Maleylated bovine serum albumin triggers cytolytic function in selected populations of primed murine macrophages

The complex algal polysaccharide fucoidan has been reported as serving as a second signal for activation of macrophages primed in vivo by BCG. To assess the potential utility of this observation in analyzing biochemical mechanisms involved in macrophage activation, we examined the triggering effects of maleylated bovine serum albumin (maleylated-BSA), a defined molecule that clears via similar mechanisms. Cytolysis of P815 mastocytoma targets was triggered by maleylated-BSA, in a dose-dependent manner, in murine peritoneal macrophages primed in vivo by BCG. Unlike bacterial LPS, which triggered cytolysis when used to pretreat the macrophages, maleylated-BSA was only effective if present throughout the period of macrophage-target cytolytic interaction. Maleylated-BSA alone did not lyse the P815 targets and did not affect the binding of such targets by macrophages. Maleylated-BSA was equally effective in triggering cytolysis in BCG-primed macrophages from C3H/HeJ or C3H/HeN mice. Macrophages primed in vitro with IFN-gamma, however, could not be triggered by maleylated-BSA, even though these macrophages bound maleylated-BSA comparably to the BCG-primed macrophages. When responsive macrophages were fully activated in vitro by IFN-gamma and LPS and then allowed to decay to the primed state, maleylated-BSA was then as effective as LPS in triggering cytolysis. Taken together, the results indicate that maleylated-BSA can trigger cytolysis in certain populations of primed macrophages but not in others.     

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).     

The cytolytic function of mononuclear phagocytes. II. Factors that determine the binding and lysis of tumor cells by activated macrophages

The effects of various modifiers upon the interaction of LPS- and BCG-activated macrophages with cells of mastocytoma P815 have been investigated. The efficiency of binding and lysis of the tumor cells is to a great extent determined by activation of the effector-cells, expression of the trypsin-sensitive receptors on the surface of macrophages, and by the type of target-cells. Introduction into the analytical system (effector-target) of unlabeled tumor cells or membrane preparations obtained from them inhibits substantially both binding and lytic activity of cytotoxic macrophages. If nontransformed cells or their membranes are applied, no significant changes in the investigated processes can be detected. Trypsinization of tumor cells as well as of activated but not resident macrophages modifies considerably the interaction of effectors with targets. The quantity of tumor cells bound with macrophages does not depend on the fact, which of the partners is subject to trypsinization, but it is much less than that of target-cells bound in the control. The incubation of activated macrophages with actinomycin D results in a substantial suppression of their lytic activity, whereas treatment of tumor cells with this inhibitor of protein synthesis leads to a considerable decrease in stability of the targets against lytic activity of the factor activated by effectors. The obtained data reveal the ways of selective binding and effective lysis of transformed targets by activated macrophages.     

Spontaneous cytotoxicity of macrophages against pancreatic islet cells

Activated peritoneal macrophages were found to lyse syngeneic [3H]leucine-labeled pancreatic islet cells or rat insulinoma cells after 15 h of coculture at 37 degrees C. Lysis was verified by electron microscopic analysis. Islet cell lysis was dependent on the T:E ratio and was comparable with P815 and L929 tumor cells used as targets. The cytotoxic activity was localized in the adherent fraction of Corynebacterium parvum activated peritoneal cells and was destroyed by incubation of cells with macrophage-toxic silica particles. Syngeneic thyrocytes and hepatocytes were found to be resistant to the cytolytic action of activated macrophages. It has been shown previously that macrophages contribute to pancreatic islet inflammation. The present in vitro analysis demonstrates that macrophages can function as effector cells in islet destruction.     

Studies on the mechanism of lymphocyte- mediated cytolysis. VIII. The use of con A to delineate a distinctive killer T cell subpopulation.

Alloimmune spleen cells (C57BL/6 anti P815), but not normal spleen cells, lyse syngeneic (EL4) target cells in the presence of Con A. Con A dependent cytotoxicity was mediated by T cells and required the continued presence of lectin. Cytolysis in the presence of a succinylated derivative was equivalent to that seen with the parent Con A molecule. In contrast to previous reports of Con A dependent cytolysis, however, we conclude that lysis is not primarily caused by directly cytotoxic T cells. The reasons for this conclusion are: 1. Removal of directly cytotoxic cells by adsorption on P815 monolayers did not alter the Con A dependent cytolysis of EL4 cells; 2. Populations in which no direct T killers were demonstrable (e.g., spleen cells harvested 5 days after alloimmunization) lysed both P815 and EL4 cells in the presence of Con A; and 3. Con A dependent cytolysis, but not direct cytotoxicity, could be induced by culturing normal C57BL/6 spleen cells for 4 days with a sonicated extract of P815 cells. We hypothesize that the cell "activated" to lyse targets in the presence of Con A is a T cell which has differentiated lytic potential following alloantigenic stimulation, but has either insufficient density or affinity of antigen receptors to serve as a directly cytotoxic cell. The role of Con A is viewed as 2-fold: i) to "bridge" killer and target cell, and ii) to "activate" the effector.     

