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The co-expression of 2B4 (CD244) and CD160 delineates a subpopulation of human CD8+ T cells with a potent CD160-mediated cytolytic effector function.

Within human CD8+ T lymphocytes, the CD27-CD45RAhigh or CD56+ phenotypes contribute to precisely define the cells with CTL effector function. Novel markers were demonstrated to correlate with CTL properties, such as the 2B4 (CD244) receptor, a member of the CD2 subset of the immunoglobulin superfamily or the glycosylphosphatidylinositol-anchored CD160 receptor. We performed a study of these markers to further define the population of effectors with CTL functions. Here we show that cytotoxic subpopulations defined by surface markers CD160, CD56 and CD57 are mostly contained in the 2B4+CD8+ T cell population. Expression of CD160 identifies two populations in the 2B4+ population. The 2B4+CD160+ subset expresses a bona fide CTL phenotype. The co-expression of 2B4 and CD160 defines T cells containing high amounts of perforin and granzyme B. During CTL ontogeny, an up-regulation of 2B4 and CD160 is observed from a naive to a terminally differentiated phenotype. Finally, we demonstrated that CD160 triggering failed to induce cytotoxicity per se, but costimulated CD3-redirected killing. We conclude that the co-expression of 2B4 and CD160 defines a CD8+ T lymphocyte subpopulation with high CTL activity.

Antigens, CD↗

Kinetics of CD8+ effector T cell responses and induced CD4+ regulatory T cell responses during Friend retrovirus infection.

Cytolytic CD8+ T cells are critical for the control of acute Friend virus (FV) infection yet they fail to completely eliminate the virus during chronic infection because they are functionally impaired by regulatory T cells (Treg). We performed a kinetic analysis of T cell responses during FV infection to determine when dysfunction of CD8+ T cells and suppressive activity of CD4+ regulatory T cells develops. At 1 week post infection, virus-specific CD8+ T cells with effector phenotype and cytolytic potential expanded. Peak expansion was found at 12 days post infection, correlating with peak viral loads. After 2 weeks when viral loads dropped, numbers of activated CD8+ T cells started to decline. However, a population of virus-specific CD8+ T cells with effector phenotype was still detectable subsequently, but these cells had lost their ability to produce granzymes and to degranulate cytotoxic molecules. Contemporaneous with the development of CD8+ T cell dysfunction, different CD4+ T cell populations expressing cell surface markers for Treg and the Treg-associated transcription factor Foxp3 expanded. Transfer as well as depletion experiments indicated that regulatory CD4+ cells developed during the second week of FV infection and subsequently suppressed CD8+ T cell functions, which was associated with impaired virus clearance.

Adoptive Transfer↗

Functional expression of chemokine receptor CCR6 on human effector memory CD8+ T cells.

Since CCR6 is a receptor for the chemokine CCL20, which is produced in tissues such as intestine and colon, it is thought that T cells expressing CCR6 are involved in mucosal immunity. The expression and function of CCR6 on human CD8+ T cells have not well been analyzed, although it is known that this receptor is expressed on a subset of human CD8+ T cells. We here characterize human CCR6+ CD8+ T cells. Multi-color flow cytometric analysis demonstrated that CCR6+ cells are predominantly found among CD8+ T cells having the memory phenotype. The expression of CCR6 is positively and negatively correlated with that of CCR5 and CCR7, respectively. CCR6+ CD8+ T cells express granzyme A and a low level of perforin but not granzyme B. In addition, a major population among these cells has the ability to produce IFN-gamma and TNF-alpha but not IL-2. These results indicate that CCR6+ CD8+ T cells have characteristics of early effector memory cells rather than effector or central memory cells. A chemotaxis assay revealed that CCR6+ CD8+ T cells have the ability to migrate in response to CCL20, suggesting that these T cells migrate to tissues such as colon and are involved in mucosal immunity.

Adult↗

Maintenance of CCL5 mRNA stores by post-effector and memory CD8 T cells is dependent on transcription and is coupled to increased mRNA stability.

