotoxic action of thymus-derived lymphocytes in vitro

Spleen cells removed from immunized mice specifically kill allogeneic lymphoma cells in vitro, but in the presence of specific antigen nonspecific target cell growth inhibition also occurs. Only the specific target cell killing was found to be θ-sensitive, the nonspecific cytotoxicity was caused by a population of θ-resistant, adherent, and AMS-sensitive cells. Nonspecific cytotoxic effects were caused by spleen cells from normal mice after incubation with endotoxin, and these effects were inhibited by removal of the adherent cells.     

Plasminogen activation and lysis of blood clots induced by cells in vitro

When human diploid fibroblasts were seeded onto the surface of blood clots, lysis of the clot occurred as a result of the release of cellular plasminogen activator. A number of aspects of this lysis were studied. 1. There was no significant difference in rates of lysis of whole blood clots, platelet-rich plasma clots, and platelet-poor plasma clots brought about by the same number of fibroblasts. 2. Clot lysis was promoted by nondividing cells and by proliferating cells. 3. Using cycloheximide to block protein synthesis it was found that the plasminogen activator released by fibroblasts had an active half-life of less than an hour. 4. When clots were washed prior to the addition of cells then lysis occurred at an increased rate. This was probably due to the removal of alpha 2-antiplasmin from the clots, since when antisera to alpha 2-antiplasmin was added to clots, lysis also proceeded at an increased rate. 5. Medium conditioned by fibroblasts did not promote clot lysis even when antiplasmin was removed by washing or by addition of antisera. 6. Cells had to be in direct contact with the clot in order to bring about lysis; when cells were separated from clots by permeable membranes there was no lysis. 7. When cross-linking of fibrin was reduced by the inhibition of transglutaminase, the rate of clot lysis was increased.     

Pretreatment of P815 mastocytoma cells with inhibitors of protein synthesis reduces their susceptibility to lysis by cytotoxic thymus-derived lymphocytes

Protein synthesis inhibitors like cycloheximide, emetine, and puromycin diminish the ability of P815, a mastocytoma of DBA/2 mice, to react with anti-H-2^d cytotoxic thymus-derived lymphocytes (CTLs). Compared to untreated P815, tumor cells incubated with the protein synthesis inhibitors exhibited a reduced sensitivity to lysis and a reduced ability to inhibit lysis of untreated P815 cells. Consistent with this reduced reactivity of cycloheximide-treated P815 cells with CTLs was the inability of anti-H-2^d CTLs to form T cell-target cell conjugates with treated P815 cells. As evaluated by the binding of an anti-H-2^d serum, treated P815 cells expressed the same amount of H-2 membrane antigen as untreated cells. However, treated cells were still lysed by CTLs in the presence of the agglutinator, concanavalin A (Con A).     

Cytotoxic cells suppress in vitro generation of cellular immunity by stimulator cell lysis

Irradiated BALB/c spleen-cell populations actively cytotoxic to BL/6 alloantigens (modulator cells) were capable of suppression of the in vitro generation of BALB/c anti-BL/6 cellular cytotoxicity. This suppression was abrogated by anti-θ serum plus complement. The suppression was dose dependent on the number of modulator cells and correlated directly with the magnitude of their cytotoxicity. By varying the number of stimulator cells, specific suppression for a relevant stimulator cell and nonspecific suppression for an irrelevant stimulator cell were demonstrated in the same cultures. These data suggest that cytotoxic cells caused specific suppression in mixed lymphocyte culture by lysing stimulator cells although evidence for other nonspecific suppressor factors was seen. A model was proposed suggesting that cell populations possessing high levels of cytotoxicity may feed back negatively on an ongoing immune response by competing with proliferating T cells for cellular antigen.     

Immune regulation by self-reactive T cells is antigen specific

Immune regulation plays an important role in the establishment and maintenance of self-tolerance. Nevertheless, it has been difficult to conclude whether regulation is Ag specific because studies have focused on polyclonal populations of regulatory T cells. We have used in this study a murine transgenic model that generates self-reactive, regulatory T cells of known Ag specificity to determine their capacity to suppress naive T cells specific for other Ags. We show that these regulatory cells can regulate the responses of naive T cells with the same TCR specificity, but do not inhibit T cell proliferation or differentiation of naive T cells specific for other Ags. These results demonstrate that immune regulation may be more Ag specific than previously proposed.     