Failure of infiltrating precursor cytotoxic T cells to acquire direct cytotoxic function in immunologically privileged sites

Minor H incompatible P815 tumor cells inoculated into the anterior chamber (AC) of the eyes of BALB/c mice grow progressively, revealing this to be an immunologically privileged site. By contrast, a similar inoculation of tumor cells is rapidly rejected from nonprivileged ocular sites (subconjunctiva). Mice with progressively growing AC-tumors and those that reject their ocular subconjunctiva tumors both have expanded clones of tumor-specific cytotoxic precursor cells (pTc) in their spleens and cervical lymph nodes. In an effort to determine why the expanded pool of primed pTc is unable to effect rejection of AC intraocular tumors, we have examined the susceptibility of the tumor cells growing within the immunologically privileged AC to lysis by cytotoxic T cells and the cytotoxic function of tumor-infiltrating lymphocytes. P815 tumor cells extracted from intraocular tumors and P815 cells maintained in routine tissue culture are equally susceptible to lysis when exposed in vitro to fully differentiated, DBA/2-specific cytotoxic T cells. Thus, progressively growing tumor cells within the AC are not insensitive to immune-mediated lysis by cytotoxic T cells. We have been able to harvest significant numbers of DBA/2-specific pTc from these same intraocular tumors. When the tumor-infiltrating lymphocytes are driven in vitro with exogenous IL-2, they acquire the capacity to lyse specifically P815 tumor cells. However, no evidence of fully cytotoxic, tumor-specific T cells was found among lymphocytes harvested from intraocular tumors, i.e., when the harvested cells were tested immediately for cytolytic activity. Inasmuch as we have reported that directly cytotoxic T cells are present during tumor rejection at nonimmunologically privileged ocular sites, such as the subconjunctival space, we conclude that progressive growth of P815 tumor cells within the anterior chamber is due in part to a failure of infiltrating pTc to differentiate in situ into fully functional cytotoxic effector cells.     

Lack of reconstitution of nude mice alloreactivity by purified interleukin 2 and induction of non-H-2-specific effector cells by crude supernatants

To verify or to challenge the reports indicating that IL-2 was the only molecule involved in the reconstitution of nu/nu mice alloreactivity in vitro, Balb/c (H-2d) nu/nu spleen cells were primed in culture against C57/B16 (H-2b) in the presence of crude IL-2-containing supernatants or purified IL-2. The generation of cytotoxic effectors was evaluated against a panel of 51Cr-labeled target cells. Although crude IL-2-containing supernatants sustained the generation of cytotoxic effectors, purified "natural" IL-2 (from different origins) and recombinant IL-2 were not able to do so. Con A or PHA were identified as cofactors synergizing with IL-2 to induce effectors from nu/nu spleen cells. These effectors efficiently lysed EL4 (H-2b, tumor line), but not mitogen-induced blast cells from the same strain. They also lysed targets bearing irrelevant allogenic H-2 specificities. Cold competition experiments confirmed the lack of H-2 specificity of such effectors: lysis of EL4 cells (H-2b) was inhibited strongly by YAC-1 cells (H-2a, very sensitive to NK lysis) or P815 cells (H-2d, autologous to the nu/nu effectors). Our results clearly challenge earlier conclusions and indicate that IL-2 alone does not reconstitute nude mice alloreactivity. Crude supernatants containing IL-2 and mitogen induce nonspecific effectors with patterns of reactivity similar to those of activated natural killers. We think that the cytotoxicity observed in these conditions in nude mice results from the mitogenic triggering of some kind of prethymic killer cells which subsequently are expanded by IL-2.     

