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PubMed1991

[Vasoactive intestinal peptide inhibits vagal neuro-effector transmission in feline airway smooth muscle but not in humans].

It has been reported that low concentrations of vasoactive intestinal peptide (VIP) suppress the release of acetylcholine (ACh) from vagal nerve terminals. However, there is little documentation of the neuro-effector transmission of VIP in human airways. In the present study, the effects of VIP on excitatory neuro-effector transmission in the human bronchus were investigated, in comparison with feline trachea. In feline trachea, VIP (10(-10) to 10(-7) M) reduced the amplitude of contractions evoked by field stimulation in the presence of indomethacin (10(-6) M). By contrast, VIP (10(-10) to 10(-9) M) had no effect on ACh-sensitivity of smooth muscle cells. These results indicate that VIP in low concentrations has a pre-junctional action inhibiting excitatory neuro-effector transmission, in addition to a post-synaptic action, presumably suppressing transmitter release from the vagal nerve terminals. On the other hand, in human bronchi, VIP (10(-9) to 10(-7) M) did not affect either contractions evoked by field stimulation or those evoked by ACh. These results indicate that VIP does not exhibit any pre-junctional effects in the human bronchus at these concentrations.

Animals↗
PubMed1990

Cognate interactions between helper T cells and B cells. IV. Requirements for the expression of effector phase activity by helper T cells.

After activation with anti-CD3, activated Th (THCD3), but not resting Th, fixed with paraformaldehyde induce B cell RNA synthesis when co-cultured with resting B cells. This activity is expressed by Th of both Th1 and Th2 subtypes, as well as a third Th clone that is not classified into either subtype. It is proposed that anti-CD3 activation of Th results in the expression of Th membrane proteins that trigger B cell cycle entry. Kinetic studies reveal that 4 to 8 h of activation with anti-CD3 is sufficient for ThCD3 to express B cell-activating function. However, activation of Th with anti-CD3 for extended periods of time results in reduced Th effector activity. Inhibition of Th RNA synthesis during the anti-CD3 activation period ablates the ability of ThCD3 to induce B cell cycle entry. This indicates that de novo synthesis of proteins is required for ThCD3 to express effector function. The ability of fixed ThCD3 to induce entry of B cell into cycle is not due to an increase in expression of CD3, CD4, LFA-1, ICAM-1, class I MHC or Thy-1. Other forms of Th activation (PMA and A23187, Con A) also induced Th effector function. Furthermore, purified plasma membranes from anti-CD3 activated, but not resting Th, induced resting B cells to enter cycle. The addition of IL-4, but not IL-2, IL-5, or IFN-gamma amplified the DNA synthetic response of B cells stimulated with PM from activated Th. Taken together these data indicate that de novo expression of Th surface proteins on activated Th is required for Th to induce B cell cycle entry into G1 and the addition of IL-4 is required for the heightened progression into S phase.

Animals↗
PubMed1991

Effector and regulatory cells in autoimmune oophoritis elicited by neonatal thymectomy.

(C57BL/6 x A/J)F1 (B6AF1) mice thymectomized between days 1 and 4 of age develop autoimmune oophoritis (D3TX oophoritis) 4 to 6 wk later. Oophoritis can be adoptively transferred to young recipients, and the disease in D3TX mice is prevented by reconstitution with normal adult spleen cells. The present study was further defined the nature of the effector and suppressor cells. Contrary to an earlier report, oophoritis is transferred to syngeneic and not allogeneic recipients. The spleen cells from D3TX mice when stimulated in vitro with Con A, also transfer oophoritis to adult recipients. The effector cells are CD4+: oophoritis transfer is abrogated by CD4 antibody and not by CD8 antibody and C. Spleen cells from D3TX male mice transfer disease less efficiently than female cells, thus endogenous ovarian Ag may be required for activation of effector T cells. T cells from normal adult spleen that suppress D3TX oophoritis also appear to be of CD4+ phenotype. These cells are likely to be derived from adult thymus because adult thymocytes also suppress D3TX disease. We were unable to substantiate the earlier claim that suppressor cells in normal mice are ovarian Ag specific. Thus male and female spleen cells suppress disease with comparable efficiency, and deprivation of endogenous ovarian Ag by neonatal ovariectomy of cell donors had no observable effect on disease suppression.

