Generation of alloreactive cytotoxic T lymphocytes: production of T cell and macrophage helper factors in addition to IL 1 and IL 2 by peritoneal cells from mice immunized to Listeria monocytogenes

We investigate the production and biological activity of soluble helper factors produced by peritoneal T cells and macrophage derived from mice primed in vivo with Listeria monocytogenes. Supernatant fluids from co-cultures of these immune T cells and activated macrophages contained Interleukin 1 (IL 1) and Interleukin 2 (IL 2), and had the ability to assist the generation of cytotoxic T lymphocytes (CTL) from a population of nylon wool nonadherent spleen cells sensitized to allogeneic heat-treated thymocytes. The ability to assist CTL development involved T cell and macrophage factors in addition to IL 1 and IL 2. Immune T cells cultured alone produced a factor, devoid of significant IL 2 activity, that assisted CTL development only if adherent cells were present in the responding population. Activated macrophage produced a 38,000 dalton component, distinct from IL 1 on the basis of m.w., that assisted the development of CTL from nylon wool nonadherent splenic cells. Supernatants fluids from co-cultures of immune T cells and allogeneic, nonactivated macrophage contained a CTL helper factor but did not contain IL 1 or IL 2 activities. In contrast, supernatant fluids from co-cultures of immune T cells and syngeneic, nonactivated macrophage contained all 3 activities. This suggests a genetic restriction for the production of IL 1 and IL 2 that does not restrict the production of a CTL helper factor. These results demonstrate that T cell- and macrophage-derived helper factors distinct from IL 1 and IL 2 participate in the development of CTL.     

Induction of cytotoxic T lymphocytes against herpes simplex virus type i: role of accessory cells and amplifying factor

Spleen cells from mice primed with herpes simplex virus type 1 (HSV-1) could be induced to differentiate into cytotoxic T lymphocytes (CTL) by in vitro culture with infectious HSV-1 but not by heat-inactivated virus. Induction of CTL failed to occur if the spleen cells were depleted of adherent cells by passage over columns of nylon wool before culture with virus. The CTL response could be restored by adding normal syngeneic peritoneal cells (PC) or L cell fibroblasts but not by allogeneic PC or BALB/c 3T3 fibroblasts. Thus, the induction of HSV-1-specific CTL was H-2 restricted. The response of HSV-1-stimulated nylon wool-depleted spleen cells could also be restored by adding amplifying factor (AF) produced in 24 hr mixed lymphocyte cultures. The addition of AF to nondepleted spleen cells also permitted the generation of CTL with heat-inactivated HSV-1 as a viral stimulant. Our results indicated that induction of a HSV-1 CTL response requires two signals, one provided by virus and a second, presumably nonspecific, by helper T cells. It was suggested that only the helper cells require H-2 restriction and need to be presented virus in the context of a macrophage.     

T-cell responses induced by the parenteral injection of antigen-modified syngeneic cells. II. Mechanisms, specificity, and cellular analysis of 2,4,6-trinitrophenol (TNP)-specific cytolytic response priming by intravenous versus subcutaneous injection with TNP-modified syngeneic cells