A microassay for the rapid and selective binding of cells from solid tumors to mouse macrophages

Summary A microassay was developed to study the rapid binding characteristics of murine macrophages activated by gamma interferon and muramyl dipeptide to adherent neoplastic or nonneoplastic target cells. The binding of tumor cells to both activated and nonactivated macrophages was time- and temperature-dependent, and independent of tumor cell type. Activated macrophages bound more tumor cells than nonactivated macrophages. The initial binding of macrophages to target cells did not necessarily lead to lysis. First, primed macrophages bound tumor cells but did not lyse them, and second, nonactivated macrophages bound nontumorigenic cells without subsequent lysis. The rapid binding assay described here could prove useful in investigating the recognition mechanism(s) between macrophages and tumor cells derived from solid primary and metastatic cancers.     

H2-restricted recognition of cloned HLA class I gene products expressed in mouse cells

Long-term syngeneic mouse cytolytic T lymphocyte (CTL) clones were obtained from DBA/2 (H2d) mice immunized with P815 (H2d) cells transfected with cloned human class I histocompatibility genes, HLA-CW3 or HLA-A24. Three distinct patterns of specificity were defined on P815 HLA transfectant target cells. One clone lysed HLA-CW3 but not -A24 transfectants, and a second lysed HLA-A24 but not -CW3 transfectant target cells. The third clone lysed P815 targets transfected with either HLA gene. None of the CTL clones lysed L cells (H2k) transfected with the same HLA genes or human targets that expressed these HLA specificities. Several lines of evidence indicated that recognition of HLA transfectants by these CTL clones was H2 restricted. First, lysis of P815 HLA transfectants could be inhibited by anti-H2Kd monoclonal antibody. In addition, the anti-P815-HLA CTL clones could lyse a (human X mouse) hybrid target that expressed both HLA class I and H2Kd antigens, but not a clonal derivative that no longer expressed H2Kd. The most direct evidence for H2-restricted recognition of P815-HLA transfectants by the syngeneic CTL clones was obtained by double transfection of mouse L cells (H2k) with both HLA and H2 class I genes. L cells transfected with HLA and H2Kd genes were susceptible to lysis by the same CTL clones that lysed the corresponding P815-HLA transfectant targets. Thus under certain conditions, CTL recognition of xenogeneic class I histocompatibility gene products can be restricted by other class I gene products.     

Lysis of C1Q-coated chicken erythrocytes by human lymphoblastoid cell lines

Human lymphoblastoid cells lysed chicken erythrocytes (E) that carried cell surface bound human C1q. Antibody to E(A) was not required for the C1q-dependent reaction. The effect of C1q was inhibited by Fab'2 anti-C1q and by the serum C1q inhibitor. The action of the lymphoblastoid cells was inhibited by anti-metabolites and by pretreatment of the cells with trypsin which is known to destroy their C1q receptor. Lymphoblastoid cell lysate was inactive. The time course of the C1q-dependent lysis was comparable to that of the antibody-dependent cellular cytotoxic reaction of human K-cells. Lysis of EA by human peripheral lymphocytes was enhanced up to 50% by human C1q.     

Macrophage binding of cells resistant and sensitive to contact-dependent cytotoxicity

We compared macrophage binding and killing of F5b cells to the binding and killing of P815 mastocytoma cells and to several other nontransformed and transformed cell lines. Formalin fixation of elicited or activated macrophages did not affect binding of F5b or 3T3 cells but did abrogate binding of P815 cells. However, formalin fixation abrogated resident macrophage binding of F5b and 3T3 cells. Therefore, depending on the type of macrophage or target cell, formalin fixation may affect binding. Only the binding of P815 cells was dependent upon activation; macrophage binding of target cells F5b and 3T3 was not. Even though macrophages bound F5b and 3T3 cells, macrophages only mediated contact-dependent cytotoxicity against F5b cells. Macrophages did not kill 3T3 cells. Experiments also compared macrophage binding and killing of the uv-light-induced tumor cell lines 1422, 2237, and 2237a46. Only the cell line 2237a46 was susceptible to contact-dependent killing. Both 1422 and 2237 cells were resistant. In contrast, cell lines 2237a46 and 1422 were bound by activated macrophages while 2237 cells were bound poorly.     

