H-2K/H-2D and Mls and I region-associated antigens stimulate helper factor(s) involved in the generation of cytotoxic T lymphocytes

This study examines the antigen that stimulate production or release of a soluble helper factor(s) involved in development of cytotoxic T lymphocytes (CTL). Antigens associated with the Mls locus, I and K/D regions of the MHC were all capable of stimulating responder cells in MLC to produce helper factor. These supernatant fluids were all capable of providing "help" for the generation of cytotoxic T lymphocytes in MLC in which spleen cells are stimulated by allogeneic heat-treated thymocytes or splenocytes. Previous reports from our laboratory as well as others have shown that heat-treated cells do not stimulate a cytotoxic response. Heat-treatment of Mls, I, and H-2K/H-2D region incompatible stimulatory cells in MLC eliminated their ability to induce responder cells to produce helper factor, suggesting this is the mechanism whereby heat-treatment reduces the ability of cells to stimulate cell-mediated lympholysis (CML). The inability of supernatant fluids, from MLCs in which heat-treated cells were the stimulators, to assist in the generation of cytotoxic T cells did not appear to be the result of any suppressive factor induced by such treatment. Further, the antigens that stimulate pre-killer cells appear functionally distinct from those heat labile antigens (Mls, I, H-2K/H-2D associated) that stimulate helper factor production since heat-treated allogeneic cells served as stimulators of cytotoxicity provided helper activity was added to the MLC.     

E rosette forming lymphoblasts fail to stimulate allogeneic cells in mixed leukocyte culture (MLC)

Summary Lymphoblasts from patients with acute lymphatic leukemia were examined for the presence of surface markers and for their capacity to stimulate allogeneic donors in MLC. Lymphoblasts from eight patients, which made E rosettes, consistently failed to stimulate allogeneic donors on at least three separate occasions despite the vigorous response of these same allogeneic donors to remission cells from the patients.There were eleven patients who had lymphoblasts with no detectable markers or null lymphoblasts. Three of these also failed to stimulate in MLC. The null lymphoblasts from the remaining eight patients produced vigorous allogeneic responses. Since serologic data is now available suggesting that null lymphoblasts from some ALL patients have serologically detectable T cell antigens [16] while others have antigens found predominantly on B cells [20], it is conceivable that the capacity of these cells to stimulate in MLC may distinguish lymphoblasts within the null category with those which fail to stimulate representing early T cell precursors and those which do stimulate being early B cell precursors.     

A functional analysis of hematopoietic growth factor production from class I and class II human alloreactive T cell clones

Class I and Class II human alloreactive T cell clones or their conditioned media were mixed with progenitor cell-enriched null cells to assess their ability to stimulate human hematopoietic progenitor cell (HPC) growth. Optimal release of erythroid, myeloid or megakaryocyte colony-stimulating factors occurred after 72 hours and required contact of cloned T cells with irradiated stimulator cells expressing the appropriate major histocompatibility complex (MHC) determinants recognized by the T cells. Individual clones were quite heterogeneous in their capacity to release hematopoietic growth factors. Clones that produced optimal levels of factors that stimulated granulocyte-macrophage colony growth did not always produce equivalent amounts of factors that stimulated erythroid colony growth and vice versa when tested against identical target cells. Class II clones released nearly twice as much interleukin 3 activity as Class I clones. Class II clones that lacked cell-mediated lympholytic (CML) activity against B or T lymphoblastoid targets were consistent stimulators of HPC growth. In contrast, Class I or Class II clones that contained CML activity either poorly stimulated or inhibited HPC growth. These CML-positive clones produced greater amounts of gamma interferon. Our findings may have important implications for HPC growth following allogeneic mismatched bone marrow transplantation.     

Polymorphism of the Interleukin- and Interleukin Receptor Genes: Population Distribution and Association with Atopic Asthma

Population distribution and pathogenetic significance for bronchial asthma (BA) of the eight polymorphic variants of six interleukin- (IL) and interleukin receptor genes, C-589T, G/C 3"-UTRIL4, C-703T IL5 T113M IL9 Q551R, 150V IL4RA, G-80A IL5RA, and G1972A IL5RB, was examined. In the population samples of Russians, Tajiks, Buryats, and Tuvinians racial and ethnic specificity of these polymorphisms was established. These specific features were manifested as population-specific genetic portraits in respect of polymorphic allele frequencies. Analysis of the BA patients and their relatives from Tomsk by use of transmission/disequilibrium test (TDT) revealed the presence of a statistically significant association between the C-703 IL5 allele and the disease (P= 0.005). This is the first evidence of an association between the IL5 gene polymorphism and BA.     