Animals↗
PubMed1991

Characterization of target injury of murine acute graft-versus-host disease directed to multiple minor histocompatibility antigens elicited by either CD4+ or CD8+ effector cells.

The precise identity of effector mononuclear cells capable of eliciting acute graft-versus-host disease (AGVHD) is controversial. In this study, highly purified subsets of donor T cells were used to produce AGVHD to multiple minor histocompatibility (H) antigens in two strain combinations of mice matched for the major histocompatibility complex (MHC). In the C3H.SW- greater than B6 strain combination, only CD8+ effector cells produced histologic evidence of AGVHD in skin and liver, which peaked 3 weeks after transplant. In the B10.D2- greater than DBA/2 strain combination, CD4+ effector cells, and to a lesser extent, CD8+ cells, mediated disease in skin, liver, and intestine, which peaked during the fourth week after transplant. Analysis of skin and liver from both combinations showed target cell injury that was phenotypically similar and resembled that previously described in human disease in other studies. In addition, prominent epithelial injury also was detected in oropharyngeal mucosa, esophagus, hepatobiliary ducts, and seminal vesicle in both transplant settings. These findings indicate that functionally different subsets of donor T cells may be capable of initiating common pathways of cellular injury in selected target sites in AGVHD, and have potential implications for strategies that seek to ablate disease development by manipulation of donor marrow before transplantation.

Acute Disease↗
PubMed1991

In vitro characterization of a murine recipient anti-donor effector cell responsible for the development of chronic graft-versus-host disease.

Injection of parental spleen cells into unirradiated F1 hybrid recipients is frequently used for the induction of murine graft-vs-host disease (GVHD). Injection of C57BL/6 spleen cells into B6D2F1 recipients results in the acute form of GVHD whereas injection of DBA/2J cells into the same recipients results in a chronic form of GVHD. In vivo studies have suggested that injection of DBA/2J cells into B6D2F1 recipients results in the chronic form of GVHD instead of acute GVHD because of recipient B6D2F1 effector cells that inhibit DBA/2J CD8+ T cell function. The present report describes the development of an in vitro assay that measures the activity of these B6D2F1 effector cells. The results obtained using this assay indicate that these B6D2F1 effector cells are found primarily in the LN, need to be activated in order to function, are inhibited by anti-CD8 antibodies, and mediate their function as a result of DBA/2J cells binding to Ag expressed on their cell surface.

Animals↗
PubMed1990

The role and specificity of guanine nucleotide binding proteins in receptor-effector coupling.

Nine distinct alpha subunits of guanine nucleotide binding proteins (G-proteins) have now been identified by cDNA cloning. Each of these functions to allow transduction of information between hormone-activated receptors in the plasma membrane and effector systems which are either ion channels or enzymes which regulate the intracellular concentration of second messengers. As the individual G-proteins are highly similar in primary sequence, it is pertinent to ask what degree of specificity of interaction each of these display with the various receptors and effector systems. Specificity of tissue location defines that the rod and cone transducins (TD1 and TD2, respectively) act as the coupling proteins between rhodopsin and cone opsins and their cyclic nucleotide phosphodiesterase effectors and that G(olf) is the G-protein which tranduces signals from odorant receptors to adenylate cyclase in olfactory sensory neurones. However, many of the other identified G-proteins are co-expressed in a single tissue or cell. Whilst sensitivity to ADP-ribosylation catalysed by bacterial toxins from Bordetella pertussis and Vibrio cholerae has allowed a further subdivision of the G-protein family, this approach is limited as these toxins have multiple G-protein substrates. As the extreme C-terminus of the alpha subunit of each G-protein appears to be a key domain for the interactions of receptors and G-proteins we have generated a series of G-protein-selective antipeptide antisera against this region and then have used these antisera to attempt to interfere with receptor-G-protein coupling. With this approach we have been able to demonstrate that a delta opioid receptor-mediated inhibition of adenylate cyclase in neuroblastoma x glioma, NG108-15, cell membranes is transduced specifically by Gi2 and in the same cell that alpha 2 adrenergic inhibition of Ca2+ currents is transduced by Go. Similar strategies are likely to be of universal significance, for example in the identification of the G-protein (Gp) which regulates the receptor-mediated activation of phosphoinositidase C. Methods to allow pharmacological manipulation of the levels of expression of various G-proteins in the membranes of cells are also discussed. Such approaches are also likely to assist in the identification of G-proteins of defined functions.

