A monoclonal antibody against altered LFA-1 induces proliferation and lymphokine release of cloned T cells

A murine monoclonal antibody (I-17, IgM) has the following functional effects on murine long-term T cell clones: inhibition of cell-mediated lysis, induction of proliferation, release of lymphokines and change of the cell morphology. The determinant detected by I-17 is expressed on long-term T lines but not on thymocytes, lymph node cells and spleen cells. I-17 precipitated proteins with apparent molecular mass of 220 kDa, 170 kDa, 150 kDa and 100 kDa. Biochemical studies indicate that the determinant recognized by I-17 is tunicamycin sensitive and that I-17 binds to the alpha chain of the lymphocyte function-associated antigen (LFA-1).     

Activation of suppressor T cells during Epstein-Barr-virus-induced infectious mononucleosis.

Infectious mononucleosis is caused by the Epstein-Barr virus (EBV), an unusual human pathogen because it preferentially infects B lymphocytes and consequently activates them to produce immunoglobulins. When cultures of lymphocytes from patients with infectious mononucleosis were stimulated with polyclonal activators, unseparated cells failed to produce immunoglobulins, whereas purified B cells responded normally. Cocultures demonstrated profound suppressor T-cell activity in blood from patients with infectious mononucleosis. Early in this disease, circulating immunoglobulin-secreting cells were elevated, but during the second week their number was strikingly depressed. These data indicate that during infectious mononucleosis, EBV causes polyclonal activation of B cells, reflected by hypergammaglobulinemia and increased circulating immunoglobulin-secreting cells. Next, suppressor T cells become activated and inhibit further B-cell activation. Thus, activation of suppressor T cells in infectious mononucleosis provides a unique additional mechanism of host defense because these T cells inhibit the activation and proliferation of an important target of the causative virus.     

Unidirectional inhibition of early signal transduction of helper T cells by cloned suppressor T cells

Early intercellular events occurring in cloned T helper (Th) cells following interaction with cloned T suppressor (Ts) cells were studied by stopped-flow fluorometry. It was found that the increase of intracellular Ca2+ ([Ca2+]i) in major histocompatibility complex (MHC)-restricted Th clones induced by the stimulation with antigen and antigen-presenting cells (APC) is inhibited by the incubation with antigen-activated Ts clones. Optimal suppression required that the two cells recognize antigen on the same APC, although the restriction element for recognition could be different. There was an absolute requirement for recognition of the same antigen by these two cell types. The inhibitory effect was unidirectional in that Ts clones could inhibit the increase of [Ca2+]i of Th clones but not vice versa. Ts clones could not suppress the [Ca2+]i response of other Ts clones. If Th and Ts clones do not share the same MHC restriction specificity, a longer co-incubation time for activation of Ts is required for the inhibition of the [Ca2+]i response of the Th clone, suggesting the presence of a non-specific suppressive mediator that selectively acts on Th.     

Reciprocal stimulation of gammadelta T cells and dendritic cells during the anti-mycobacterial immune response

Gammadelta T cells and dendritic cells (DC) are two distinct cell types of innate immunity that participate in early phases of immune response against Mycobacterium tuberculosis infection. Here we show that a close functional relationship exists between these cell populations. Using an in vitro coculture system, Vgamma1 T cells from Tcrb(-/- )mice were found to be activated by DC infected in vitro with BCG, as indicated by the elevated CD69 expression, IFN-gamma secretion and cytotoxic activity. This activation process was due to a non-cognate mechanism since it required neither cell to cell contact nor interaction between the TCR and a specific antigen, but was mediated by DC-derived IL-12. Reciprocally, Vgamma1 T cells provided a key cytokine, IFN-gamma, which increased IL-12 production by BCG-infected DC. Moreover, exposure of BCG-infected DC to Vgamma1 T cells conditioned the former to prime a significantly stronger anti-mycobacterial CD8 T cell response. Consequently, stimulation of gammadelta T cells and their non-cognate interaction with DC could be applied as an immune adjuvant strategy to optimize vaccine-induced CD8 T cell immunity.     

