Measles virus-specific human T cell clones. Characterization of specificity and function of CD4+ helper/cytotoxic and CD8+ cytotoxic T cell clones

PBMC from healthy adult individuals seropositive for measles virus (MV) were tested for their capacity to proliferate to UV-inactivated MV (UV-MV) or to autologous MV-infected EBV-transformed B cell lines (EBV-BC). MV-specific T cell responses were observed in 11 of 15 donors tested (stimulation index greater than 2), when optimal doses of UV-MV were used in proliferative assays. T cell clones were generated from PBMC of three donors responding to MV, by using either UV-MV or MV-infected autologous EBV-BC as APC. Stimulation with UV-MV generated exclusively CD3+ CD4+ CD8- MV-specific T cells, whereas after stimulation of PBMC with MV-infected EBV-BC, both CD3+ CD4+ CD8- and CD3+ CD4- CD8+ MV-specific T cell clones were obtained. Of 19 CD4+ T cell clones tested so far, 7 clones reacted specifically with purified fusion protein and 1 with purified hemagglutinin protein. Seven clones proliferated in response to the internal proteins of MV. Three clones reacted to whole virus but not to one of the purified proteins, whereas one clone seemed to recognize more than one polypeptide. Some of the T cell clones, generated from in vitro stimulation of PBMC with UV-MV, failed to recognize MV Ag when MV-infected EBV-BC were used as APC instead of UV-MV and PBMC. CD3+ CD4+ CD8- T cell clones recognized MV in association with HLA class II Ag (HLA-DQ or -DR), and most of them displayed CTL activity to autologous MV-infected EBV-BC. All CD4+ HLA class II-restricted CTL clones thus far tested were capable of assisting B lymphocytes for the production of MV-specific antibody. The CD4- CD8+ T cell clone MARO 1 recognized MV in association with HLA class I molecules and displayed cytotoxic activity toward MV-infected EBV-BC.     

Acetyl glyceryl ether phosphorylcholine (PAF-acether) and leukotriene B4-mediated neutrophil chemotaxis through an intact endothelial cell monolayer

Human endothelial cell monolayers were grown on nucleopore filters, and used to partition the two halves of a modified Boyden chamber. Human neutrophil chemotaxis through the monolayer was studied in response to leukotriene B4 and acetyl glyceryl ether phosphorylcholine (PAF-acether). Both leukotriene B4 and PAF-acether concentration-dependently stimulated neutrophil chemotaxis through intact monolayer. The biologically inactive lyso-PAF, and leukotriene C4 and D4 were inactive as chemotactic agents. Leukotriene A4 was weakly chemotactic. In the absence of chemotaxin, little penetration of the monolayer by neutrophils was observed. Agents that elevate neutrophil cyclic AMP levels inhibit both leukotriene B4 and PAF-acether-stimulated chemotaxis through the endothelial cell monolayer. The specific 5-lipoxygenase inhibitor, 6,8-de-epoxy-6,9-(phenylimino) delta 6,8-prostaglandin I1 (U-60257), inhibits PAF-acether, but not leukotriene B4-mediated chemotaxis. These data suggest that an intact 5-lipoxygenase may be required for normal PAF-acether-mediated chemotaxis, but leukotriene B4-mediated chemotaxis is independent of 5-lipoxygenase activity. This system may prove to be a useful model for the study of neutrophil-endothelial cell interactions.     

Different roles of the CD2 and LFA-1 T-cell co-receptors for regulating cytotoxic, proliferative, and cytokine responses of human V gamma 9/V delta 2 T cells