A novel cytotoxic T-cell clone that exhibits differential modes of recognition in the proliferative and cytolytic phase

A T-cell clone (1G8-H7) cytotoxic to P815Y mastocytoma (H-2d) has been established from spleen cells of a C3H/He mouse (H-2k) primed with P815Y cells by means of in vitro stimulation with irradiated C3H.H-2o(H-2KdDk) spleen cells. The clone 1G8-H7 was an interleukin 2 (IL-2)-dependent and H-2Kd antigen-dependent CTL clone and it killed P815Y cells but not Concanavalin A-induced spleen blast cells bearing H-2Kd antigen. The involvement of H-2Kd antigen in the cytolytic recognition mechanism was shown by the inhibition of lysis by anti-H-2Kd monoclonal antibody and also by the cold inhibition experiment that employed H-2Kd-bearing spleen cells. Comparison of cytotoxic activities between 1G8-H7 and Kd-specific CTL clones showed that the killing of P815Y cells by clone 1G8-H7 was not explained by the susceptibility to cell-mediated cytolysis of P815Y cells. These results suggest that H-2Kd antigen on the stimulating cell is sufficient to deliver a proliferation signal in the proliferative phase of this clone, but in the cytolytic phase an additional interaction with surface structure on the target cell other than that with H-2Kd antigen is required for the induction of cytolysis. Possible elucidations for the differential modes of recognition are discussed.     

Resistance of primary CD8+ cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines

Recent evidence has shown that cloned, murine CTL cell lines are resistant to the cytotoxic components of the toxic granules they release upon specific interaction with their target cells. Inasmuch as the resistance might be due to selection in culture over many months by repeated exposure to these cytolytic components (which are released repeatedly as a result of the cultured CTL being periodically stimulated by target cells), we asked whether primary CTL are also resistant. The primary CTL were elicited in vivo by i.p. injection of allogeneic tumor cells or in vitro by 5- to 6-day MLC or by 48-h exposure to the lectin Con A. The responding cells were separated into purified CD8+ (i.e., CD4-, CD8+) and purified CD4+ (i.e., CD4+, CD8-) T cell populations that were analyzed for cytolytic activity and for resistance to lysis by toxic secretory granules derived from cloned CTL cell lines. The CD8+ T cells were highly cytolytic and relatively resistant; they retained their cytolytic activity and were lysed to a minimal extent (0 to 10%) by quantities of isolated granules that lysed 80 to 90% of the P815 tumor cell line (tested as a representative standard cell line). The CD4+ T cells, in contrast, had only minimal cytolytic activity and were far more susceptible to granule-mediated lysis. Although the resistance of primary CD8+ T cells is impressive, it is not as pronounced as the resistance of the cloned CTL cell lines, indicating that during long-term culture there is some selection for increased resistance to granule-mediated lysis. In contrast to T cells (especially CD8+ T cells), Ia+ macrophages, isolated from primary immune peritoneal exudates, were highly susceptible to granule-mediated lysis.     

DNA of human Raji target cells is damaged upon lymphocyte-mediated lysis

Human Raji target cells DNA is degraded by the introduction of single-strand breaks (alkali-sensitive sites) upon lymphocyte-mediated lysis. This type of DNA degradation appears earlier and is more extensive in lymphocyte-than in antibody + complement-mediated lysis of Raji cells, regardless of the species of effector lymphocytes (human or mouse). Mouse P815 target cell DNA is extensively fragmented (yielding 200 base pair fragments) when human or mouse lymphocytes are used to lyse P815. Thus, these observations indicate that both human and mouse target cell DNA are affected during lymphocyte-mediated lysis. Moreover, the pattern of DNA degradation in target cells lysed by effector lymphocytes is characteristic of the target cell species, suggesting that DNA degradation proceeds through the activation of target cell endonuclease(s).     

Natural cytotoxicity against mouse hepatitis virus-infected target cells. I. Correlation of cytotoxicity with virus binding to leukocytes