Immunological memory is associated with the display of improved effector functions by cells of the adaptive immune system. The storage of untranslated mRNA coding for the CCL5 chemokine by CD8 memory cells is a new process supporting the immediate display of an effector function. Here, we show that, after induction during the primary response, high CCL5 mRNA levels are specifically preserved in CD8 T cells. We have investigated the mechanisms involved in the long-term maintenance of CCL5 mRNA levels by memory CD8 T cells. We demonstrate that the CCL5 mRNA half-life is increased in memory CD8 T cells and that these cells constitutively transcribe ccl5 gene. By inhibiting ccl5 transcription using IL-4, we demonstrate the essential role of transcription in the maintenance of CCL5 mRNA stores. Finally, we show that these stores are spontaneously reconstituted when the inhibitory signal is removed, indicating that the transcription of ccl5 is a default feature of memory CD8 T cells imprinted in their genetic program.

3T3 Cells↗

Dysregulation of phosphatidylinositol 3-kinase and downstream effectors in human breast cancer.

Phosphatidylinositol 3-kinase (PI3-K) is a growth factor-activated transforming lipid (and protein) kinase, involved in cell motility and invasion, that has multiple effectors. Relatively little is known about its expression and enzymatic activity in human breast cancer. Since growth factor receptors are amplified in breast cancer, and the tumor suppressor PTEN may be mutated in human breast cancer, it was hypothesized that PI3-K and its downstream effectors would be activated in this disease. In 11 resected tumors analyzed for expression of this kinase, a mean 3-fold increase in protein expression was observed over the corresponding adjacent control tissue. Using an in vitro lipid kinase assay of the immunoprecipitated PI3-K protein, a greater than 2-fold increase in activation was observed. These changes were observed in the absence of an activation of either protein kinase B (PKB, akt1) or p70 S6 kinase (p70 S6K). However, p21-activated kinase (Pak), p38 mitogen-activated protein kinase (p38 MAPK) and mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK 2) were all overexpressed and demonstrated increased enzyme activity. It may be concluded that aberrant mitogenic signaling in human breast cancer in vivo involves Pak, p38 MAPK and MAPKAPK2 downstream of PI3-K, but neither of PKB or p70 S6K. It is proposed that this pathway may serve as a useful targeting nexus for investigation of small molecule inhibitors in human breast cancer.

Breast Neoplasms↗

Membrane-associated antigen from the SV40-induced hamster fibrosarcoma, para-7. I. Role in immune complex formation and effector cell blockade.

PARA-7 membrane-associated antigen was prepared by treatment of the tumor cells with pH 9.4 glycine buffer, or by concentration of spent cell culture medium. When admixed with sensitized effector cells, both preparations could specifically block cellular cytotoxicity for PARA-7 target cells. Pretreatment of target cells with antigen did not result in blocking. Incubation of antigen extract with simian virus 40 (SV40) anti-serum caused neutralization of antibody-dependent cellular cytotoxicity (ADCC) with concomitant formation of a factor which blocked at the target cell level but not at the effector cell level. Serum from tumor-bearing hamsters exhibited blocking characteristics comparable to those of the antigen-SV40 teristics comparable to those of the antigen-SV40 antiserum preparation. Washing experiments indicated that PARA-7 antigen was more efficient than PARA-7 antigen-antibody complexes in blocking cell-mediated immunity in vitro. Material extracted from untransformed hamster embryo fibroblasts either by itself or when admixed with SV40 antiserum exhibited no significant blocking activity. These observations support the concept that loss of serum ADCC during progressive tumor growth is due to immune complex formation.

Animals↗

Immunologic responses to a murine mammary adenocarcinoma. II. Monocyte effector activation by humoral factors.

Sera from DBA/2 mice bearing a syngeneic T1699 mammary adenocarcinoma not only induced specific antibody-dependent cellular cytotoxicity (ADCC) mediated by monocytes (and/or macrophages) but also armed (activated) such effector cells into specific killer cells in vitro. Removal of specific antitumor antibody (IgG2a subclass) by absorption with T1699 cells abolished ADCC activity at the level of target cell coating but did not alter the level of effector-arming activity. Fractionation on Sephadex G-20 indicated more than one factor capable of inducing specific monocyte-mediated lysis of target cells in vitro, including immune complexes, free IgG2a 7S antibody (ADCC), and another factor(s) with a molecular weight very close to that of 7S immunoglobulins.