Adenylyl Cyclases↗
PubMed1990

Growth factors, lipid mediators and effector cells.

Hematopoetic growth factors stimulate the proliferation of leukocyte precursors in bone marrow cultures, but some also augment the responsiveness of mature effector cells. For example, granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances superoxide production and cytotoxicity of neutrophils stimulated by diverse agonists. We show that preexposure of neutrophils to GM-CSF is absolutely required for the induction of leukotriene B4 and platelet-activating factor (PAF) synthesis by a soluble agonist, C5a or N-formyl-methionyl-leucine-phenylalanine (FMLP). Lipid mediator synthesis occurs very rapidly after triggering with the second signal, and under identical conditions superoxide release is enhanced. Interleukin-3 (IL-3), another hematopoetic growth factor, enhances granule release and more profoundly leukotriene C4 (LTC4) synthesis in basophils stimulated by immunoglobulin-E (IgE)-dependent or -independent agonists. Sequential stimulation with IL-3 and C5a results in the production of large quantities of LTC4, while neither factor alone induces the release of lipid mediators. We conclude that a major function of these cytokines is to allow lipid mediator synthesis in effector cells after triggering with agonists which by themselves do not induce the production of bioactive lipids. We also propose that lipoxygenase metabolites and PAF represent an autocrine response amplification pathway in effector cells which might explain the enhanced responsiveness of cells primed by these cytokines.

Animals↗
PubMed1990

Characterization and genetic restriction of suppressor-effector cells induced by staphylococcal enterotoxin B.

The capacity of the staphylococcal enterotoxins to stimulate all T cells bearing certain (but not all) TCR has generated a great deal of interest. This stimulation appears to involve specific binding of the toxin to class II Ags and subsequent stimulation via the TCR. Previous studies from this laboratory have demonstrated that staphylococcal enterotoxin B (SEB) induces multiple T suppressor cell populations that inhibit both primary and secondary plaque-forming cell responses. Efforts to characterize these suppressor cell populations have demonstrated that the suppressor population active early in the antibody response expresses the Lyt-1-2+ cell surface phenotype, whereas depletion analysis suggests that the population active late in an ongoing response bears the Lyt-1+2+ cell-surface markers. In the present study, enrichment for this late acting effector population with the use of sequential panning with anti-Lyt mAb reveals significant suppressive activity at both the initiation and effector phases of a 5-day Mishell-Dutton coculture. Additional experiments using I-J disparate strains of mice have demonstrated a genetic restriction at the "I-J" gene locus between the cells mediating SEB-induced suppression and their target. Depletion of SEB-primed splenocytes with anti-I-J mAb suggests that both the early and late effector cells bear I-J molecules on their surface. Taken together, these results show that SEB induces suppressor cell populations with properties similar to those exhibited by Ag-specific cell circuits.

Animals↗
PubMed1990

Alloantigen-specific T suppressor-inducer and T suppressor-effector cells can be activated despite blocking the IL-2 receptor.