DQw3 variants defined by cloned alloreactive T cells

The polymorphism of HLA class II molecules expressing the serologically defined alloantigen DQw3 was studied using cloned proliferative T lymphocytes. Two clones, IG9 and IC3, were selectively primed against DQw3-associated determinants and tested against a panel of 92 HLA-D homozygous cells. Both clones were specific for DQw3, but each showed a distinct response pattern. Clone IG9 recognized a DQw3-associated determinant expressed on a subset of DR4 and DR5 haplotypes and on all DRw6, 7, w8, and w9 haplotypes tested. In contrast, clone IC3 recognized a distinct DQw3-associated determinant expressed only on a subset of DR4 haplotypes. In monoclonal antibody inhibition experiments, anti-DQ, but not anti-DR or anti-DP antibodies, blocked reactivity of both clones IG9 and IC3, further demonstrating that the determinants defined by these clones are associated with DQ molecules. In DNA hybridization studies using a DQ beta probe, a correlation was observed between restriction site polymorphisms in the DQ beta gene, designated DQw"3.1" and "3.2," and the expression of the T-cell-defined IG9 and IC3 determinants. It is, thus, possible to demonstrate by cloned T-cell reactivity functionally relevant recognition sites on DQw3+ molecules that are associated with structural polymorphisms defined by molecular and genomic analysis.     

Activation of influenza-specific memory cytotoxic T lymphocytes by Concanavalin A stimulation

Traditionally, the in vitro activation of virus-specific memory cytotoxic T lymphocytes (CTLs) has been achieved by stimulating the CTLs with antigen-presenting cells (APCs) infected with an appropriate virus or pulsed with virus-specific antigenic peptides. Here, we describe the utilization of the polyclonal activator Concanavalin A (ConA) for in vitro restimulation of memory CTLs from virus-primed mice. Using this simple method, the activation of splenocytes with ConA for 3 days (i) eliminates the need to stimulate with virus-pulsed APCs and (ii) generates CD8⁺ CTLs that exhibit virus specificity and MHC-restricted lytic activity similar to CTLs obtained by conventional viral restimulation. In vitro ConA stimulation of splenocytes from BALB/c mice primed with the A/Texas/77 or A/Japanese/57 strain of influenza virus and from C57L/J mice infected with the A/Texas strain, generated CTLs with specific lytic activity. Hence reactivation of memory CTLs by this method is a general phenomenon rather than a mouse or viral strain-specific one. The ConA stimulation method used here had a recall of long-term (1 year) memory CTLs that effectively lysed virally infected targets. Further ConA-stimulated effector lymphocytes from virally primed animals have been shown to recognize and subsequently lyse target cells pulsed with virus or virus-derived peptides. The ConA reactivation of specific anti-viral CTLs may facilitate (i) studying anti-viral CTL responses and (ii) identifying of viral epitopes when unknown or when appropriate viral stimulation is impossible.     

Influenza peptide-induced self-lysis and down-regulation of cloned cytotoxic T cells.

Virus-specific cytotoxic T-cell (Tc) clones can lyse target cells in vitro in the presence of their specific peptide epitopes. The lytic potency of murine influenza nucleoprotein (NP)-specific Tc clones was investigated after observing that target cell killing was reduced in the presence of high (greater than 0.2 microM) concentrations of specific NP peptide antigen. Following incubation of Tc for 16 hr in the presence of a range of peptide concentrations, two effects were observed; (i) a peptide dose-dependent mortality of Tc, which has been attributed to self-lysis by clonal Tc in the presence of specific peptide; (ii) and a reduced ability to specifically lyse NP-expressing target cells whilst retaining lectin-dependent lytic activity in the surviving Tc. This functional down-regulation was reversible after 24 hr incubation in the absence of peptide. Toxic effects were excluded, since inhibition of specific target lysis by Tc was mediated only be pretreatment with specifically recognized peptide.     