Adenocarcinoma↗

Heterogeneity among human lymphocyte effector cells mediating spontaneous lymphocyte-mediated cytotoxicity.

The spontaneous lymphocyte-mediated cytotoxicity (SLMC) of lymphocytes from normal individuals was studied using the lymphocyte titration assay. Various techniques were used to study selectively several lymphocyte subpopulations. The activity of these subpopulations was tested in both the antibody-dependent cellular cytotoxicity (ADCC) and lymphocyte titration assays. Surface marker studies were performed on each lymphocyte subpopulation. The results from these studies indicate that effector cells mediating SLMC are a heterogeneous lymphocyte population composed of Fc-receptor-positive lymphocytes both with and without C3 receptors that consistently segregate with cells that do not form rosettes with sheep red blood cells. Our data suggest that, in many respects, the effector cells in SLMC and ADCC are identical. However, the mechanism appears to be different.

Adult↗

Interferon production and activation of non-specific effector cells by stimulation with lymphoblastoid cell lines in vitro.

A population of non-specific effector lymphocytes is generated in response to stimulation with lymphoblastoid cell lines (LCL). These cytotoxic cells have an activity analogous to that exhibited by natural killer (NK) cells. When lymphoid cells lines established by transformation with Epstein-Barr virus (EBV) are used to stimulate autochthonous T-cell-enriched lymphocytes, the activity against the NK-sensitive target, K562, is increased up to 14-fold. The stimulated T lymphocytes produce interferon, and this factor augments the cytotoxic activity of unstimulated cells. The size distribution of cytotoxic lymphocytes after stimulation with the autochthonous EBV line is unlike that of either T-cells or interferon-augmented T-cells. Autochthonous stimulated lymphocytes which kill K562 are of several size classes, and are included in populations containing large blast cells. In contrast, the K562 activity of both unstimulated T-cells and interferon-augmented T-cells is contained in a more discrete population of cells, just slightly larger than the majority of lymphocytes. Thus, the generation of non-specific effector cells during stimulation with lymphoblastoid cell lines appears to involve the activation of a population of blast-size cells in addition to those initially responsive to interferon augmentation.

Antibody-Dependent Cell Cytotoxicity↗

Abrogation of tumor-inhibitory MRC-OX8+ (CD8+) effector T-cell generation in rats by selective depletion of neutrophils in vivo using a monoclonal antibody.

These studies were designed to examine the role of neutrophils in transplantation immunity to syngeneic rat tumors. We have earlier reported that specific transplantation immunity to syngeneic transplanted tumors is abrogated by selective depletion of peripheral-blood neutrophils by administration of a monoclonal antibody (MAb) (RP-3) at the time of immunization with X-irradiated tumor cells. In order to elucidate the mechanisms of this phenomenon, we have now examined whether induction of the sensitized spleen cells that inhibit the growth of tumor cells is inhibited by RP-3 treatment at the time of in vivo priming with tumor-associated antigen (TAA). Neutrophils were required to induce the sensitized T cells responsible for tumor inhibition with Winn's assay. In addition, CD8+ T cells are proved to be effector cells in an assay system of tumor-growth inhibition using a diffusion chamber. Our results indicate that neutrophils are required for priming rats with TAA to induce CD8+ effector T cells in tumor inhibition.

Animals↗

Expression of mRNAs for pore-forming protein and two serine esterases in murine primary and cloned effector lymphocytes.

The cDNAs encoding several proteins present in the granules of cytolytic effector lymphocytes have now been cloned. These include the cytolytic pore-forming protein (PFP) or perforin, and at least six serine esterases (SE), also called granzymes. The cDNA probes for PFP, SE-1, and SE-2 are used here to study the expression of these proteins in murine primary effector lymphocytes. Among the stimuli effective in inducing the expression of PFP, SE-1, and SE-2 were recombinant interleukin-2, the lectin concanavalin A in the presence of phorbol esters, and allogeneic cells in mixed lymphocyte cultures. Some correlation was seen between the levels of PFP and SE mRNAs and cytotoxicity measured in a standard 51Cr release assay. We also examined a panel of 13 cloned cytotoxic T lymphocyte (CTL) lines and found that mRNAs for PFP and SE-2 were expressed in all CTL lines, including some that were previously considered not to produce PFP. Twelve of the 13 CTL lines also proved to possess the mRNA for SE-1. One thymoma cell line, TIMI.4, did not express mRNA for PFP, although it expressed mRNA for SE-1 and SE-2.