We have examined the underlying mechanisms accounting for the enhanced in vitro TNP-specific cytotoxic T-lymphocyte (CTL) response following the parenteral injection of syngeneic hapten-modified lymphoid cells. Augmented CTL activity noted following parenteral injection (iv vs sc) of 2,4,6-trinitrophenol-modified syngeneic spleen cells (TNP-SC) is most apparent when limiting numbers of TNP-modified stimulator cells are used in the in vitro sensitization phase. Enhanced CTL responses seen following sc and iv priming is due to distinct mechanisms. Spleen and lymph node (LN) cells from sc primed mice were found to contain significant levels of radioresistant helper activity upon coculture with either viable normal spleen cells in bulk culture or with thymocytes as the source of precursor CTLs in a limiting dilution assay. The helper activity was found to be mediated by a Lyt 1+2- T cells. In addition, Lyt 2-depleted spleen and LN cells from sc primed BALB/c mice could restore the ability of tolerant spleen cells from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-injected BALB/c mice to generate TNP-specific CTLs. Conversely, Lyt 2-depleted spleen and LN cells from iv primed mice provided no measurable helper activity either in bulk culture or in the limiting dilution assay and did not restore the ability of TNBS-tolerant BALB/c spleen cells to generate TNP-specific CTLs. CTL priming via the iv route was found to be completely antigen specific as iv injection of either 2,4-dinitrophenol (DNP)- or fluorescein isothiocyanatel (FITC)-modified cells caused no enhanced CTL activity. Priming via the sc route exhibited a unique specificity pattern as it was shown that sc injection of both TNP-SC and DNP-SC, but not FITC-SC, resulted in enhanced TNP-specific CTL responses. CTL T-helper (Th)-cell induction via the sc route was correlated with (1) the presence of H-2 I region determinants on the inducer cells as the sc injection of TNP-modified erythrocytes led to no enhanced CTL responses or CTL Th activity (while iv injection of TNP-erythrocytes did lead to enhanced CTL responses without detectable helper activity) and (2) the detection of both hapten-specific T-cell proliferation and Interleukin 2 (IL-2) production upon restimulation in culture. We conclude that the sc injection of TNP-SC leads preferentially to an increase of specific Lyt 1+ helper activity, while iv injection leads preferentially to an apparent expansion of Lyt 2+ prelytic effector CTLs.     

Further studies on Th-B, a cell surface antigenic determinant present on mouse B cells, plasma cells and immature thymocytes.

A goat antiserum (Goat anti-M104E) has been produced which contains antibodies selectively cytotoxic for mouse B cells and a subpopulation of thymus cells. It reacts with the Th-B antigenic determinant which has been shown by us (1–3) to be present on B cells and on plasma cells and on some cells in the thymus. It also is very cytotoxic for mouse B cells while a previously developed rabbit antiserum was not. The antiserum was obtained by immunization with cells of the BALB/c mouse myeloma MOPC-104E. When the antiserum was purified by in vivo absorption in mice, antibodies remained which were cytotoxic for cells of all of several myelomas at a titer between 1:128 and 1:1024 as determined by an in vitro complement dependent cytotoxicity test. The in vivo purified antibodies were also cytotoxic for about 70% of thymus cells, for about 70% of spleen cells, for about 50% of lymph node cells and for about 20% of bone marrow cells. They were very cytotoxic for splenic or lymph node B cells separated from T cells by a nylon wool column and only slightly cytotoxic for splenic or lymph node T cells. The antibodies were only weakly cytotoxic for one out of five T cell tumors tested and not cytotoxic for the remaining four. Irrespective of target cells used, the cytotoxicity of purified Goat anti-M104E was easily removed by absorption with cell suspensions from tissues which contain B cells, plasma cells or thymus cells. In order to confirm that the same anti-Th-B antibodies recognize the determinant present on spleen cells and on some thymocytes, the purified Goat anti-M104E serum was absorbed with either spleen cells or thymus cells. The absorbed sera were tested for ability to label thymocytes or spleen cells using the fluorescence activated cell sorter (FACS). Either absorption removed essentially all the antibody capable of binding to either cell population. In addition it was shown, using the FACS, that only B cells and not T cells of the spleen contain the Th-B determinant. The anti-Th-B antibodies have now been used for the rapid elimination of B cells from a mixed population of lymphocytes without affecting the function of mature T cells. Thus in vitro treatment of spleen cells from SRBC-immunized donors with purified Goat anti-M104E plus complement results in the killing of a high proportion of the B memory cells as shown by the reduction of PFC produced when the treated cells are transferred to irradiated recipients. The T cell helper function of the transferred cells is not affected by Goat anti-M104E treatment as shown by appropriate cell transfer experiments in which effective B cells are provided by an AKR anti-Thy-1.2-treated spleen cell population and effective T cells are provided by the Goat anti-M104E-treated spleen cell population. Antibodies detecting Th-B may serve as an approach to understanding the ontogeny of lymphocytes. Our results suggest that Th-B is a cell surface marker appearing early in the development of lymphoid cells, on the common precursor of B and T cells and that it is lost from T cells as they mature in the thymus.     