Immunologic functions of isolated human lymphocyte subpopulations. IV. Stimulation of MLC and CML by human T cells.

Surface immunoglobulin positive and immunoglobulin negative human lymphocyte populations were obtained by immunoabsorbent column chromatography. Both cell populations were effective as stimulating and target cells in allogeneic MLC and CML reactions. The immunoglobulin negative population was further depleted of both EAC rosette forming cells and nylon wool adherent cells. The resulting highly purified T cell population was also able to stimulate allogeneic cells in MLC, and induce the generation of specifically cytotoxic killer cells in CML.     

Detection of melanoma-reactive CD4+ HLA-class I-restricted cytotoxic T cell clones with long-term assay and pretreatment of targets with interferon-γ

Twenty-five CD4⁺ cytotoxic T lymphocyte (CTL) clones were obtained from the peripheral blood or tumor tissues of melanoma patients undergoing active specific immunotherapy. Melanoma-reactive T cells were cloned by limiting dilution using either autologous or allogeneic melanoma cells to stimulate their proliferation. Sixteen of the clones reacted against autologous melanoma cells but not against the autologous lymphoblastoid cell line, which we defined as melanoma-specific. Optimal demonstration of the lytic activity of CD4⁺ CTL required a 16-h incubation period and an effectortarget cell ratio of 401. In addition, a 24-h pre-incubation of the target melanoma cells with 100 U interferon (IFN) consistently augmented lysis by these CD4⁺ CTL, increasing it from a mean level of 20% to one of 52%. Lysis by 8 of the 11 melanoma-reactive CD4⁺ T cell clones was exclusively HLA-class-I-restricted, as judged by blocking with monoclonal antibodies (mAb). Five of these HLA class-I-restricted clones were reactive only with the autologous melanoma cells, while the other 3 clones were also reactive with allogeneic melanoma cells. In all cases, the T cells and melanoma targets shared at least one HLA class I allele, usually HLA-A2, HLA-C3 or HLA-B62. Interestingly, lysis by 2 of the 11 clones was inhibited by both anti-HLA-class-I or -HLA-class-II mAb, while lysis by 1 other clone was inhibited by neither. HLA class I molecules and several accessory molecules were maximally expressed by the melanoma target cells, both in terms of distribution and copy number before IFN treatment. Thus, IFN may have acted by increasing the expression of melanoma-associated epitopes as presented by HLA class I (or HLA class II) molecules. A proportion of human CD4⁺CTL appeared to recognize melanoma-associated epitopes presented by the HLA class I molecule, although their lytic potency may be less than that of their CD8⁺ counterparts.This work was supported by USPHS grant R01-CA 36233, and a grant from the Concern Foundation for Cancer Research.     

T cell responses to allogeneic human mesenchymal stem cells: immunogenicity,tolerance, and suppression

Human mesenchymal stem cells (MSCs) were evaluated for their ability to activate allogeneic T cells in cell mixing experiments. Phenotypic characterization of MSCs by flow cytometry showed expression of MHC Class I alloantigens, but minimal expression of Class II alloantigens and costimulatory molecules, including CD80 (B7-1), CD86 (B7-2), and CD40. T cells purified from peripheral blood mononuclear cells (PBMCs) did not proliferate to allogeneic MSCs. Lack of response was not due to a deficiency of costimulation, since retroviral transduction of MSCs with either B7-1 or B7-2 costimulatory molecules did not result in lymphoproliferation. Although these results suggested that MSCs were immunologically inert or potentially tolerogenic, T cells cultured with MSCs produced IFN- and displayed secondary kinetics to restimulation with PBMCs, indicating alloantigen priming rather than tolerance induction by the MSCs. To determine whether MSCs suppressed alloreactive T cells, MSCs were added to primary mixed lymphocyte reaction (MLR) cultures. MSCs suppressed cell proliferation when added at the initiation of culture or when added to an ongoing MLR culture. Suppression was dose-dependent, genetically unrestricted, and occurred whether or not MSCs were pretreated with IFN-. MSCs in transwell chambers suppressed primary MLR cultures, indicating that suppression was mediated by soluble molecules. Analysis of cytokines in suppressed MLR cultures demonstrated up-regulation of IFN- and IL-10, and down-regulation of TNF- production relative to control cultures. We conclude that MSCs can initiate activation of alloreactive T cells, but do not elicit T cell proliferative responses due to active suppressive mechanisms.     