Spleen cells from uninfected control mice selectively lysed BALB/c 3T3 fibroblasts infected with mouse hepatitis virus (MHV), a murine coronavirus. Lysis of infected cells occurred within 3 hr, and histocompatibility between effector and target cells was not required. This natural, cell-mediated, virus-associated cytotoxicity differed from NK cell- and T cell-mediated lysis. Spleen cells from animals infected with MHV were enriched in NK activity and were more cytotoxic to YAC-1 target cells, but did not show enhanced cytotoxicity for MHV-infected target cells. Spleen cells from beige mice, which are deficient in NK cell activity, were able to lyse MHV-infected target cells, as were spleen cells from nude mice, which are deficient in T cell activity. Lysis of MHV-infected target cells could be mediated by cells from the spleen and, to a lesser extent, by cells from the bone marrow, but not by resident peritoneal cells or thymocytes. We suggest the term "virus killer (VK) activity" for this phenomenon. VK activity of splenocytes from different mouse strains correlated with the ability of the splenocytes to bind purified radiolabeled MHV virions. MHV virions caused agglutination of spleen leukocytes from susceptible mouse strains, indicating that leukocyte agglutination or adsorption may provide a useful assay for coronaviruses such as MHV which lack hemagglutinating activity. SJL mouse splenocytes did not bind MHV and did not lyse infected targets. MHV bound relatively well to splenocytes of other mouse strains, but poorly to thymocytes and erythrocytes. Binding of MHV to leukocytes was not influenced by 6 mM EDTA or EGTA, indicating a lack of requirement for Mg++ or Ca++. VK activity was also resistant to EDTA and EGTA, in contrast to NK activity, which was sensitive to those chelating agents. VK activity was also unaffected by actinomycin D, cycloheximide, or puromycin, indicating that new protein synthesis was not required for lysis. Antibody to interferon-alpha/beta did not block lysis, nor was there substantially enhanced lysis mediated by leukocytes from mice infected with virus and thus exposed to high levels of interferon. VK activity was blocked by antibody directed against the peplomeric glycoprotein E2 of MHV. VK activity required infected target cells, because cells with adsorbed MHV virions were not lysed by splenocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of Bacillus Calmette--Guérin-activated macrophages and neoplastic cells in vitro. I. Conditions of binding and its selectivity

The initial interaction in vitro between Bacillus Calmette-Guérin-activated, peritoneal macrophages from C57B1/6J mice and two nonadherent neoplastic targets (P815 and EL-4) was found to represent firm physical binding of the targets to the macrophages. Binding between the Bacillus Calmette-Guérin macrophages and the EL-4 or P815 targets was greater than that between these two targets and inflammatory macrophages elicited by thioglycollate broth or between lymphocytes and either type of macrophage. Bacillus Calmette-Guérin MΦ also selectively bound three other neoplastic targets (P388, L1210, and RBL-5). The binding, which rose progressively for 60 min of cocultivation at 37 °C, was linear with respect to both the number of interacting targets and macrophages and required the presence of divalent cations and trypsin-sensitive structures on the macrophages. Binding was temperature dependent and required living, metabolically active macrophages. H-2 differences between targets and activated MΦ were not required for binding and did not prevent it. Finally, binding of the P815 targets to the Bacillus Calmette-Gue?in MΦ could be saturated by the addition of excess targets.     

Cloned Listeria monocytogenes specific non-MHC-restricted Lyt-2+ T cells with cytolytic and protective activity

Mice were infected with Listeria monocytogenes and Lyt-2+ T cell clones capable of lysing Ag-primed bone marrow macrophages were established. In accordance with earlier findings obtained at the population level, some T cell clones were identified which lysed bone marrow macrophages of different MHC type provided the relevant Ag was present. This unusual target cell recognition was further analyzed using a T3+, L3T4-, Lyt-2+, F23+, KJ16+ T cell clone, designated L-28. Target cell lysis by this clone was Ag specific, apparently non-MHC restricted. In contrast, YAC cells and P815 cells were not lysed by clone L-28. However, lysis of irrelevant targets could be induced by anti-T3, F23, or KJ16 mAb. Furthermore, Ag-specific lysis was blocked by anti-Lyt-2 mAb and by F(ab)2 fragments of F23 mAb. In addition to its cytolytic activity, clone L-28 produced IFN-gamma after co-stimulation with accessory cells, Ag, and rIL-2 and conferred significant protection on recipient mice when given together with rIL-2. These data suggest that non-MHC-restricted Lyt-2+ killer cells generated during listeriosis are cytolytic T lymphocytes that interact with their target Ag via the T cell receptor/T3 complex and the Lyt-2 molecule and, furthermore, that these cells play a role in anti-listerial resistance. The possible relevance of IFN-gamma secretion and target cell lysis for antibacterial protection is discussed.     