Capacity of different cell types to stimulate cytotoxic T lymphocyte precursor cells in the presence of interleukin 2

Plastic-adherent cells enriched for dendritic cells (AC) were found to be among the most potent stimulator cells for the activation of cytotoxic T lymphocytes (CTL) in vitro in the presence of interleukin 2 (IL 2) and a constant second set of allogeneic stimulator cells. Concanavalin A-activated nylon wool-nonadherent spleen cells ( CNWT ), concanavalin A-activated unfractionated spleen cells ( Cspl ), and some variants of the ESb T lymphoma line were equally effective as stimulator cells, however, and provoked a substantial cytotoxic response at concentrations of 10(4) cells per culture or less. In contrast, nonactivated nylon wool-nonadherent spleen cells ( NWT ) or unfractionated spleen cells (Spl) and cells of the P815 mastocytoma, the Meth A fibrosarcoma, and the T cell lymphomas Ly 5178 Eb and ESb did not stimulate cytotoxic responses at these cell concentrations. The strong stimulatory potential of the Cspl preparation was reduced by treatment with anti-Thy-1 antibody plus complement, whereas the stimulatory activity of the AC preparation was resistant to this treatment. All cell types tested expressed class I major histocompatibility antigens. Nonactivated NWT cells, in contrast to the CNWT preparation, showed no detectable staining with anti-I-E or anti-I-A antibodies and also a slightly weaker staining with class I antisera. Experiments with the tumor cell lines revealed, however, that there was no strict correlation between stimulatory potential and density of class I alloantigens or the expression of I-E determinants. Experiments on primary cytotoxic responses in vivo gave similar results. Experiments in cultures with a single set of stimulator cells and I region-compatible responder cells indicated that AC and Cspl or CNWT also have a markedly stronger capacity than NWT to induce IL 2-dependent DNA synthesis.     

Prethymic nylon wool-passed bone marrow cells, substituting for helper T cells, can augment the generation of cytotoxic T lymphocytes from their precursors.

Nylon wool-passed bone marrow (NW-BM) cells treated with anti-Thy.1 monoclonal antibody and complement were added to a mixed lymphocyte culture which contained a limiting number of lymph node cells, as responder cells, and a sufficient number of mitomycin-c-treated allogeneic spleen cells as stimulator cells. NW-BM cells of the same MHC haplotype as responder cells enhanced the generation of allo-specific cytotoxic T lymphocytes (CTL) not only at a relatively high dose (3 x 10(3) cells/well) of responder cells, but also at an extremely dilute dose (1 x 10(3) cells/well). NW-BM cells which had a third-party MHC haplotype, a haplotype different from both responder and stimulator cells, also enhanced the generation of CTL at relatively high doses, but not at low doses, of responder cells. NW-BM cells which had MHC haplotypes identical with those of responder cells induced CTL from helper T cell-depleted responder cells, but NW-BM cells which had the third-party haplotype did not. These results showed that the enhancing effects of NW-BM cells of the same MHC haplotype as responder cells might be due to a specific helper effect and the enhancing effect of NW-BM cells of the third-party haplotype might be due to a nonspecific filler effect, which only conditioned the cultured cells. It was also found that, to exhibit the helper effect, NW-BM cells had to possess MHC class II, but not MHC class I, molecules in common with CTL precursors. This study showed that in the induction of CTL, prethymic NW-BM cells had a capability comparable to that of mature helper T cells.     