CD4+ T cell responses elicited by different subsets of human skin migratory dendritic cells

Skin dendritic cells (DC) are professional APC critical for initiation and control of adaptive immunity. In the present work we have analyzed the CD4+ T cell stimulatory function of different subsets of DC that migrate spontaneously from human skin explants, including CD1a+CD14- Langerhans' cells (LC), CD1a-CD14- dermal DC (DDC), and CD1a-CD14+ LC precursors. Skin migratory DC consisted of APC at different stages of maturation-activation that produced IL-10, TGF-beta1, IL-23p19, and IL-12p40, but did not release IL-12p70 even after exposure to DC1-driving stimuli. LC and DDC migrated as mature/activated APC able to stimulate allogeneic naive CD4+ T cells and to induce memory Th1 cells in the absence of IL-12p70. The potent CD4+ T cell stimulatory function of LC and DDC correlated with their high levels of expression of MHC class II, adhesion, and costimulatory molecules. The Th1-biasing function of LC and DDC depended on their ability to produce IL-23. By contrast, CD1a-CD14+ LC precursors migrated as immature-semimature APC and were weak stimulators of allogeneic naive CD4+ T cells. However, and opposite of a potential tolerogenic role of immature DC, the T cell allostimulatory and Th1-biasing function of CD14+ LC precursors increased significantly by augmenting their cell number, prolonging the time of interaction with responding T cells, or addition of recombinant human IL-23 in MLC. The data presented in this study provide insight into the function of the complex network of skin-resident DC that migrate out of the epidermis and dermis after cutaneous immunizations, pathogen infections, or allograft transplantation.     

Capacity of B cells to function as stimulators of a primary mixed leukocyte reaction

The capacity of B cells to serve as stimulator cells for a primary mixed leukocyte reaction (MLR) was evaluated. Percoll-fractionated B cells were stimulated with lipopolysaccharide and dextran sulfate (L/D) or a B cell stimulatory factor (BSF-1)-containing culture supernatant, and then were fixed before being used as stimulator cells to more precisely define the state of activation associated with MLR stimulatory capacity. It was found that unstimulated B cells or B cells stimulated for 1 day with L/D or BSF-1 were incapable of initiating a primary MLR, whereas B cells incubated for 3 days in L/D were potent stimulators. The differential activity of 1 day L/D- and BSF-1-activated B cells compared with 3 day L/D-activated B cells was not related to the amount of the relevant MHC class I or class II alloantigens on these cell populations, because all three groups had large increments in MHC class II expression in the following order: BSF-1 greater than 3 day L/D greater than 1 day L/D, and had little difference in MHC class I expression. Also, all three populations were capable of stimulating both MHC class I- and class II-specific T cell hybrids. It was concluded that the capacity of 3 day L/D-activated cells to stimulate a primary MLR was due to the elaboration of necessary co-stimulator molecules. We evaluated whether interleukin 1 (IL 1) was the co-stimulator involved. That this was not the case was indicated by two findings. First, 3 day-activated L/D cells failed to express IL 1 activity as measured by a highly sensitive IL 1 assay that utilizes the T cell line D10.G4.1. Second, recombinant IL 1 added to MLR cultures containing 1 day L/D- or BSF-1 activated B cells failed to function as a co-stimulator. In contrast, the phorbol ester PMA was a potent co-stimulator in this system. We conclude from these experiments that appropriately activated B cells can function as stimulators of a primary MLR, and that they elaborate critical co-stimulator molecules, distinct from IL 1, that enable them to function in this regard.     