BACKGROUND: Human V gamma 9/V delta 2 T lymphocytes recognize nonpeptidic antigens in a manner distinct from the classical antigen recognition by alpha beta T cells. The apparent lack of major histocompatibility (MHC) restriction and antigen processing allows very fast responses against pathogenic insults. To address the potential functional requirement for accessory molecules, we investigated the roles of the CD2 and lymphocyte function-associated antigen (LFA)-1 T-cell co-receptors in antigen-induced activities of human V gamma 9/V delta 2 T-cell clones. MATERIALS AND METHODS: Human peripheral blood V gamma 9/V delta 2 T lymphocytes were cloned and their cytotoxicity against Daudi lymphoma was measured by a standard 51Cr-release assay. The responses of V gamma 9/V delta 2 T lymphocytes to nonpeptidic antigens were assessed using DNA synthesis and cytokine ELISA assays. Monoclonal antibodies specific for various molecules with potential T-cell accessory functions were utilized in blocking assays. RESULTS: All of our V gamma 9/V delta 2 T-cell clones displayed the Th1 phenotype. The anti-LFA-1 antibody strongly inhibited the cytotoxicity of V gamma 9/V delta 2 T cells against Daudi B-cell lymphoma; whereas, it had no influence on the antigen-induced cytokine release or proliferation. In contrast, antibodies against CD2 and LFA-3 had no effect on the lytic activity of V gamma 9/V delta 2 T cells, but strongly inhibited the cytokine release and proliferation. However, the CD2-LFA-3 interaction was not an absolute requirement for the cytokine release and the DNA synthetic activity of antigen-stimulated V gamma 9/V delta 2 T cells, since the inhibitory effect could be reversed by addition of exogenous interleukin 2 (IL-2). CONCLUSIONS: These novel observations indicate that the signals generated by different accessory molecules and IL-2 can contribute in an integrated fashion to the regulation of V gamma 9/V delta 2 T cells. These interactions may be important for the effectiveness of V gamma 9/V delta 2 T-cell responses.     

Cyclophosphamide stimulates lung fibroblasts to release neutrophil and monocyte chemoattractants

Cyclophosphamide is an alkylating antineoplastic agent used in several conditions. However, little is known about the mechanism of its pulmonary toxicity. In the present study, we determined that human lung fibroblasts release activity for neutrophils and monocytes in response to cyclophosphamide in a dose- and time-dependent manner. Checkerboard analysis revealed that both neutrophil and monocyte activities were chemotactic. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular-sieve column chromatography revealed that both neutrophil (NCA) and monocyte (MCA) chemotactic activities had multiple peaks. NCA was inhibited by a leukotriene B(4) receptor antagonist and anti-interleukin-8 and anti-granulocyte colony-stimulating factor antibodies. MCA was attenuated by a leukotriene B(4) receptor antagonist and anti-monocyte chemoattractant protein-1 and anti-granulocyte-macrophage colony-stimulating factor antibodies. The concentrations of interleukin-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, and granulocyte-macrophage colony-stimulating factor significantly increased in response to cyclophosphamide. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to cyclophosphamide.     

The generation of cytolytic activity in mixed leukocyte cultures: cortisone-resistant thymocytes are deficient in helper T lymphocytes

Cortisone-resistant (CR) thymocytes did not generate cytolytic activity toward H-2 K or D alloantigen unless they were also stimulated by H-2 I or non-H-2 alloantigens, even though spleen cells generated brisk cytolytic activity toward H-2 K or D alone. Backstimulation by stimulating strain T lymphocytes accounted for neither the response of spleen cells toward H-2 K or D alloantigen nor the response of CR thymocytes to a full set of alloantigens. In addition, lack of non-T accessory cells did not account for the CR thymocyte pattern of reactivity. Rather, CR thymocytes appeared to be relatively deficient in helper T lymphocytes (HTL). CR thymocytes generated specific cytolytic activity toward H-2 D alloantigen when T cell growth factors (TCGF) or cloned alloreactive helper T lymphocytes were added to culture. CR thymocytes contained fewer HTL precursors detected at limit dilution than spleen cells did. Thus spleen cells generated cytolytic activity toward class I alloantigens alone, but under the same culture conditions CR thymocytes had to be stimulated by both class I and class II alloantigens. Class II alloantigens may be required to stimulate cytolytic activity only under culture conditions in which class I-reactive HTL are not sufficient to provide a minimal threshold of help.     