Antigen-presenting T cells. I. Class I alloantigen (bm1)-bearing T lymphoblasts efficiently stimulate a primary clonal response of Lyt-2+ cytotoxic lymphocyte precursors of B6 origin

The cytotoxic response of splenic Lyt-2+ T cells to class I H-2 alloantigen-bearing stimulator cells was analyzed under limiting dilution conditions. One of 50 to one of 200 nylon wool-nonadherent (FACS-purified), small Lyt-2+ spleen cells of B6 origin gave rise in vitro to a cytotoxic T lymphocyte clone that specifically lysed targets bearing bm1 alloantigen. This population of alloantigen-specific cytotoxic lymphocyte precursors (CLP) was activated by different types of bm1 stimulator cells with different efficiency: 2 X 10(5) nonfractionated spleen cells, 5000 normal peritoneal cells, 400 to 10(4) L3T4+ helper T blasts, or 2000 to 10(4) Lyt-2+ T blasts induced clonal growth of this CLP pool. Irradiated or mitomycin-treated small (L3T4+ or Lyt-2+) bm1-derived T cells were inefficient stimulator cells for this response. Supplementation of culture medium with (recombinant) interleukin 2 was necessary and sufficient to support clonal development of alloantigen-triggered CLP in the presence of allogeneic T blasts. Under these limiting dilution conditions, we observed comparable cloning efficiencies for (wild-type) Kb-allospecific splenic Lyt-2+ CLP from bm1 mice generated in response to either irradiated B6 spleen cells or inactivated B6-derived T cell lines (EL4 and RBL-5 lymphoma cells). The data indicate that normal T lymphoblasts as well as tumor T cell lines stimulate clonal development in vitro of class I H-2-allospecific cytotoxic T lymphocytes in the presence of interleukin 2.     

Requirements for stimulation of T cell responses against virus-infected cells: nature of ectromelia virus-infected cells capable of stimulating cytotoxic T cells in a secondary response in vitro.

The nature of infected stimulator cells in the in vitro secondary cytotoxic T cell response to ectromelia infection was investigated. It was found that macrophages were better stimulator cells than spleen cells. B cells (Ig-positive cells) were superior to T cells (Ig-negative cells) both on a relative proportion and on a cell-to-cell basis. Concanavalin A and lipopolysaccharide-stimulated lymphocytes were also effective stimulator cells but appeared to be slightly inferior to spleen cells. Spleen cells depleted of Ia-positive cells were markedly inferior to normal spleen cells as stimulators. It was also found that primary and secondary cytotoxic T cells were largely Ia-negative. These findings are discussed in relation to the likely events during T cell responses to infection in vivo.     

Effect of L-ornithine on proliferative and cytotoxic T-cell responses in allogeneic and syngeneic mixed leukocyte cultures

The effect of L-ornithine on cytotoxic and proliferative responses in mixed leukocyte cultures has been analyzed. The activation of cytotoxic T lymphocytes (CTL) was strongly inhibited when 9 X 10(-3) M L-ornithine was added at the initiation of the cultures. The CTL precursor cells were not completely and irreversibly inactivated, however, since the cells generated normal cytotoxic activity if resuspended after 6 days in fresh culture medium together with a fresh set of stimulator cells. Experiments in microcultures with nylon-wool-nonadherent T-cell-enriched spleen cells as responder cells and "plastic adherent cells" as stimulator cells revealed that the cytotoxic responses were almost completely suppressed if ornithine was added within the first 20 hr but were only partially suppressed if ornithine was added after 48 hr. Also, ornithine had only a mild suppressive effect on proliferative responses in allogeneic and syngeneic mixed leukocyte cultures. The strong suppressive effect of the cytotoxic response was therefore not explained by a general toxic effect of L-ornithine on the responding cells in the culture. The addition of interleukin 2 (IL-2)-containing EL-4 supernatants did not prevent but rather enhanced the suppressive effect of L-ornithine. This indicated that the inhibitory effect was not (exclusively) expressed at the level of the IL-2-producing helper T cells. Since activated macrophages have been reported to secrete arginase, it appears that L-ornithine may be part of a regulatory circuit that selectively regulates the development of cytotoxic effector T cells.     