Flare up of joint inflammations induced by cloned helper T cells. Retention of the helper T cells

Joint inflammations were induced in mice by intra articular (ia) injection of cloned helper T cells specific for methylated bovine serum albumin (mBSA) together with mBSA. Local injection of mBSA several weeks after waning of a joint inflammation induced by cloned helper T cells caused a flare up reaction. This indicates that the helper T cells persisted in the joint after the primary inflammation. In the present paper we show that the helper T cells can also persist for some time in a knee joint in the absence of the specific antigen and/or an inflammatory reaction.     

Change in functional phenotype of cloned human alloreactive cytolytic T cells

Alloreactive T cell clones primed in vivo were tested for the expression of T cell differentiation antigens CD2, CD3, CD4, and CD8. Each of 29 different clones were found to express CD2 and CD3, but were variable in their expression of CD4 (7 positive clones) and CD8 (15 positive clones). Six clones were positive for both CD4 and CD8. One of the 29 clones expressed neither CD4 or CD8. Over a period of 12–18 weeks of culture, these clones began to lose their alloreactivity but acquired NK-like activity. By changing the concentration of TCGF, the “allo” and “NK-like” lytic activities could be modulated. After 18 weeks of culture, these clones lost their alloreactive specificity, but not their NK activity. The expression of surface markers was unchanged. CD2 and CD3 molecules were determined to play a role in both the alloreactive and NK activity of these clones.     

Production of a histamine-releasing lymphokine by antigen- or mitogen-stimulated human peripheral T cells.

Human peripheral blood leucocytes (PBL), cultured in the presence of mitogen or antigen, yielded supernatants which contained a factor with histamine-releasing activity (HRA). This factor, analogous to the putative lymphokine HRA described by Thueson et al. (1979a), was shown to promote the rapid release of histamine from PBL preparations containing basophils. HRA production by PBL in response to co-culture with the skin test antigens Candida and SK-SD was shown to reflect the in vivo immune status of the PBL donor. Experiments with isolated cell populations from PBL implicated T cells as the prime source of HRA, production of the factor being macrophage-dependent. HRA activity failed to synergize anti-IgE-mediated histamine release, and was not species-restricted.     

Activation of extrathymic T cells in the liver during liver regeneration following partial hepatectomy.

Partial hepatectomy was performed in C57BL/6 mice to investigate whether extrathymic T cells in the liver are activated during liver regeneration. This study is based on the finding that in mice with malignant tumours, extrathymic T cells in the liver are activated and yet the intrathymic pathway is suppressed (i.e. thymic atrophy). Attention was therefore focused on whether a similar phenomenon is induced during benign cell regeneration. Extrathymic T cells were identified using the two-colour immunofluorescence test for CD3 and interleukin-2 receptor beta-chain (IL-2R beta) [or lymphocyte function-associated antigen-1 (LFA-1)] antigens. They were estimated to be intermediate CD3+ [or T-cell receptor (TcR)] cells with high expressions of IL-2R beta and LFA-1. It was demonstrated that the proportion and number of intermediate CD3+ cells increased in the early phase (days 2-4 after partial hepatectomy), and that the thymus was inversely atrophic at the same time. This raised the possibility that extrathymic T cells may also be responsible for regulation of normal cell regeneration.     