Biphasic Modulation of GABAA Receptor Binding by Steroids Suggests Functional Correlates

Neuroactive steroids and other positive modulators of GABA_A receptors showed regional variation in both the efficacy and potency for modulation of [³⁵S]TBPS binding to rat brain membrane homogenates, with biphasic concentration-dependence. GABA present in the binding assays prevented the enhancement phase of the steroid concentration-dependence plot while the antagonists bicuculline and RU5135 prevented the inhibition phase. Using recombinant GABA_A receptors, expressed in insect cell line Sf9 using baculovirus, enhancement by steroids of [³⁵S]TBPS binding was sensitive to the presence of the 2 subunit and the nature of the subunit (122S > 12, 62, 622S, and 62). As in cerebellum, addition of RU5135 reduced the inhibitory phase and revealed a small enhancement of TBPS binding by neuroactive steroids. The subunit-dependent interactions of steroid and GABA site ligands are consistent with a three-state model in which the receptor mono-liganded by GABA or steroid has a different affinity for TBPS than the resting state, and the receptor biliganded by GABA, steroid, or both has little affinity for TBPS.     

Antigen presentation by interferon-gamma-treated endothelial cells and fibroblasts: differential ability to function as antigen-presenting cells despite comparable Ia expression

The effect of interferon-gamma (IFN-gamma) on endothelial cell (EC) and fibroblast (FB) class II major histocompatibility complex (MHC) gene product expression and antigen presenting ability was examined. Control FB did not express class II MHC gene products, whereas a small (less than 1%) population of passaged EC expressed class II gene products. IFN-gamma induced a comparable density of HLA-DR expression on nearly all EC and FB. IFN-gamma-treated EC and FB also expressed HLA-DP but at a lower density, whereas HLA-DQ expression was barely detectable on either cell type. Control FB were not able to stimulate allogeneic T4 cell DNA synthesis or function as antigen-presenting cells (APC). Control EC were also unable to stimulate allogeneic T4 cell DNA synthesis unless large numbers of stimulator cells were used. Small numbers of IFN-gamma-treated EC were able to stimulate allogeneic T4 cell DNA synthesis, whereas larger numbers were markedly more effective than control EC. In contrast, IFN-gamma-treated FB were ineffective stimulators of allogeneic T4 cell DNA synthesis. IFN-gamma-treated FB were able to present the exogenous antigen SKSD to autologous but not allogeneic T4 cells, but they were extremely inefficient APC. The inability of IFN-gamma-treated FB to function as APC could not be explained by FB-mediated immunosuppression, Ia density, or HLA-DQ expression. This limited capacity of IFN-gamma-treated FB to participate in Ia-restricted functional interactions with T4 cells correlated with a similar diminished capacity to support nonspecific mitogen-induced proliferation of T4 cells before IFN-gamma-induced Ia expression. This accessory cell function was not enhanced by IFN-gamma treatment. Monocytes syngeneic to the responding T4 cells but not interleukin 1 (IL 1) permitted IFN-gamma-treated FB but not control FB to stimulate allogeneic T4 cell DNA synthesis, but they remained markedly less effective stimulators than monocytes. Moreover, IFN-gamma-treated FB were effective stimulators of alloprimed T4 cells, in contrast to their inability to stimulate fresh T4 cells. Furthermore, monocytes and IFN-gamma-treated FB were comparably effective stimulators of alloreactive T cell lines. These data suggest that accessory cells perform functions unrelated to Ia and IL 1 that are necessary for mitogen-, alloantigen-, and antigen-induced proliferation of freshly isolated T cells. Monocytes and EC effectively perform this function, but FB do not. This accessory cell function does not seem to be as important for the activation of primed T cells.     

Reversal of phorbol ester-mediated reduction of cloned T lymphocyte cytolysis by interleukin 2

During previous studies on the regulation of cloned T lymphocyte function, we observed that murine cytotoxic T lymphocyte (CTL) clones progressively lose the ability to lyse appropriate target cells during prolonged (24 to 48 hr) incubation with the tumor promoter phorbol myristic acetate (PMA). We further observed that the cytolytic function of PMA-treated CTL clones can be restored by incubation with secondary MLC supernatant (2 degrees MLC SN), a potent source of cytokines. We now report our observations on the nature of the cytokine(s) responsible for recovery of CTL activity. Like 2 degrees MLC SN, the lectin-induced SN from a cloned helper T cell and the lectin-induced SN from a T cell hybridoma can restore cytolytic activity to cloned CTL treated with PMA. In contrast, supernatants from L929 cells, WEHI-3 cells, and P388D1 cells fail to restore cytolytic activity to similarly treated cloned CTL. These data suggest that IL 2 and/or gamma-IFN, but not CSF-1, CSF-GM, IL 3, or IL 1, can influence expression of cytolysis by cloned CTL. Furthermore, highly purified IL 2 can restore cytolytic activity, even when cytosine arabinoside is present to inhibit clonal expansion. Our studies indicate that cytolysis is a reversible function of cloned CTL, and that cytolysis may not necessarily represent an end-stage feature of CTL maturation. Our studies further show that IL 2 is both necessary and sufficient for resumption of cytolytic function by "deactivated" CTL. As such, these observations suggest that IL 2 can regulate not only T cell proliferation but also the expression of cytolysis by some cytolytic T cell populations.     