Enhancement by monokines of leukotriene generation by human eosinophils and neutrophils stimulated with calcium ionophore A23187

Human blood eosinophils and neutrophils that had been incubated with the supernatants of cultures of lipopolysaccharide (LPS)-stimulated blood mononuclear cells demonstrated respective enhanced abilities to produce immunoreactive leukotriene C4 (LTC4) and immunoreactive leukotriene B4 (LTB4) after activation by the calcium ionophore A23187. Under optimal conditions, the enhancing effect was observed with the eosinophils (n = 21) and the neutrophils (n = 14) from all but one donor of each type of granulocyte. Enhancement was maximum when granulocytes were preincubated with a 1/3 dilution of LPS-stimulated mononuclear cell culture supernatants for 1 to 2.5 min and were then stimulated with 2.5 microM ionophore for 1 to 2 min (neutrophils) or 15 min (eosinophils). Maximal enhancement ranged from 20 to 4500% for LTC4 generation by eosinophils (geometric mean, 87%) and from 30 to 1600% for LTB4 generation by neutrophils (geometric mean, 105%). There was no enhancement of leukotriene biosynthesis when the LPS-stimulated mononuclear cell culture supernatants and ionophore were added simultaneously to the granulocytes. The enhancing activity for LTC4 generation by eosinophils was removed by washing the cells after the addition of the LPS-stimulated mononuclear cell culture supernatants and before the introduction of ionophore. This enhancing activity was produced by Ig-, Leu-1- adherent blood mononuclear cells, which are presumed to be monocytes; supernatants of adherent cells augmented A23187-induced LTC4 generation by eosinophils from 21 to 2300% (geometric mean, 402%) in 11 experiments and LTB4 generation by neutrophils from 7 to 200% (geometric mean, 60%) in 10 experiments. There was an inverse correlation between the percent enhancement and the LTC4 levels produced by stimulated eosinophils in the absence of the monokine(s) (r = -0.79, p less than 0.01), but not between percent enhancement and the LTB4 levels generated by ionophore-activated neutrophils in the control buffer. The activity of the monocyte-derived enhancing material on each type of granulocyte was relatively heat stable. Enhancement of eosinophil production of LTC4 was associated with an acidic group of monocyte-derived molecules having isoelectric points of 4.2 to 4.3, 4.5 to 4.6, and 4.9, and exhibiting marked heterogeneity in size.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytosolic phospholipase A2-alpha is necessary for platelet-activating factor biosynthesis, efficient neutrophil-mediated bacterial killing, and the innate immune response to pulmonary infection: cPLA2-alpha does not regulate neutrophil NADPH oxidase activity

The role of a cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA(2)-alpha activity was blocked with the specific cPLA(2)-alpha inhibitor, Pyrrolidine-1 (human cells), or by cPLA(2) -alpha gene disruption (mice). cPLA(2)-alpha inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcgammaII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA(2)-alpha inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B(4). Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA(2)-alpha(-/-) mice compared with wild type littermates. These studies identify a novel role for cPLA(2)-alpha in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.     

Mast cell mediators stimulate synthesis of arachidonic acid metabolites in macrophages

Mast cells and macrophages were isolated from human lung tissues by using density gradient centrifugation, cell sorter, and adherence techniques. Passively sensitized mast cells in the absence of exogenous arachidonic acid (AA) released leukotriene (LT)C4, LTD4, PGD2, and thromboxane-B2 when challenged with Ag, and in the presence of AA, released 5-hydroxyeicosatetraenoic acid (HETE) and 15-HETE in addition to the above metabolites. Passively sensitized macrophages did not release significant amounts of AA metabolites when challenged with Ag. However, these cells released LTB4, LTC4, LTD4, LTE4, 5-HETE, PGE2 and 6-keto-PGF1 alpha when co-incubated with activated mast cells. During co-incubation, mast cells also generated greater amount of AA metabolites than when they were activated alone. The stimulatory action of mast cells on macrophages was shown to be due to the extracellular factor(s) present in the supernatant of the activated mast cells. Both heat and trypsin inhibited the biologic activity of mast cell-derived stimulatory factor. In addition, extraction of mast cells' materials with chloroform or ether showed no activity associated with the organic phase, suggesting it possibly possesses a protein nature, such as peptides, protease, or peptidase. These results suggest that mast cell-macrophage interaction might be important in the generation of multiple mediators in the airways during immediate hypersensitivity reactions.     