Inhibition of cytotoxic responses by prostaglandin E2 in the presence of interleukin 2

Experiments are described which provide direct evidence for a strong suppressive effect of prostaglandin E₂ (PGE₂) on the activation of cytotoxic T lymphocytes (CTL). The experiments also tested the hypothesis that PGE₂ inhibits cytotoxic responses exclusively by preventing the helper T cells from producing interleukin 2 (IL-2). In support of this hypothesis we found that indomethacin enhanced cytotoxic responses in the absence but not in the presence of concanavalin A-activated spleen cell supernatant (CSCS) indicating that the endogenously produced prostaglandin(s) inhibited primarily the production of IL-2. However, the addition of PGE₂ to microcultures was found to inhibit in a dose-dependent manner the activation of cytotoxic responses against I-region compatible stimulator cells even in the presence of CSCS. This inhibition was not observed in the presence of I-region incompatible stimulator cells, indicating that the inhibitory effect of PGE₂ did not result from a general toxic effect on the culture. The results argue against the notion that PGE₂ inhibits cytotoxic responses exclusively by preventing the production of IL-2.     

Mitomycin C-treated or irradiated concanavalin A-activated T cells augment the activation of cytotoxic T cells in vivo

The activation of cytotoxic T lymphocytes (CTL) in vivo after immunization of normal or cyclophosphamide-treated mice with allogeneic cells was strongly augmented by the administration of mitomycin C-treated or irradiated concanavalin A-activated spleen cells (Con A-spl). This effect of the Con A-spl was abrogated by treatment with Anti-Thy 1 antibody plus complement, and was therefore presumably mediated by activated "helper" T cells. (The term "helper" cell is only operationally defined in this context and indicates that the augmenting irradiation resistant T cells are obviously not CTL precursor cells). These observations indicated (i) that even the cytotoxic response against allogeneic stimulator cells suffers in vivo from insufficient "helper" T cell activity, and (ii) that the injection of Con A-spl may serve as a simple procedure to apply this "helper" activity in vivo. This procedure was at least as effective as the repeated injection of interleukin 2 (IL-2)-containing cell supernatants with up to four 30-unit doses of IL-2 per mouse. IL-2-containing cell supernatants were found to mediate similar effects only if injected into the footpads but not intravenously. This was in line with the reported observation that IL-2 has an extremely short half-life in vivo. The injection of Con A-spl was also found to augment the proliferative response in the regional lymph nodes.     

Helper T cells against tumor-associated antigens (TAA): preferential induction of helper T cell activities involved in anti-TAA cytotoxic T lymphocyte and antibody responses

This study establishes assay systems for helper T cell activities assisting cytotoxic T lymphocyte (CTL) and antibody responses to tumor-associated antigens (TAA) and demonstrates the existence of TAA that induce preferentially anti-TAA CTL helper and B cell helper T cell activities in two syngeneic tumor models. C3H/HeN mice were immunized to the syngeneic X5563 plasmacytoma or MH134 hepatoma. Spleen cells from these mice were tested for anti-TAA helper T cell activity capable of augmenting anti-trinitrophenyl(TNP) CTL and anti-TNP antibody responses from anti-TNP CTL and B cell precursors (responding cells) by stimulation with TNP-modified X5563 or MH134 tumor cells. The results demonstrate that cultures of responding cells plus 85OR X-irradiated tumor-immunized spleen cells (helper cells) failed to enhance anti-TNP CTL or antibody responses when in vitro stimulation was provided by either unmodified tumor cells or TNP-modified syngeneic spleen cells (TNP-self). In contrast, these cultures resulted in appreciable augmentation of anti-TNP CTL or antibody response when stimulated by TNP-modified tumor cells. Such anti-TAA helper activities were revealed to be Lyt-1+2- T cell mediated and TAA specific. Most interestingly, immunization with X5563 tumor cells resulted in anti-TAA helper T cell activity involved in CTL, but not in antibody responses. Conversely, TAA of MH134 tumor cells induced selective generation of anti-TAA helper T cell activity responsible for antibody response. These results indicate that there exists the qualitative TAA-heterogeneity as evidenced by the preferential induction of anti-TAA CTL- and B cell-helper T cell activities. The results are discussed in the light of cellular mechanisms underlying the preferential anti-TAA immune responses, and the interrelationship between various types of cell functions including CTL- and B cell-help.     