Conditions for the generation of autoreactive human T cell clones: requirement for lymphokines other than interleukin 2 alone

In an attempt to determine whether factors other than interleukin (IL) 2 alone were necessary for the generation of autoreactive suppressive T cell clones, lymphocytes from HLA-Dw-matched allogeneic mixed leukocyte cultures (MLC) were propagated and cloned in purified IL 2, partially purified IL 2, conditioned medium (CM) from stimulated peripheral blood mononuclear cells (PBMC), or with purified IL 2 plus IL 3, IL 4, or interferon-gamma (IFN-gamma). Cloning efficiencies were very low in all cases (less than 10%) and of 48 clones tested only 6 were capable of autocrine proliferation after stimulation with autologous PBMC. Four of these clones were derived from populations expanded and cloned in CM, one from cultures with partially purified IL 2, and one with purified IL 2. All were CD4+ alpha/beta-T cell receptor. Their stimulation was blocked by anti-DR and broadly reactive MHC class II-specific monoclonal antibodies (mAb) but not by anti-DQ or anti-DP mAb. One clone was blocked exclusively by broad mAb but not by anti-DR, -DQ, or -DP mAb, and this was the only clone to suppress lymphocyte proliferation in allogeneic MLC, a property previously described for autoreactive clones derived under similar conditions detecting potentially novel lymphocyte activating determinants designated "DY." These results therefore suggest that DY-specific autoreactive suppressive clones are produced under these conditions only at a low frequency and that an unidentified factor other than IL 2, IL 3, IL 4, or IFN-gamma is involved in their generation.     

Differential effect of anti-beta 2-microglobulin on IL 2 production and IL 2 receptor expression in the primary mixed lymphocyte culture reaction

The mechanism of inhibition of the proliferative response in primary mixed lymphocyte culture (1 degree MLC) by antibodies to beta 2-microglobulin (beta 2m) was investigated. It is demonstrated that anti-beta 2m antibodies inhibit the production of interleukin 2 (IL 2). In contrast, the expression of IL 2 receptor is not affected by anti-beta 2m. The addition of purified exogenous IL 2 to the antibody-treated 1 degree MLC can completely restore the proliferative response, indicating that anti-beta 2m does not interfere with IL 2 binding to its receptor. Similarly, anti-beta 2m does not interfere with the capacity of IL 2-dependent T cell lines or T cell clones to respond to exogenous IL 2. The inhibition of cell proliferation and IL 2 production by anti-beta 2m is maximal when the antibody is added at the beginning of 1 degree MLC culture, and no effect of anti-beta 2m is seen when added after 3 days of culture. Anti-beta 2m has no effect on mitogen-induced cell proliferation and IL 2 production. Anti-beta 2m acts on the responder cell population, as demonstrated in experiments in which responder cells or stimulator cells are treated separately with the antibody. The expression of HLA-class II antigens (i.e., HLA-DR and DQ (DC) on the T cells activated on 1 degree MLC is not affected by anti-beta 2m. These studies indicate that the HLA-beta 2m class I antigen complex plays a role in T lymphocyte activation via release of IL 2, and suggest the existence of different mechanisms for activation of IL 2 producers and IL 2 responders in 1 degree MLC.     

Auto-tumor recognition following in vitro induction of MHC antigen expression on solid human tumors: stimulation of lymphocytes and generation of cytotoxicity against the original MHC-antigen-negative tumor cells

Summary Expression of major histocompatibility complex (MHC) class I antigens was induced in eight out of nine freshly prepared tumor cell suspensions by exposure to interferon (IFN) and tumor necrosis factor (TNF) in vitro. The untreated, class-I-antigen-negative, and the treated, antigen-positive, cells of three tumors (one breast carcinoma, one plasmocytoma and one ovarian carcinoma) were compared for the capacity to stimulate autologous and allogeneic blood lymphocytes, to generate auto-tumor cytotoxicity and for sensitivity to the lytic effect induced in autologous mixed lymphocyte tumor cell culture (MLTC). The MHC class I-negative cells did not stimulate, while the cells induced for expression of antigens did. On the other hand, when the autologous cytotoxic cells were generated in the MLTC by the class I antigen-positive tumor cells the class I-negative tumor cells were also damaged. Lysis of the class-I-positive tumor cells was abrogated by the W6/32 monoclonal antibody directed against the monomorphic part of the class I molecules.     