To determine IL-2 requirement for activation of suppressor cells, PBMC were primed in one-way MLR in the presence of 10 micrograms/ml anti-IL-2R beta-chain antibody 2A3 (CD25) or control antibody, then irradiated and added as regulators in a fresh MLR. Cells primed in the presence of antibody 2A3 suppressed the proliferative response to fresh autologous lymphocytes to specific alloantigen but had no effect on the response to cells from third party donors. Priming in the presence of an antibody of irrelevant specificity induced only limited suppressor activity. Activated suppressor cells did not show cytolytic activity specific for the stimulators when tested at the time of the suppressor cell assay. To identify the subset(s) responsible for suppression, cells primed in the presence of antibody 2A3 were separated into CD4+/CD45RA+, CD4+/CD45RA-, and CD8+ subsets, which were irradiated and then tested. The suppressive activity was found predominantly in the CD4+/CD45RA+ subset, whereas CD8+ cells had some activity and CD4+/CD45RA- cells had none. No subset suppressed the response of autologous cells to third-party cells. When primed CD4+/CD45RA+ cells were cocultured with fresh autologous lymphocytes depleted of CD8+ cells, no suppression was observed, indicating that, although the CD4+/CD45RA+ cells can function as inducers of suppressors, they cannot function as suppressor-effectors. Conversely, CD8+ cells activated in MLR in the presence of 2A3 caused suppression, regardless of whether the fresh autologous responder population contained CD8+ cells. CD4+/CD45RA+ and CD8+ subsets isolated after priming in the presence of 2A3 also demonstrated Ag-specific suppression in the generation of cytotoxic T lymphocytes whereas CD4+/CD45RA- cells had no activity. Our data are consistent with the model that suppression of alloreactivity requires the cooperation of two types of cells, a CD4+/CD45RA+ suppressor-inducer and a CD8+ suppressor-effector population. Activated Tsi and fresh Tse or activated Tse alone can suppress lymphocyte proliferation and generation of CTL in response to specific Ag. Activation of Ag-specific T suppressor-inducer and T suppressor-effector cells appears to be relatively IL-2 independent and presumably require one or more other growth factors.

Antibodies, Monoclonal↗
PubMed1990

Cytokines alter target cell susceptibility to lysis: I. Evaluation of non-major histocompatibility complex-restricted effectors reveals differential effects on natural and lymphokine-activated killing.

Interferon-gamma (IFN) and tumor necrosis factor-alpha (TNF) were examined for their ability to enhance major histocompatibility complex (MHC) expression on a variety of human tumor and normal tissue targets. Enhanced expression of MHC correlated with decreased target susceptibility to lysis by fresh peripheral blood mononuclear cells (PBMCs) and IL-2-augmented PBMCs (aPBL) but not as clearly with cells with lymphokine-activated killer (LAK) activity. These studies revealed maximal MHC enhancement after 48-72 h of incubation in IFN. Resistance to lysis by natural killer (NK) cells was best demonstrated after 72 h. Further, IFN and TNF were synergistic in their effects on MHC expression and induction of resistance of the cultured leukemias K562 and Molt-4 to aPBL effectors. Conversely, LAK susceptibility was usually unaltered after target IFN and TNF treatment. Incubation of fibroblasts and vascular endothelial cells with IFN also consistently resulted in MHC class I enhancement and resistance to NK lysis, whereas LAK susceptibility was variably affected. The brief incubation of fresh PBL in IL-2 (4-6 h) resulted in effectors highly lytic toward cultured cells, but with no activity against fresh tumor. Cultured cell lines treated with IFN and TNF were rendered relatively resistant to lysis by these activated cells. Fresh tumor MHC expression and LAK susceptibility was unchanged after IFN incubation. Additionally, there was no correlation between the level of MHC class I or class II expression and LAK susceptibility to any fresh, uncultured melanoma studied. These data suggest that LAK effectors possess different mechanisms of tumor recognition or lysis than cells with NK activity or cells briefly incubated (4-6 h) in IL-2. The ability of tumor-infiltrating lymphocytes to lyse the cultured autologous tumor target was markedly increased by preincubation of the targets with IFN and TNF. Finally, it appears that IL-2 treatment and the resultant endogenous production of IFN by T-lymphocytes should not adversely affect tumor susceptibility to current immunotherapy using IL-2.

Cytotoxicity, Immunologic↗
PubMed1990

Colony-stimulating factors: enhancement of effector cell function.