Lymphocyte responses to syngeneic antigens. I. Enhancement of the murine autologous mixed lymphocyte response by polyethylene glycol

The normally weak murine T-cell proliferative response against autologous non-T stimulator cells (the autologous mixed lymphocyte culture (MLC) was enhanced markedly by inclusion of the hydrophilic polymer, polyethylene glycol (PEG), into the culture medium. Potentiation of the autologous MLC was indicated on the basis of increased [³H]TdR incorporation by responding cells, as well as by the numbers of viable cells recovered from mixed cell cultures. PEG is not a polyclonal activator of T and/or B lymphocytes, since nylon wool nonadherent lymphoid cells (T cell-enriched fraction), nylon wool adherent cells (B cell-enriched fraction) and T cell-deficient “nude” spleen cells were not stimulated into DNA synthesis when cultured separately with PEG. Inclusion of 4% PEG into the culture medium was found to optimally enhance autologous MLC, although concentrations between 2 and 5% also significantly elevated responsiveness. At a responder/stimulator ratio of 1:2, autologous MLC yielded peak [³H]TdR incorporation after 5 days of culture. At lower ratios (1:1 and 2:1), however, Δ cpm of autologous MLC continued to increase over a culture period of 7 days. Enhanced responsiveness in the presence of PEG was observed in strains of mice representing a variety of H-2 haplotypes, indicating that at least the potential for autoreactivity of this type is a naturally occurring and widespread characteristic of murine species. An absolute requirement for purified T responder cells was necessary in the autologous MLC, since unseparated lymphoid cell responder LN or spleen cells demonstrated marked proliferation when cultured alone in medium containing PEG. The proliferation of T cells to autologous non-T cells within the same unseparated lymphoid cell preparation appears to be responsible for this phenomenon. Ia antigens expressed by the stimulator cells are involved in the induction of T-cell response, since anti-Ia sera added directly to the cultures inhibited the autologous MLC, but did not affect other T-cell responses to alloantigens or mitogens. Despite the marked proliferation observed in the autologous MLC performed in the presence of PEG, there was no generation of cytotoxic effector cells. Thus, PEG does not appear to add, or alter determinants on stimulator cells to an extent that they are recognized as foreign by precursor cytotoxic T cells. Although the mechanism of enhancement of autologous MLC by PEG is not totally defined, it appears, at least functionally, to promote cellular interactions that occur normally between T cells, B cells, and macrophages. In this respect, PEG will be a powerful and useful probe to dissect the cellular interactions that take place in autologous responses.     

Thymectomized, irradiated, and bone marrow-reconstituted chimeras have normal cytolytic T lymphocyte precursors but a defect in lymphokine production

A model system has been developed to study extrathymic T cell differentiation; mice have been thymectomized, lethally irradiated, and reconstituted with bone marrow cells depleted of Thy-1+ cells. After 8 wk, the spleen cells of these athymic, bone marrow-reconstituted chimeras contain Thy-1+ precytolytic T lymphocytes (CTL) that are able to respond to antigen only if supernatant from Con A-activated T cells is added to culture. The phenotype of these pre-CTL is similar to that of thymocytes, suggesting that they may be immature T cells. Initial evaluation of the CTL repertoire of these athymic mice demonstrated that the CTL generated to trinitrophenyl-modified syngeneic cells are H-2-restricted, and that the CTL generated to alloantigens have many of the cross-reactivities observed in normal mice but not in nude mice. In this report, we demonstrate a helper T cell defect in these thymectomized chimeras. These chimeras lack an Ly-1+ helper cell required for thymocytes to differentiate to CTL. Further studies revealed that when spleen cells from these thymectomized chimeras were stimulated with Con A, they produced normal levels of interleukin 2. However, these splenocytes were defective in the production of another factor needed for CTL differentiation.     