Diacylglycerols enhance human neutrophil degranulation responses: relevancy to a multiple mediator hypothesis of cell function

At 10 microM, 1-0-oleoyl-, 1-0-palmitoyl-, and 1-0-myristoyl-2-0-acetyl-glycerol weakly stimulated neutrophils to release lysozyme, an enzyme in secondary granules, but had no such effect on the release of a primary granule enzyme, beta-glucuronidase. The glycerides (1-10 microM) had a second effect on both granule populations: they enhanced the degranulating potencies of leukotriene B4, platelet-activating factor, a formylated oligopeptide, and C5a by 10- to 30-fold. In contrast, they were much less effective in enhancing responses to ionophore A23187 and partially inhibited responses to phorbol myristate acetate. The diether analogue, 1-0-hexadecyl-2-0-ethylglycerol was inactive in these regards. We suggest that diacylglycerols are a novel class of bioactive products mobilized from phosphoglycerides in stimulated neutrophils; as co-products of this mobilization, platelet-activating factor and leukotriene B4 may interact with diacylglycerols to promote cell function.     

Polymorphonuclear leukocytes modulate tissue factor production by mononuclear cells: role of reactive oxygen species

To determine whether polymorphonuclear leukocytes (PMN) modulate the production of tissue factor (TF) by monocytes, PBMC were incubated with increasing concentrations of PMN. PMN did not express any procoagulant activity. After 20-h cocultures, PMN enhanced or inhibited the TF production of PBMC, and this effect depended on the PMN/PBMC ratio. When the ratio increased from 1/1000 to 1/5, without or with LPS, the TF activity of PBMC increased to peak at 2.5-fold the baseline value (p < 0.01). The TF Ag and TF mRNA also increased. This potentiating effect was mediated by reactive oxygen species (ROS) released by PMN during the coculture; it did not require direct cell contact between PMN and PBMC, it was enhanced when PMN were stimulated by fMLP (a chemotactic peptide), and it was inhibited by two antioxidants, N-acetyl cysteine and pyrrolidine dithiocarbamate. In contrast, when the PMN/PBMC ratio was further increased from 1/2 to 2/1, the PBMC TF activity, Ag, and mRNA decreased and were inhibited compared with those of PBMC cultured alone (p < 0.01). This inhibitory effect required direct cell contact between PMN and PBMC, and it was not due to a PMN-mediated cytotoxicity. To confirm the role of ROS, H2O2 enhanced then inhibited the TF activity of PBMC in a dose-dependent manner, similarly to PMN. Thus, PMN may play an important role in the pathogenesis of thrombosis and atherosclerosis by exerting concentration-dependent regulatory effects on the TF production by PBMC via the release of ROS.     

The predominance of CD8+ T cells after infection with measles virus suggests a role for CD8+ class I MHC-restricted cytotoxic T lymphocytes (CTL) in recovery from measles. Clonal analyses of human CD8+ class I MHC-restricted CTL

Stimulation of PBMC, in children recovering from acute measles, with autologous EBV-transformed and measles virus (MV)-infected lymphoblastoid cell lines (B-LCL) expanded primarily MV-specific CD8+ T cells. A large number of CD8+ T cell clones were obtained either by passaging of bulk cultures at limiting dilutions or by direct cloning of PBMC without previous stimulation in bulk culture. The MV-specific CD8+ T cell clones responding in a proliferative and a CTL assay were found to be class I MHC restricted. In contrast, CD4+ MV-specific T cell clones, which were generated by the same protocol, recognized MV in association with class II MHC molecules. Analysis of processing requirements for Ag presentation to CD8+ and CD4+ T cell clones, measured by the effect of chloroquine in a proliferative T cell response, revealed that both types of T cells recognized MV Ag processed via the endogenous/cytoplasmic pathway. Thus, these studies indicate that, as in most other viral infections and in contrast to previous suggestions, the class I MHC-restricted CTL response by CD8+ T cells may be an important factor in the control and elimination of MV infection. Therefore, the role proposed for CD4+ class II-restricted T cells in recovery from measles needs to be reevaluated.     