Antimetastatic effect of immunomodulators from Nocardia opaca in mice and rats activation of peritoneal macrophages by these fractions

Summary Nocardia delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, injected i.p. in an oil/water emulsion to F6 rhabdomyosarcoma-bearing rats, inhibited the development of pulmonary metastases; 6 out of 10 rats were protected. Repeated i.p. administration of emulsified NDCM and of two other compounds, a Nocardia water soluble mitogen (NWSM a hydrosoluble fraction) and purified cell walls (CW, an insoluble macromolecular fraction) in Lewis lung carcinoma (LLC)-bearing mice resulted in a significant reduction of lung metastases. The efficiency of these fractions was enhanced by association with monokines. A combination regimen of NDCM, NWSM, and CW (100 g/0.1 ml) and monokines (0.1 ml), injected i.p. in LLC-bearing mice, yielded a greater antimetastatic effect than either therapy alone. Peritoneal macrophages from mice which had been injected i.p. with NWSM or CW, when triggered either by TPA (tetradecanoyl phorbol acetate) or by zymosan, released large quantities of hydrogen peroxide and had a high rate of glucose consumption. These macrophages were activated as judged by their cytostatic activity against syngeneic P815 mastocytoma growth; they expressed biochemical markers which have been reported to characterize the activated state. Incubation of thioglycollate-elicited peritoneal macrophages with NWSM, and monokines for 72 h resulted in a cytotoxic activity against labeled LLC cells; addition of macrophage activating factor significantly increased the cytotoxic capacity of these macrophages. In view of this we postulate that the antimetastatic effect of soluble and insoluble N. opaca fractions and monokines might be mediated by activated peritoneal macrophages.     

Activation of the alternative complement pathway by Leishmania promastigotes: parasite lysis and attachment to macrophages

When exposed to normal human or guinea pig sera, promastigotes of Leishmania enriettii and L. tropica activate the complement cascade by the alternative pathway and fix C3 on their surfaces. In high (25%) serum concentrations, the result of complement activation is parasite lysis. At lower concentrations (4%), complement fixation results in enhanced parasite binding and uptake into murine peritoneal macrophages. Parasites are lysed in normal guinea pig, C4-deficient guinea pig, normal human, and C2-deficient human sera when they are incubated at 37 degrees C for 30 min. Fetal calf and normal mouse sera are poorly lytic. Lysis requires Mg++ but not Ca++, is mediated by heat labile (56 degrees C, 30 min) component(s), and does not occur when the incubations are maintained at 4 degrees C. Guinea pig serum preadsorbed with promastigotes of L. tropica in EDTA at 4 degrees C for 30 min is fully lytic. Immunofluorescence studies with anti-C3 antibodies show that under these conditions C3 is deposited on the surface of the parasite. The serum-dependent binding of parasites to macrophages is also mediated by heat-labile, nonadsorbable factor(s) present in normal guinea pig and mouse sera, as well as C2-deficient and C4-deficient sera. The serum-dependent macrophage recognition mechanism is trypsin sensitive but relatively resistant to chymotrypsin. Parasites but not macrophages can be presensitized at room temperature with low levels (8%) of serum to enhance their binding to macrophages. Presensitization does not occur at 4 degrees C. These results show that Leishmania promastigotes of several species can fix complement by activating the alternative complement pathway. This may then result either in parasite lysis or in an accelerated uptake of the parasite into phagocytic cells. In vivo, the biologic outcome of infection may reflect a balance between extracellular lysis and enhanced uptake into phagocytic cells.     

Characteristics of cytotoxic thyroglobulin-specific T cell hybridomas

Clones of cytotoxic thyroid-specific T cell hybridomas were generated by fusion of thyroglobulin-primed, "in vitro"-boosted CBA lymph node cells with the AKR-derived lymphoma cell line BW 5147. One hundred and thirty one clones were obtained. Among them, 15 were able to induce the lysis of 51Cr-labeled syngeneic thyroid epithelial cells after 5 h of incubation at 37 degrees C. Two T cell clones, HTC1 and HTC2, were further studied. These clones, which exhibit cell surface characteristics of cytotoxic cells, were specific for only syngeneic thyroid cells (allogeneic thyroid cells or syngeneic epithelial cells were never lysed by these hybridomas). Moreover, by using Ag-pulsed syngeneic macrophages as targets, syngeneic cytotoxicity was shown to be specific for thyroglobulin and not for a nonrelated Ag. The lysis obtained with these autoreactive thyroid-specific T cell clones is restricted to class I major histocompatibility Ag. This property is assessed by both the blocking of syngeneic cytotoxicity toward thyroid epithelial cells or thyroglobulin-pulsed macrophages only by anti-class I mAb and by the detection of specific lysis of target cells exclusively when effector hybrid cells and target thyroid epithelial cells or thyroglobulin-pulsed macrophages shared at least class I major histocompatibility Ag.