Haemopoietic growth factors both stimulate the growth and maturation of marrow precursor cells and enhance the functional activities of mature myelomonocytic effector cells. These biological effects are consistent with the expression of functional colony stimulating factor receptors on granulocytes and monocytes as well as on those immature marrow cells with proliferative potential. As the number of defined haemopoietic growth factors increases, both general perspectives of effector cell function and pathways of growth factor-induced signal transduction become more complex and less clearly understood. Although most known haemopoietic growth factors are briefly discussed, an effort has been made to focus on granulocyte-macrophage colony stimulating factor as a prototypic inducer of enhanced neutrophil, eosinophil and macrophage effector cell function.

Animals↗
PubMed1990

Graft rejection by cytolytic T cells. Specificity of the effector mechanism in the rejection of allogeneic marrow.

Cellular effector mechanisms of allograft rejection remain incompletely described. Characterizing the rejection of foreign-marrow allografts rather than solid-organ grafts has the advantage that the cellular composition of the marrow graft, as a single cell suspension, can be altered to include cellular components with differing antigen expression. Rejection of marrow grafts is sensitive to lethal doses of radiation in the mouse but resistant to sublethal levels of radiation. In an effort to identify cells mediating host resistance, lymphocytes were isolated and cloned from spleens of mice 7 days after sublethal TBI (650 cGy) and inoculation with allogeneic marrow. All clones isolated were cytolytic with specificity for MHC encoded gene products of the allogeneic marrow donor. When cloned cells were transferred in vivo into lethally irradiated (1025 cGy) recipients unable to reject allogeneic marrow, results utilizing splenic 125IUdR uptake indicated that these MHC-specific cytotoxic clones could suppress marrow proliferation. In order to characterize the effector mechanism and the ability of the clones to affect final engraftment, double donor chimeras were constructed so that 2 target cell populations differing at the MHC from each other and from the host were present in the same marrow allograft. Results directly demonstrated an ability of CTL of host MHC type to mediate graft rejection and characterized the effector mechanism as one with specificity for MHC gene products.

Animals↗
PubMed1986

Enhanced lysis of herpes simplex virus type 1-infected mouse cell lines by NC and NK effectors.

Spontaneously cytotoxic murine lymphocytes lysed certain cell types infected by herpes simplex virus type 1 (HSV-1) better than uninfected cells. The levels of virus-directed lysis varied widely from target to target, and we found that differences in virus-directed lytic efficiency could be attributed both to the characteristics of HSV-1 replication in the different targets and to the subgroup of natural effector cells which mediated lysis. Although HSV-1 adsorbed to the surface of all the target cells, those in which the virus replicated more efficiently were lysed to a greater extent. As targets, we used cell lines that, when uninfected, were spontaneously lysed by NK cells (YAC-1) or by NC cells (WEHI-164). We also used a fibroblastoid cell line (M50) and a monocytic tumor line (PU51R), which were not spontaneously killed. Using complement-mediated elimination of Qa-5-positive or asialo-GM1-positive NK cells to distinguish NK from NC activity, we found that NK cells lysed HSV-1-infected YAC cells better than uninfected cells, and an NC-like activity selectively lysed HSV-1-infected WEHI cells. In addition, we showed that both NK and NC cytotoxicities contributed to the lysis against the HSV-1-infected fibroblastoid line, M50, but the infected PU51R cells were killed by only NK effectors. These findings were consistent with the results of experiments performed to define the role of interferon in induction of virus-augmented cytolysis. Increased lysis of YAC-HSV and PU51R-HSV was entirely due to interferon activation and was completely abolished by performing the 51Cr-release assay in the presence of anti-interferon serum. Because NC activity was not augmented by interferon, virus-enhanced NC lysis of M50-HSV and WEHI-HSV was not due to this nonspecific mechanism. Together, our data show that HSV-1 infection of NK/NC targets induces increased cytotoxicity, but the effector cell responsible for lysis is determined by the uninfected target, or by an interaction between the virus and target cell, rather than by a viral determinant alone.