Stimulator cell requirements for allospecific T cell subsets: specialized accessory cells are required to activate helper but not cytolytic T lymphocyte precursors

Murine cortisone-resistant thymocytes were separated by staining with monoclonal anti-Lyt-2 antibody and FMF into Lyt-2- and Lyt-2+ subsets in order to analyze the nature of stimulator accessory cells required to activate each of these functionally distinct T cell subpopulations. The Lyt-2- fraction was able to proliferate but not to generate cytotoxic cells when stimulated by irradiated allogeneic spleen cells. Fractionation of the stimulator population showed that low numbers of dendritic cells and splenic macrophages, but not equivalent numbers of whole spleen cells or peritoneal macrophages, were able to stimulate the Lyt-2- population. On the other hand, the Lyt-2+ population, which showed little if any proliferation in response to irradiated spleen cells, contained all the precursors of cytolytic T lymphocytes. In contrast to the highly specific stimulator requirement of the Lyt-2- fraction, allospecific cytotoxic cells were generated from Lyt-2+ cells by any alloantigen-bearing stimulator cell provided interleukin 2 was present. This was confirmed by limiting dilution analysis: alloreactive CTL-P frequencies in spleen and thymus were not influenced by the nature of the stimulator cell. These data collectively indicate that heterogeneous Ia+ accessory cells are required to stimulate helper but not cytolytic T cell precursors.     

Cellular requirements for the generation of primary cell-mediated lympholysis responses to Qa-1 antigens

Mixed leukocyte cultures (MLC) between NZB responder spleen cells and Qa-1-disparate stimulator spleen cells were employed to determine the cellular requirements for the generation of primary anti-Qa-1 cell-mediated lympholysis (CML) responses. Although primary anti-Qa-1 cytotoxic lymphocytes (CTL) were generated during H-2-homologous stimulation, anti-Qa-1 CTL were not detectable from MLC in which the stimulators were H-2 allogeneic. Anti-Qa-1 CTL also were not generated from MLC in which the stimulators were semiallogeneic. Thus, H-2 identity between responder and stimulator cells was not sufficient to permit the generation of primary anti-Qa-1 CTL when H-2 disparity was also present. The capacity for H-2 disparity to prevent anti-Qa-1 CML responses was further demonstrated in MLC containing both H-2-allogeneic and H-2-homologous stimulator cells. Therefore, in subsequent studies we employed NZB responders and H-2-homologous, Qa-1-disparate stimulators. When various subpopulations of stimulator cells were studied for their ability to induce anti-Qa-1 CTL, nylon wool-adherent cells were found to be potent stimulators, but nylon wool-nonadherent cells were not. Furthermore, depletion of macrophages from the stimulator population abrogated the generation of anti-Qa-1 CML responses, despite the presence of responder macrophages in the culture. In contrast, all fractionated subpopulations stimulated anti-H-2 CML responses. When macrophage-enriched cells were used as stimulators, anti-Qa-1 CTL could be generated with approximately 80-fold fewer stimulator cells than when unfractionated splenocytes were used as stimulators. These findings indicated that stimulator macrophages were essential for the generation of primary anti-Qa-1 CTL. Direct evidence for macrophage expression of Qa-1-antigens was obtained by using a Qa-1b-specific CTL clone. These studies provide i) the first evidence for Qa-1 expression on macrophages, ii) a basis for comparison of the cellular interactions necessary to generate CTL against H-2K/D-encoded vs Qa-1-encoded class 1 antigens, and iii) a model for investigating the mechanisms responsible for the immunodominance of H-2K/D alloantigens.     

The requirement of Ia-positive accessory cells for the induction of tumor-specific cytotoxic T lymphocytes in vitro

The mechanism for the induction of cytotoxic T cells specific for tumor-associated antigens was studied by using fractionated responder T cells, tumor cells, and accessory cells in vitro. The tumor-specific cytotoxic T cells were induced by culturing immunized spleen cells with the tumor cells in vitro for 5 days. Nylon-column-purified T cells alone did not induce cytotoxic T cells upon culture with tumor cells, but the addition of normal spleen cells as accessory cells did successfully induce the cytotoxic T cells, suggesting that the presence of accessory cells is required for the activation of tumor-specific cytotoxic T cells in vitro. The accessory function was associated with spleen cell populations adhering to a plastic dish, a Sephadex G-10 column or a nylon wool column, and was sensitive to anti-Ia serum and C treatment, but was resistant to anti-Ig serum or anti-Thy 1 serum and C treatment, suggesting that the accessory cells are Ia-positive macrophages. Not only syngeneic but also allogeneic macrophages had the accessory function and the allogeneic macrophages were also Ia positive. These results suggest that Ia-positive macrophages play a crucial role in the induction of tumor-specific cytotoxic T cells in vitro. The possible role of Ia-positive accessory cells in the induction of tumor-specific cytotoxic T cells is discussed from the standpoint of cellular interactions.     