Cerebral vascular endothelial cells are effective targets for in vitro lysis by encephalitogenic T lymphocytes.

Lysis of cerebral vascular endothelial cells (EC) by CD4-positive, myelin basic protein-specific encephalitogenic T cell lines was investigated. Unstimulated EC were not lysed, but culture in the presence of murine rIFN-gamma resulted in the expression of class II MHC (Ia) molecules and the concomitant ability to function as effective target cells for lysis. The possible requirement for Ia molecules was further demonstrated by antibody-blocking experiments. Lysis of EC targets also required the presence of specific Ag (myelin basic protein); PPD-specific T cell lines also lysed the PPD-pulsed EC. In all cases, lysis was directly proportional to E:T ratios. In addition, continuous passage of T cell lines resulted in the concomitant loss of encephalitogenicity and ability to affect EC lysis, indicating a possible relationship between these two factors. These results demonstrate that CD4+ T cells interact with cerebral vascular EC. It is suggested that such interactions may be important in the pathogenesis of diseases involving migrations of these cells across the blood-brain barrier.     

Auto-tumor lysis by blood lymphocytes in vitro

Summary Selectivity of the lysis of the tumor cells by autologous blood lymphocytes and its various subsets was investigated by means of the cold target competition assay. The effectors were autologous lymphocytes passed through a nylon-wool column (unfractionated: U) and their low-and high-density subsets, either without or after activation. The lymphocytes were activated (a) in autologous mixed lymphocyte tumor cell culture in autologous (MLTC), (b) in mixed lymphocyte culture (MLC), without and with interleukin-2, for 6 days, or (c) by phytohaemagglutinin for 3 days. Autologous-lymphocyte-mediated cytotoxicity (auto-tumor lysis: ALC) by the unfractionated, unmanipulated blood lymphocyte (U) population, its high-density fraction and those induced for auto-tumor lysis in the MLTC is regularly weak and affects only the autologous tumor cells. Their ALC function was inhibited only by the target identical unlabelled cells while the effect of separated low-density lymphocytes was inhibited also by allogeneic tumor cells.The cold-target competition assay indicated that several subsets with different specificities exist simultaneously in the effector populations activated in MLC, because the various targets did not cross-compete or did so only partially. Whenever interleukin-2 was added, at the start of the mixed cultures (MLTC or MLC), the lytic effects were no longer selective. Phytohaemagglutinin-activated effectors lysed several targets. These targets were inhibitory in a criss-cross fashion. Generally, populations showing auto-tumor selectivity had weak lytic effects, while the strongly activated effectors, with strong cytotoxic function, were not selective.     

Complement-dependent lysis of tumor cells by a baboon IgM antibody to a tumor-associated antigen

Summary We developed a high-titer polyclonal antiserum to a glycoprotein tumor-associated antigen (TAA) by immunization of a baboon with the purified glycoprotein antigen. The baboon serum was fractionated into IgG and IgM components by DEAE Affi-Gel blue chromatography. The ability of the baboon IgM anti-TAA antibody to effect tumor cell lysis in the presence of complement was tested using a chromium-release assay. The baboon antibody was able to lyse melanoma target cells (20.8%–71.4% cytolysis), breast carcinoma cells (36.5%–38.9% cytolysis), and a neuroblastoma cell line (35.5% cytolysis) in the presence of complement but did not effect significant lysis of autologous lymphoblastoid cell lines (4.9% cytolysis) or peripheral blood lymphocytes from healthy volunteers (12.6% cytolysis). Cytolysis of melanoma target cells was completely inhibited by preabsorption of the IgM anti-TAA antibody with UCLA-SO-M14 (M14) cells and partially inhibited by preabsorption with several other melanoma cell lines. There was no significant inhibition of tumor cell lysis after preabsorption of the antibody with lymphoblastoid cell lines. Complement-dependent lysis of M14 targets could be blocked by addition of the purified antigen to the antibody prior to incubation with the tumor cells. Our results suggest that the glycoprotein TAA resides on the tumor cell surface and that the baboon IgM anti-TAA antibody recognizes the antigen on the cell surface and is able to fix complement and effect the lysis of the tumor cells.     