Animals↗
PubMed1987

Induction by killed Listeria monocytogenes of effector T cells mediating delayed-type hypersensitivity but not protection in mice.

Using a local passive transfer system, we found that effector T cells mediating delayed-type hypersensitivity (DTH) but not acquired cellular resistance (ACR) to Listeria monocytogenes (strain EGD) were generated in mice immunized with killed Listeria, although immunized mice did not express DTH or ACR. When non-adherent cells of peritoneal, lymph node, or spleen cells from mice immunized with killed Listeria were transferred into the footpad of naive recipient mice along with eliciting antigen, positive delayed footpad reaction (DFR) was elicited. However, there was no evident protection against challenge at the site of the local transfer. Cells from mice immunized with viable Listeria conferred significant degrees of DFR and ACR on the recipients. DFR transferred by cells immunized with killed Listeria was mediated by L3T4+ T cells in an antigen-specific manner. The antigen-specific proliferative response of T cells from mice immunized with killed Listeria was much lower than that of T cells from mice immunized with viable Listeria. The production of macrophage chemotactic factor (MCF) by cells from killed Listeria-immune mice was much the same as that by cells from viable Listeria-immune mice. In contrast, the production of interleukin-2 (IL-2) and macrophage activating factor (MAF) was much lower in cells from killed Listeria-immune mice. The elimination of L. monocytogenes (strain L461), a strain of low virulence, was enhanced at the site of DFR transferred with cells from killed Listeria-immune mice. These results suggest that stimulation with killed bacteria is effective for the generation of DTH-mediating effector T cells, and that different effector T cells mediating DTH or ACR are involved in cell-mediated immunity to L. monocytogenes.

Animals↗
PubMed1988

Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells.

Previous studies show that cytotoxic activated macrophages cause a reproducible pattern of metabolic inhibition in viable tumor target cells. This includes inhibition of DNA synthesis, two oxidoreductases of the mitochondrial electron transport chain (NADH: ubiquinone oxidoreductase and succinate: ubiquinone oxidoreductase), and the citric acid cycle enzyme aconitase. This pattern of metabolic inhibition is induced by a cytotoxic activated macrophage associated biochemical pathway with L-arginine deimination activity that synthesizes L-citrulline from L-arginine and oxygenated nitrogen derivatives from the imino nitrogen removed from the guanido group of L-arginine. Here we report that macrophages activated in vivo by infection with bacillus Calmette-Guérin or in vitro by murine rIFN-gamma or murine IFN-alpha/beta (in the presence of the second signal LPS in all cases) develop inhibition of aconitase and the same two oxidoreductases of the mitochondrial electron transport chain as was documented earlier in target cells of cytotoxic activated macrophages. In addition, this pattern of metabolic inhibition which develops in cytotoxic activated macrophages is caused by the L-arginine-dependent effector mechanism. Inhibition of mitochondrial respiration by effectors of the L-arginine-dependent cytotoxicity system results in a compensatory increase in activity of the glycolytic pathway. We speculate that the pattern of metabolic inhibition induced in cytotoxic activated macrophages by the L-arginine-dependent effector system causes changes in the macrophage intracellular environment that increases resistance to certain facultative and obligate intracellular pathogens.

Aconitate Hydratase↗
PubMed1989

The molecular basis of granuloma formation in schistosomiasis. II. Analogies of a T cell-derived suppressor effector factor to the T cell receptor.

During Schistosoma mansoni infection, Ts cells regulate granulomatous modulation via antigenically and genetically restricted suppressor inducer and suppressor effector factors. The T suppressor effector factor (TseF) directly suppresses granuloma formation both in vitro and in vivo. In this study, we probe the molecular basis of these TseF properties. Using techniques of heterodimeric chain reduction with DTT and in vitro functional complementation, chimeric molecules were constructed. By analyzing genetic restrictions, antigenic specificities, and phenotypic markers, the contributions of the component chains to 72 kDa TseF reactivity were determined. One chain bore an Ag receptor and imparted antigenic specificity. The other chain bore an IJ determinant, a TCR beta-chain allotypic determinant, a suppressor effector phenotypic determinant, and imparted functional genetic restriction. Functional activity required covalent, probably sulfhydryl mediated, linkage as succinylation prevented the separated component chains from reconstituting functional activity. Additional studies demonstrated that anti-serum directed against either the T cell receptor or the T3 epsilon-chain could abrogate functional activity. However, TseF bore no T3 epsilon-chain phenotypic marker per se suggesting that TseF effects T lymphocytes via transmembrane signal transduction. These studies suggest that a regulatory network is operative in granuloma modulation. This regulatory network is mediated by a soluble TseF that bears significant structural homologies to the classic TCR.