Structural differences in cell surface T25 polypeptides from thymocytes and cloned T cells

The molecule bearing the Thy-1.2 antigen, T25, has been isolated from fractionated thymocytes as well as from cloned cytolytic and helper T cells by immune precipitation with monoclonal antibodies. Using NaDodSO4/polyacrylamide gels cross-linked with N,N'-diallytartardiamide, electrophoretic analyses of the precipitates revealed a heterogeneous pattern of polypeptide bands, ranging in apparent molecular weight from 28,000-36,000 daltons. Thymocytes separated on the basis of Thy-1.2 surface antigen density were found to differ not only in amount of Thy-1.2 expressed, but also in the structure of the surface T25 molecules isolated from each subpopulation. This structural variation was evidenced in changes detected in the relative density and electrophoretic mobility of T25 band patterns obtained on SDS gels. Differences in T25 band patterns were also observed in polypeptides isolated from cloned cytolytic or helper T cells. These data suggest that although surface T25 is structurally heterogeneous, the pattern of heterogeneity can differ from cell to cell, or from population to population. Changes in T25 structure appear to correlated with differences in either T lymphocyte maturation or T cell function.     

Importance of oxidative metabolism in T cell cytotoxicity: a comparison of cloned T cells and spleen cells.

To distinguish between direct and indirect involvement of oxygen metabolites in CTL cytotoxicity a comparison was made of cloned CTLs and mixed cells from mouse spleen. Tumour cells could be protected from cloned CTLs and from spleen CTLs by the thiol protecting reducing agents DTT and DETC. Cytotoxic activity was inhibited by diversion of reducing power from NAD(P)H to artificial electron acceptors and by the inhibitor of NAD(P) linked enzymes cibacron blue. Although H2O2 formation could be detected during the lysis of P815 by spleen CTLs it did not prove to be a necessary requirement for cytolysis since it was not formed when glutaraldehyde-treated P815 cells were lysed. Of the scavengers of toxic oxygen metabolites tested only the hydroxyl radical scavenger sodium benzoate inhibited cytotoxicity.     

Activation of cholera toxin-specific T cells in vitro.

Cholera toxin (CT) and its B subunit (CT-B) are potent oral immunogens in vivo, although both strongly inhibit polyclonal lymphocyte activation in vitro. In order to help understand this paradox, we have studied the activation and proliferation of CT-specific T cells in vitro, by using CT-B-primed lymph node T cells as responders, concanavalin A-stimulated peritoneal macrophages as antigen-presenting cells (APCs), and various forms of CT-B as antigen. The results indicate that in many ways CT-specific T cells respond in a manner similar to that of T cells specific for other protein antigens: the degree of proliferation was proportional to the dose of antigen and APCs in the cultures, was antigen specific, and was H-2 restricted. APCs from genetic high-responder strains to CT stimulated significantly more proliferation in F1 (high x low) responder T cells than did APCs from low responder strains. However, there was a marked difference in the activation of CT-specific T cells when different forms of CT-B were used. Native CT-B stimulated little or no T-cell proliferation, whereas denatured CT-B or CT-B blocked by its ligand, GM1 ganglioside, stimulated T cells well. Addition of native CT-B to cocultures of primed T cells, APCs, and these latter stimulatory forms of CT-B inhibited the specific proliferative response to CT-B to varying degrees, depending on the ratio of the two forms in culture. We conclude that the ability of CT-B to inhibit T cells extends even to T cells specific for CT itself. Because of these inhibitory properties, processing of CT to nonbinding molecular forms or fragments must be an important prerequisite for the immune response to CT to occur in vivo, and such processing is likely to be important in the immune response to a variety of other enterotoxins as well.     