IL-4 regulates IL-2 induction of lymphokine-activated killer activity from human lymphocytes

IL-4 is a pluripotent lymphokine acting on various cell types. We investigated the role of human IL-4 on the generation of lymphokine-activated killer (LAK) activity. Human IL-4 alone did not induce LAK activity and inhibited IL-2 induction of LAK activity from unstimulated PBMC, peripheral blood null cells, spleen cells, and lymph node cells in a dose-dependent manner. IL-4 also inhibited several phenomena induced by IL-2 such as cell proliferation, augmentation of NK activity, increase of Leu-19+ cells, and expression of IL-2R(p55) on either CD3+ or Leu-19+ cells. IL-4, however, augmented cell proliferation with other T cell mitogens including PHA, Con A, PMA, or allo-MHC Ag with or without IL-2. In contrast to unstimulated cells, IL-4 alone induced marked cell proliferation and LAK activity as well as Leu-19+ cells from in vitro IL-2 preactivated PBMC or null cells, and did not inhibit IL-2 induced cell proliferation, LAK activity, Leu-19+ cells and IL-2R(p55) expression, but rather augmented them with low doses of IL-2. Although IL-4 alone induced LAK activity from peripheral blood of some patients previously given IL-2, IL-4 inhibited in vitro LAK generation with IL-2 from these cells in most cases. Therefore, IL-4 appears to directly inhibit the IL-2 activation pathway via IL-2R(p70) and prevent resting LAK precursors from proliferating and differentiating into final effector cells. However, once cells were sufficiently preactivated by IL-2, IL-4 induced LAK activity and did not inhibit IL-2 activation of these cells. These data suggest an immunoregulatory role of IL-4 on human null cells and T cells.     

Combination of vaccination and chimeric receptor expressing T cells provides improved active therapy of tumors

We have generated murine T cells expressing chimeric immune receptors (CR) against human 5T4 oncofetal Ag (h5T4) and evaluated their tumor therapeutic efficacy alone and in combination with immunization using a replication-defective adenovirus encoding h5T4 (Rad.h5T4) and bone marrow-derived dendritic cells (BMDC). The h5T4-specific engineered T cells demonstrated Ag-specific, non-MHC-restricted cytolysis of h5T4-positive B16 and CT26 tumor cells in vitro by cytotoxicity assay and antitumor activity in vivo using a Winn assay. In the s.c. injected B16h5T4 melanoma model, early local but not systemic i.v. administration of syngeneic h5T4-specific CR T cells significantly increased mice survival. This improvement was further enhanced when combined with immunization with Rad.h5T4, followed by post-CR T cell treatment with BMDC in the active therapy model, possibly through mechanisms of enhancing Ag-specific cellular immune responses. This synergistic effect was lost without delivery of the BMDC. Our findings suggest that combining engineered T cells with specific vaccination strategies can improve the active tumor therapy.     

The role of the accessory cell in mitogen-stimulated human T cell gene expression

The role of the accessory cell in optimizing T cell proliferative responses to mitogens is a well known but poorly understood phenomenon. To further dissect the function of the accessory cell in allowing T cell proliferation, we compared mitogen-induced c-myc, interleukin 2 (IL 2), and IL 2 receptor gene expression in peripheral blood mononuclear cells (PBMC) and in T cells rigorously depleted of accessory cells through differential adherence and anti-Dr (anti-class II major histocompatibility antigen) monoclonal antibody complement-directed cytotoxicity. In cultures stimulated with phytohemagglutinin (PHA), a mitogen that requires accessory cells to induce T cell proliferation, expression of all measured genes was accessory cell dependent, since accumulation of their mRNA in PBMC was greater than that in cultures depleted of accessory cells. These genes varied in their accessory cell dependence, with IL 2 expression most dependent, c-myc expression least dependent, and IL 2 receptor expression intermediate in dependency. Use of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or ionomycin, mitogens that stimulate T cell proliferation independent of accessory cells, induced equal levels of gene expression in PBMC and in T cells depleted of accessory cells. These results suggest that PHA-stimulated T cells are dependent on an accessory cell signal(s) for optimal expression of the genes for c-myc, IL 2, and IL 2 receptor, and for proliferation. In addition, this signal(s) appears to be delivered early in the course of T cell activation events, since it can be bypassed by mitogens that directly activate protein kinase C (TPA) or induce calcium translocation (ionomycin). In addition, these data provide further evidence that expression of the c-myc protooncogene is insufficient for T cell mitogenesis, since PHA-induced accumulation of c-myc mRNA was only partially accessory cell dependent, whereas proliferation was completely accessory-cell dependent.     