Alteration of allospecific t-cell receptors after differentiation from prethymic precursor cells in semiallogeneic environments

Murine bone marrow cells (strain A) have been allowed to differentiate in vivo in syngeneic (A) or semiallogeneic hosts (A × B) to produce mature splenic T lymphocytes. After stimulation of these cells with irradiated allogeneic (C) spleen cells in tissue cultures, the cytotoxic T-cell blasts (CTL) were purified by velocity sedimentation and used to immunize (A × C) F₁ hybrid mice, to produce antisera recognizing the receptor structure (for C) on the relevant A cytotoxic cells (and their precursors). Using these sera we have been able to show that the T-cell receptor for alloantigen C on strain A cytotoxic precursor lymphocytes (CTLp) seems to differ according to the host environment in which those T cells differentiate from immature bone marrow precursors.     

Murine CD8+ cytotoxic T lymphocytes lyse Toxoplasma gondii-infected cells

Studies were performed to determine whether CTL against Toxoplasma gondii-infected cells could be induced in a murine model of T. gondii infection in which CD8+ T lymphocytes have been shown to play a major role in resistance against this parasite. In 51Cr release assays, nylon wool nonadherent spleen cells from BALB/c (H-2d) mice immunized with the temperature-sensitive (ts-4) mutant strain of T. gondii were cytotoxic for T. gondii-infected P815 (H-2d) mastocytoma cells but not for uninfected cells. This cytotoxic activity was remarkably increased after in vitro stimulation with T. gondii-infected syngeneic spleen cells. The effector cells were shown to be CD8+ T lymphocytes, because the cytotoxicity was significantly inhibited by depletion of CD8+ T lymphocytes but not by depletion of CD4+ T lymphocytes. This cytotoxic activity was genetically restricted. Spleen cells from T. gondii-immune BALB/c mice were not cytotoxic for T. gondii-infected EL4 (H-2b) thymoma cells, whereas spleen cells from T. gondii-immune C57B1/6 (H-2b) mice were cytotoxic for T. gondii-infected EL4 cells but not for T. gondii-infected P815 cells. The cytolytic activity of CD8+ T lymphocytes against T. gondii-infected cells might be a mechanism whereby these cells confer resistance against T. gondii.     

Helper factor production in murine secondary syngeneic mixed leukocyte reactions

Culture supernatants of murine thymocytes or spleen cells responding in a secondary syngeneic mixed leukocyte reaction (SMLR) were studied for their biologic effects on cell-mediated immune responses in vitro. Such supernatants contained helper factor(s) that facilitated the development of alloantigen-specific cytotoxic T lymphocyte (CTL) responses from thymocyte precursors. Thymocytes, but not spleen cells, required activation by allogeneic effect factor (AEF) in primary culture in order to proliferate and produce biologically active mediator(s) during a secondary SMLR. The same culture supernatants possessed, in some instances, weak T cell growth factor (TCGF; IL 2) activity. However, TCGF activity could be dissociated from helper factor(s) active in the CTL induction assay because some culture supernatants that had potent helper activity were devoid of TCGF activity. This lack of TCGF activity was not due to a lower degree of sensitivity of the TCGF assay or to the presence of a selective TCGF inhibitor in the SMLR-derived supernatants, indicating that the helper factor(s) studied is distinct from TCGF. Production of immunoregulatory lymphokines during the SMLR may serve as a physiologically relevant model for studying the role of T cell-derived lymphokines in immunoregulation.