Natural killer-type lysis of thymocytes from young mice by normal spleen cells in vitro

Normal spleen cells from 6- to 10-week-old mice, enriched for natural killer (NK) cells on a discontinuous polyvinylpyrrolidone-silica (Percoll) gradient, lyse thymocytes of young mice (less than or equal to 19 days old) in a short-term 51Cr release assay. The highest NK-type activity was found in band 3 (density less than or equal to 1.077 g/ml) of a four-step gradient. In some experiments band 2 (density less than or equal to 1.070 g/ml) also showed NK activity. Activity was not unequivocably detectable in cells before separation or in bands 1 and 4. These results also show that the thymocyte sensitivity is dependent on the age of the target cell. Sensitivity of very young thymocytes (less than or equal to 7 days old) was higher than that of thymus cells from 8- to 19-day-old donors. Moreover, it seemed that syngeneic target thymocytes were lysed more effectively than allogeneic. Thus, an NK-type cell population may have the ability to lyse immature thymic target cells at an early stage of their differentiation. This could be of importance as a physiological mechanism for controlling the T cell repertoire and its reactivity.     

Lysis of tumor cells by antibody and complement. III. Lack of correlation between antigen movement and cell lysis.

The increase in susceptibility to killing by rabbit antibody and guinea pig complement of guinea pig hepatoma cells (line-10), after treatment with certain metabolic inhibitors, did not correlate with the mobility of antigen on the cell surface as measured by indirect immunofluorescence.     

Soluble trinitrophenylated proteins and trinitrophenylated cells as specific inhibitors of target cell lysis by cytotoxic T lymphocytes.

Bovine serum albumin (BSA) substituted in 12 to 15 amino groups with 2,4,6-trinitrophenyl (Tnp-BSA) or carbobenzoxy (Cbz-BSA) or acetyl (Ac-BSA) groups was tested as inhibitor of the reaction in which anti-Tnp cytotoxic T lymphocytes (CTLs) lysed syngeneic ⁵¹Cr-labeled Tnp-modified spleen cells [concanavalin A (Con A) blasts]. Inhibition was observed with some consistency only with Tnp-BSA at extremely high concentration (50 mg/ml). To explore the significance of this observation, inhibition of anti-Tnp CTLs was also tested with Tnp-modified cells on which products of the major histocompatibility loci H-2K and H-2D were lacking or different from those on the stimulator cells used to elicit the CTLs. Only those Tnp cells with the same H-2 products as the stimulators were inhibitory, even though all the Tnp cells tested had essentially the same surface density of Tnp (ca 1 × 10⁸ groups/cell). It is concluded that effective specific inhibitors of anti-Tnp CTLs have both Tnp groups and the correct H-2 products on the same particle and that the specific inhibitory activity of soluble Tnp-BSA was probably due to its adsorption onto cells in the suspensions used to assay cytotoxicity.     

Cell-mediated immunity to murine tumor allografts. Increase in the activities of activated thymus-derived cells following in vitro incubation

The immunity of CS7B1 spleen cells to the allogeneic tumor P815 has been studied using a direct cytotoxic assay and an assay of macrophage migration inhibition. Both assays principally measure activities of thymus-derived cells. The activity of spleen cells in either assay is markedly enhanced by overnight incubation in the absence of deliberately added antigen, and, for the MIF cell assay at least, this enhancement apparently does not depend upon cell division or change in cell size during that incubation. The effect of overnight incubation is to some degree mimicked by short-term exposure to trypsin; furthermore, this effect can be blocked by incubation in the presence of serum from the spleen cell donors. These results suggest that blocking factors exist on the surface of some small T lymphocytes taken from thes animals, and that these factors can suppress T cell activity in both of the assay systems used.