Differential ability of fixed antigen-presenting cells to stimulate nominal antigen-reactive and alloreactive T4 lymphocytes

The capacity of paraformaldehyde-fixed human antigen-presenting cells (APC) to induce responses by autologous, freshly isolated peripheral blood T4 cells was examined and was compared with their ability to stimulate allogeneic T4 cell DNA synthesis. Fixation of glass-adherent cells (AC) with as little as 0.06% paraformaldehyde abolished leucine incorporation, whereas fixation with 0.75% paraformaldehyde caused death of greater than 98% of the AC. Control APC were able to take up and present the soluble antigens streptokinase-streptodornase (SK-SD), tetanus toxoid, or tuberculin-purified protein derivative to autologous Ia-depleted T4 cells. Fixation with greater than 0.06% paraformaldehyde eliminated such ability. When AC were incubated with antigen overnight and were then fixed, however, they were able to present nominal antigen to autologous T4 cells in a genetically restricted manner that was blocked by monoclonal antibodies directed against monomorphic determinants on class II major histocompatibility complex (MHC) molecules. Despite the ability to present nominal antigen, paraformaldehyde-fixed AC were unable to induce allogeneic T4 cell proliferation. Similar results were observed when non-T cells or spleen cells were used as stimulators. The inability of fixed APC to stimulate allogeneic T4 cell DNA synthesis was not reversed by increasing the number of fixed APC or by the addition of control AC autologous to the responding cells. Moreover, interleukins 1 and 2 either alone or in combination also failed to permit maximal T cell proliferation in response to fixed allogeneic APC. The differential effects of fixation on nominal antigen and alloantigen presentation could not be explained by the loss of membrane thymocyte stimulatory activity on fixed AC. These results indicate that antigen-bearing fixed APC are competent to stimulate proliferation by antigen-reactive T4 cells, but are deficient at inducing allogeneic T4 cell DNA synthesis. The differential sensitivity of these two Ia-restricted functions of APC to chemical denaturation (reductive methylation) by paraformaldehyde suggests that the allodeterminants and restriction elements for nominal antigen on MHC class II molecules can be functionally dissociated.     

Effect of Adriamycin on liposomal muramyl tripeptide's ability to up-regulate monocyte cytokine expression

Liposomal muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) is a biological agent in phase I and II trials for osteosarcoma and melanoma. Its mechanism of action has been linked to its ability to activate monocyte tumoricidal function and to stimulate monocyte production of tumor necrosis factor (TNF) and interleukins(IL)-1, –6, and –8. Our ultimate goal is to combine L-MTP-PE with chemotherapy. The purpose of this study was to determine whether doxorubicin (Adriamycin) interfered with the ability of L-MTP-PE to activate monocyte cytokine production. Human monocytes were cultured with or without 5–500 ng/ml of Adriamycin for 3 h and washed before being exposed to 2 g/ml L-MTP-PE for 16 h. Cultured supernatants were collected and assayed for TNF, IL-1, IL-6, and IL-8. The messenger RNA expression of IL-1, IL-1, TNF, IL-6, and IL-8 was quantified with northern blot analysis. Adriamycin did not suppress the upregulation of any of these cytokines. We concluded that combination therapy with L-MTP-PE and Adriamycin is feasible and that this combination warrents further investigation in a clinical setting.     