Animals↗
PubMed1989

Lymphocytes infiltrating human ovarian tumors: synergy between tumor necrosis factor alpha and interleukin 2 in the generation of CD8+ effectors from tumor-infiltrating lymphocytes.

Tumor-infiltrating lymphocytes (TIL) were isolated by enzymatic digestion and gradient centrifugation from 18 human ovarian carcinomas. These cells were cultured in a complete medium supplemented with recombinant interleukin 2 (IL2) alone or recombinant IL2 plus recombinant tumor necrosis factor alpha (TNF-alpha), and their growth and antitumor cytotoxicity were determined. TIL cultured in the presence of IL2 plus TNF-alpha (1000 units/ml each) for 6 days showed significantly higher cytotoxicity against fresh autologous tumor targets than did TIL cultured with IL2 alone (e.g., mean lytic units/10(7) cells for 8 TIL preparations were 290 versus 74; P less than 0.05). No differences in [3H]thymidine uptake or natural killer cell activity were observed among these TIL cultures. In titration experiments, optimal synergistic concentrations of IL2 and TNF-alpha were determined as 10(2) and 10(3) units/ml, respectively. Using these concentrations for culturing the TIL, effector cells developed which preferentially lysed autologous tumor and displayed a CD8+ phenotype (up to 75% positive). However, the autologous tumor cytotoxicity mediated by these cultured TIL on day 6 was short lived. By day 12, it was replaced by non-major histocompatibility complex-restricted, lymphokine-activated killer cell-like activity mediated by CD3-CD56+ effector cells. Simultaneously, the production of gamma-interferon and interleukin 1 decreased in these cultures. In contrast to TNF-alpha, anti-CD3 antibody synergized with IL2 to increase 2-3-fold TIL proliferation but not their cytotoxic activity against autologous tumor cell targets. These data suggest that TNF-alpha and IL2 synergize early in culture to induce tumor-reactive CD8+ effectors, some of which may be specific for autologous ovarian tumor cells. However, the conditions needed to sustain the specific autologous tumor responses in long-term cultures of human TIL remain to be determined.

Antigens, Differentiation, T-Lymphocyte↗
PubMedDate not supplied

Autocytotoxic activity of lymphokine-activated killer cells: characterization of effector cells and susceptible targets.

The specificities and surface markers of murine autocytotoxic cells induced by in vitro culture with interleukin 2 (IL2) were studied. Culturing murine spleen cells with recombinant human IL2 resulted in the generation of cytotoxic cells which killed syngeneic lymphoblasts and syngeneic activated macrophages (M phi). Both lectins and protein antigens were capable of inducing lymphoblasts recognized by lymphokine-activated killer (LAK) cells. B-lymphoblasts as well as T-lymphoblasts were sensitive to lysis by these effector cells. In addition, peritoneal M phi activated in vivo with Bacille Calmette-Guérin (BCB), Corynebacterium parvum (C. parvum), thioglycollate (TG) or lipopolysaccharide (LPS) were shown to be susceptible to lysis by LAK cells. In contrast, neither unstimulated T cells nor resident peritoneal M phi were sensitive to lysis by LAK cells, suggesting that normal cells have to be activated in order to be sensitive to lysis by these effector cells. Surface marker analysis indicated that majority of effector cells which killed syngeneic lymphoblasts and activated M phi were Thy1+, asialo GM1+, L3T4-, Ly2-.

Animals↗