Enhancing effect of anti-HLA class I monoclonal antibodies on T cell proliferation induced via CD2 molecule

The mAb 131 to a determinant preferentially expressed on the gene products of the HLA-A locus, the mAb Q6/64 and 4E to determinants preferentially expressed on the gene products of the HLA-B locus, the anti-HLA-A2,A28 mAb CR11-351, HO-2, HO-3, HO-4, and KS1, and the anti-HLA-B7 cross-reacting group mAb KS4 enhanced proliferation of T cells in most, if not all, the PBMC preparations stimulated with the anti-CD2 mAb 9-1 + 9.6. The mAb CR10-215, W6/32, and 6/31 to distinct monomorphic determinants of HLA class I antigens enhanced CD2-induced T cell proliferation in at most 30% of the PBMC preparations tested. The anti human beta 2-microglobulin (beta 2-mu) mAb NAMB-1 displayed no detectable effect on the proliferation of T cells stimulated with the mAb 9-1 + 9.6. The enhancing effect of anti-HLA class I mAb is specific, is dose dependent, is not abrogated by the addition of exogenous IL-1 and IL-2 to the cultures, and reflects the interaction of anti-HLA class I mAb with T cells. Enhancement of CD2 mediated proliferation of T cells is not a unique property of anti-HLA class I mAb, since the anti-HLA class II mAb Q5/6 and Q5/13 also had a similar effect. Analysis of the kinetics of the enhancing effect of anti-HLA class I mAb suggests that they modulate an early event of T cell activation and may affect the interaction of T cells with mAb 9-1. Phenotyping of T lymphocytes activated by mAb 9-1 + 9.6 in the presence of anti-HLA class I mAb suggests that the enhancing effect of anti-HLA class I mAb may reflect the recruitment of a higher percentage of T cells. The present study has shown for the first time that certain, but not all, the determinants of the HLA class I molecular complex are involved in the proliferation of T cells stimulated with the anti-CD2 mAb 9-1 + 9.6. Furthermore, the inhibitory effect of mAb CR11-351, KS1, Q6/64, and W6/32 on the proliferation of T cells stimulated with mAb OKT3 or with mAb BMA 031 indicates that the same determinants of HLA class I antigens play a differential regulatory role in T cell proliferation induced via the CD2 and CD3 pathway.     

An antigen-specific helper T cell hybridoma produces an antigen-specific suppressor inducer molecule with identical antigenic fine specificity. Implications for the antigen recognition and function of helper and suppressor inducer T cells

We have previously described a T cell hybridoma, A.1.1, that responds to specific Ag (P18, a synthetic polypeptide of defined sequence) in the context of I-Ad by producing lymphokines. Herein we report that this cell also releases, into culture supernatants and ascites fluid, an Ag-specific activity that functions in the induction of suppression of anti-SRBC PFC responses. This suppressive activity requires a) Ag-non-specific accessory molecules from a T suppressor inducer factor, b) Ly-2+ T cells in the assay cultures, and c) the specific Ag (P18) conjugated to the SRBC in the assay cultures. The specificity of the A.1.1-derived activity was demonstrated by the absence of suppression in cultures containing SRBC, BSA-SRBC, or conalbumin-SRBC rather than P18-SRBC. Further, the A.1.1-derived activity bound to, and could be eluted from, P18 but not conalbumin. Using a panel of synthetic variant peptides, we have mapped the critical residues in P18 required for Ag/I-Ad induced activation of A.1.1. These peptides were tested for their ability to act as targets for the A.1.1-derived suppressive activity when conjugated to SRBC and added to assay cultures. All peptides capable of stimulating the A.1.1 T cells to release lymphokines were similarly effective in the suppressor assay. Thus, the recognition of Ag by the T cells and by the T cell-derived activity appeared to be identical. The A.1.1-derived molecule was found to be capable of inducing L3T4- T cells to act as suppressor T cells following culture. These suppressor cells were active in inhibiting anti-SRBC responses in the absence of P18 and bore the Ly-2 surface marker. Thus, it is likely that the function of this Ag-specific molecule is to induce Ly-2+ suppressor T cells and thereby cause the inhibition of the response. This function is distinct from that normally associated with helper T cells and may shed new light on the possible relationship between the cell surface T cell receptor for Ag and Ag-specific T suppressor inducer molecules.     