Graft-versus-host reaction-induced immune modulation. I. Donor-recipient genetic disparity and the differential expression of Lyt-2, L3T4, and Ly-6 during acute reactions in the host thymus

To investigate the effects of graft-vs-host reactions (GvHR) on cells in a central lymphoid compartment, GvHR were induced across class I/II or class II only major histocompatibility complex differences utilizing the parent into nonirradiated F1 hybrid (P----F1) model. Thymocytes were subsequently examined during the acute stage of GvHR for the expression of Lyt-2, L3T4, and Ly-6 cell surface molecules. Class I/II "suppressive" GvHR resulted in a dramatic decrease (greater than 85%) in total thymocyte numbers by 2 wk after parental cell injection. Although a dramatic decrease in the percentages of Lyt-2 (85%----30%) and L3T4 (95%----50%) expression was observed, the percentage of thymocytes expressing Ly-6 was markedly increased compared to uninjected controls (5%----greater than 80%). This increased percentage was not due solely to a selective loss of Ly-6 negative cells from the thymus, since the actual number of Ly-6 positive cells was greater in GvHR mice than in controls. Class II GvHR during the same time interval resulted in a less dramatic decrease (20 to 60%) in total thymocyte numbers. In addition, the effect on the percentages of Lyt-2 (85%----approximately 70%) and L3T4 (95%----approximately 85) expression was subtle and transient. However, intrathymic Ly-6 expression was again clearly enhanced (5%----20 to 30%). Class I/II "proliferative" or "stimulatory" GvHR differ from "suppressive" reactions in that they are characterized by stimulatory pathologic symptoms and the appearance of autoimmune abnormalities. Such GvHR were found to result in minimal alteration of total thymocyte numbers. Similarly, the percentage expression of Lyt-2 and L3T4 was marginally affected. However, the percentage of Ly-6 expression was increased from 5%----20 to 30% and thus these intrathymic lymphocyte profiles more closely resemble those of class II as compared to class I/II "suppressive" GvHR. The present findings therefore demonstrate that major histocompatibility complex differences alone do not necessarily determine the effects of GvHR on recipient thymocytes and that Ly-6 is a useful marker for the early detection of GvHR-associated immunologic events.     

Qualitative and quantitative studies of antigen-presenting cell function by using I-A-expressing L cells

I-A-expressing transfected murine L cells were analyzed as model antigen-presenting cells. Four features of accessory cell function were explored: antigen processing, interaction with accessory molecules (LFA-1, L3T4), influence of Ia density, and ability to stimulate resting, unprimed T lymphocytes. I-A+ L cells could present complex protein antigens to a variety of T cell hybridomas and clones. Paraformaldehyde fixation before but not subsequent to antigen exposure rendered I-A+ L cells unable to present intact antigen. These results are consistent with earlier studies that made use of these methods to inhibit "processing" by conventional antigen-presenting cells. The ability of anti-L3T4 antibody to inhibit T cell activation was the same for either B lymphoma or L cell antigen-presenting cells. In striking contrast, anti-LFA-1 antibody, which totally blocked B lymphoma-induced responses, had no effect on L cell antigen presentation, measured as interleukin 2 (IL 2) release by T hybridomas, proliferation, IL 2 release, or IL 2 receptor upregulation by a T cell clone. I-A+ L cell transfectants were found to have a stable level of membrane I-A and I-A mRNA, even after exposure to interferon-gamma-containing T cell supernatants. In agreement with earlier reports, a proportional relationship between the (Ia) X (Ag) product and T cell response was found for medium or bright I-A+ cells. However, dull I-A+ cells had a disproportionately low stimulatory capacity, suggesting that there may be a threshold density of Ia per antigen-presenting cell necessary for effective T cell stimulation. Finally, I-A-bearing L cells were shown to trigger low, but reproducible primary allogeneic mixed lymphocyte responses with the use of purified responder T cells, indicating that they are capable of triggering even resting T cells. These studies confirm the importance of antigen processing and I-A density in antigen-presenting cell function, but raise questions about the postulated role of the LFA-1 accessory molecule in T cell-antigen-presenting cell interaction. They also illustrate the utility of the L cell transfection model for analysis and dissection of antigen-presenting cell function.     

Tumor cytotoxicity of human monocyte membrane-bound interleukin-1α induced by synergistic actions of interferon-γ and synthetic acyltripeptide,FK-565

Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [¹²⁵I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon- (rIFN-), but not other cytokines [rIFN-A, rIFN-, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN- and then with FK-565. FK-565 also acted synergistically with rIFN- to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1) antiserum, but not by a specific anti-(IL-1) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 of human blood monocytes can be induced by two activation signals (rIFN- then FK-565) at their suboptimal concentrations.Abbreviations IL interleukin - IFN interferon - TNF tumor necrosis factor