Activation of T cells by carbamazepine and carbamazepine metabolites

BACKGROUND: T-cell-mediated hypersensitivity is a rare but serious manifestation of drug therapy. OBJECTIVES: To explore the mechanisms of drug presentation to T cells and the possibility that generation of metabolite-specific T cells may provoke cross-sensitization between drugs. METHODS: A lymphocyte transformation test was performed on 13 hypersensitive patients with carbamazepine, oxcarbazepine, and carbamazepine metabolites. Serial dilution experiments were performed to generate drug (metabolite)-specific T-cell clones to explore the structural basis of the T-cell response and mechanisms of antigen presentation. 3-Dimensional energy-minimized structures were generated by using computer modeling. The role of drug metabolism was analyzed with 1-aminobenzotriazole. RESULTS: Lymphocytes and T-cell clones proliferated with carbamazepine, oxcarbazepine, and some (carbamazepine 10,11 epoxide, 10-hydroxy carbamazepine) but not all stable carbamazepine metabolites. Structure activity studies using 29 carbamazepine (metabolite)-specific T-cell clones revealed 4 patterns of drug recognition, which could be explained by generation of preferred 3-dimensional structural conformations. T cells were stimulated by carbamazepine (metabolites) bound directly to MHC in the absence of processing. The activation threshold for T-cell proliferation varied between 5 minutes and 4 hours. 1-Aminobenzotriazole, which inhibits cytochrome P450 activity, did not prevent carbamazepine-related T-cell proliferation. Substitution of the terminal amine residue of carbamazepine with a methyl group diminished T-cell proliferation. CONCLUSION: These data show that carbamazepine and certain stable carbamazepine metabolites stimulate T cells rapidly via a direct interaction with MHC and specific T-cell receptors. CLINICAL IMPLICATIONS: Some patients with a history of carbamazepine hypersensitivity possess T cells that cross-react with oxcarbazepine, providing a rationale for cross-sensitivity between the 2 drugs.     

Cytokine secretion profiles of cloned T cells from human aortic atherosclerotic plaques

T cells take part in the chronic inflammatory reaction in atherosclerotic plaques, but their specific role in atherosclerosis has not yet been fully elucidated. Nevertheless, one may anticipate that activated T cells may secrete cytokines capable of modulating the morphology and hence the stability of plaques by regulating cell proliferation, lipid metabolism, and extracellular matrix (ECM) synthesis and/or degradation. This study has been designed to investigate the functional properties of T cells in atherosclerotic lesions. For this purpose, T-cell clones were generated from atherosclerotic plaques isolated from human aortas obtained at autopsy from six subjects. Cloned cells were activated with PMA and OKT-3 to initiate cytokine production and cytokine profiles of CD4-positive clones were measured by ELISA. The majority of the T-cell clones (125/155, 81 per cent) produced both interferon (IFN)-γ and interleukin (IL)-4 (type 0 cytokine profile). Moreover, the production of IFN-γ was dominant in the majority of these clones. A type 1 cytokine profile (high levels of IFN-γ and low levels of IL-4) was found in 17 per cent of the clones (27/155). Only three clones (2 per cent) showed a type 2 cytokine secretion pattern (high levels of IL-4 and low levels of IFN-γ). No cytolytic activity could be established in plaque-derived T cells. Our results show that the T-cell population in atherosclerotic lesions is heterogeneous, but the most dominant T cell by far is the one with a type 0 cytokine profile. The dominant secretion of IFN-γ by T-cell clones suggest an important role for plaque T cells in modulating the growth and differentiation of other cells, such as macrophages and smooth muscle cells in atherosclerotic plaques. Copyright © 1999 John Wiley & Sons, Ltd.     

Activation or destruction of T cells via macrophages.

Macrophages (MPhi) affect the T cell response in two mutually exclusive ways: activation or deletion. A MPhi type with T cell activating functions (M1) is able to express and upregulate receptors of the B7 family. IFN-gamma favours this MPhi differentiation pathway via upregulation of CD80 (B7-1) and CD86 (B7-2). The treatment of MPhi with IFN-gamma enhances the alphaCD3-mediated T cell blast transformation and reduces the fraction of deleted T cells. This MPhi type may prevent antibody-mediated T cell destruction by the expression of costimulatory receptors. An IL-10-induced MPhi type (M2) fails to express costimulatory molecules of the B7 family but is an effective cell for T cell destruction. Forming cellular conjugates with T cells through antibodies or immune complexes, M2-MPhi preferentially delete targeted cells in vitro and in vivo.