Phorbol myristate acetate-activated keratinocytes stimulate proliferation of resting peripheral blood mononuclear lymphocytes via a MHC-independent, but protein kinase C- and intercellular adhesion molecule-1-dependent, mechanism.

Recent attention has focused on the role keratinocytes (KC) may play in the induction of T cell-mediated inflammatory responses in skin, particularly because KC, when activated by immunologic stimuli, express MHC class II Ag and secrete immunomodulatory cytokines. We tested the capacity of normal human KC that were stimulated with PMA to induce PBMC proliferation. PMA-treated, but not untreated, KC induced proliferation of allogeneic as well as autologous PBMC; in addition, when purified CD4+ or CD8+ T cells were used as responders, each subset proliferated. PBMC proliferation was not due to direct action of PMA on PBMC, nor to contamination of KC cultures with Langerhans cells (LC) or dermal APC. Pretreatment with different protein kinase C inhibitors abrogated the capacity of PMA-stimulated KC to induce proliferation. Paraformaldehyde-fixed PMA-KC stimulated PBMC proliferation, whereas supernatants from PMA-treated KC failed to do so, indicating that a membrane-associated activity on PMA-KC contributes to the induction of PBMC proliferation. PMA induced intercellular adhesion molecule-1 (ICAM-1) expression on KC; furthermore, mAb against ICAM-1 or against its ligand lymphocyte function-associated Ag (LFA-1) (CD11a/CD18) significantly, but incompletely, reduced the stimulatory capacity of PMA-treated KC, indicating that ICAM-1/LFA-1 interaction contributed to PBMC proliferation. IFN-gamma or TNF-alpha also induced ICAM-1 on KC, but these KC failed to stimulate proliferation, suggesting that PMA induces additional signals from KC, which act in concert with ICAM-1 to promote proliferation. Finally, mAb against HLA-ABC or HLA-DR did not inhibit proliferation. We conclude that PMA can activate KC to stimulate T cell proliferation in a MHC-independent fashion. This activation is mediated by protein kinase C and in part by the induction of ICAM-1 expression on KC.     

IL-1 is an autocrine growth factor for T cell clones

Activation of Th lymphocytes requires that Ag be presented on the surface of accessory cells displaying Ia Ag. A number of studies have concluded that the T cell also requires IL-1 from accessory cells. However, we recently reported that one murine T cell clone (D10.G4.1) produced its own IL-1-like activity after encountering APC (9). In this report, we demonstrate that 1) IL-1 production is a common property of murine T cell clones, 2) T cell IL-1 activity is blocked by anti-IL-1-alpha antiserum, 3) IL-1-alpha mRNA can be directly visualized in individual cloned T cells using in situ hybridization techniques, and 4) IL-1 appears to serve an autocrine role in the activation of T cell clones inasmuch as anti-IL-1-alpha antiserum blocks cell proliferation when the T cell is the only IL-1 source.     

Human T lymphotrophic virus type I may not be associated with multiple sclerosis in Japan.

To study the possible involvement of human T lymphotrophic virus type I (HTLV-I) or a related retrovirus in Japanese cases of multiple sclerosis (MS), we first performed a Western blot analysis with purified Ag of HTLV-I. Ten out of 31 MS patients (32.2%), 19 of 66 patients (28.8%) with other neurologic diseases, and 2 of 64 healthy blood donors (3.1%) had antibodies reactive with Ag corresponding to the group-specific Ag (gag) proteins (p15, p19, p24) on their sera. There were no significant differences between MS and other neurologic diseases concerning the patterns and the frequency. Second, we tried to establish T cell lines from PBMC of 22 MS patients with crude IL-2 without accessory cells, because HTLV-I-infected T cells can be immortalized in a high ratio under those conditions. Only one T cell line (MS-14C), however, could be maintained in long term culture. MS-14C and cultured T cells for 3 to 5 wk derived from MS patients were examined by Southern blot analysis under both stringent and low stringent conditions with HTLV-I as a probe. No HTLV-I related bands could be detected. By polymerase chain reaction examination, we also could not detect HTLV-I provirus genome in the fresh PBMC from 20 MS patients, although some of them had gag-reactive antibodies. Our data do not favor the hypothesis of HTLV-I or an HTLV-I-related human retrovirus in the etiology of MS.     

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.