Animals↗

What it takes to become an effector T cell: the process, the cells involved, and the mechanisms.

When activated, CD4(+) T cells differentiate into two major sub-populations differing in their profiles of secreted cytokines. Type One, or TH1, cells secrete IL-2, IFNgamma, and TNFbeta and mediate a cellular immune response. Type Two, or TH2, cells secrete IL-4, IL-5, IL-6, IL-10, and IL-13 and potentiate a humoral response. The nature of any specific immune response depends on the interaction of antigen-presenting cells and T cells. The role of antigen-presenting cells is to respond to the nature of the immune challenge and signal differentiation of CD4(+) T cells. A number of factors are involved in the effector phenotype of T cells-nature and affinity of antigen, co-receptors signals, and cytokine environment. T-cell differentiation is a complex process comprising four defined developmental stages: activation of particular cytokine genes, commitment of the cells, silencing of the opposing cytokine genes, and stabilization of the phenotype. In each of these stages, the cells respond to the products of many signaling cascades from many membrane-bound receptors. The stages in development are mediated by different molecular mechanisms, involving control of gene expression and chromatin remodeling. This review centers on the factors, cellular interactions, and molecular mechanisms involved in the maturation of naïve CD4(+) T lymphocytes into fully effector cells.

Animals↗

Tumoricidal effector mechanisms of murine BCG-activated macrophages: role of TNF in conjugation-dependent and conjugation-independent pathways.

The roles of secreted and membrane-associated TNFs were investigated in activated macrophage cytolysis of L929, EMT-6, and P815 targets. While all three targets were susceptible to cytolysis in coculture, an anti-TNF antiserum blocked lysis of L929 and EMT-6 but not of the P815 targets. Of the three targets, recombinant human or mouse TNF could only lyse the L929 target; despite the fact that a role for TNF was invoked in lysis of EMT-6 targets in coculture, the latter was strongly resistant to soluble rTNF, even at concentrations 30-40-fold higher than the Ka for its TNF-receptor. Cytolysis of the L929 target occurred when it was cocultured with BCG-activated macrophages even when these effector cells did not secrete TNF, either due to prior chemical crosslinking or to lack of exposure to a triggering level of lipopolysaccharide. Furthermore, by introduction of the anti-TNF antiserum over a dose-range, it was shown that macrophage cytolysis both of L929 and EMT-6 targets occurred in the absence of bioavailable, fluid-phase TNF. Thus, even for targets susceptible to fluid-phase TNF, TNF-dependent, direct macrophage-mediated cytolysis appears to be a function independent of secreted TNF and one that utilizes effector-target contact to express the action of a membrane form of the molecule.

Animals↗

Proteases and protease receptors in modulation of leukocyte effector functions.

Cellular immune responses depend on regulated pathways of intracellular signal transduction and leukocyte activation. Although these mechanisms are coordinated by a variety of leukocyte-restricted effector molecules, recent observations have uncovered a novel role of proteases in transducing outside-in signals of leukocyte activation. Through regulated, receptor-mediated recognitions, coagulation and fibrinolytic enzymes or effector cell granular proteases influence monocyte motility and chemotaxis, modulate pleiotropic cytokine responses, contribute to mononuclear cell proliferation, or induce target cell apoptosis. Overall, these mechanisms define a novel interface between general inflammatory reactions, invariably characterized by activation of blood protease cascades, and specialized aspects of cellular immune functions.

Animals↗

Effector molecules in expression of the antimicrobial activity of macrophages against Mycobacterium avium complex: roles of reactive nitrogen intermediates, reactive oxygen intermediates, and free fatty acids.

We studied microbicidal activities of reactive nitrogen intermediates (RNI), free fatty acids (FFA), and reactive oxygen intermediates (ROI) against Mycobacterium avium complex (MAC) and the mode of macrophage (mphi) production of these effectors. (1) Intracellular growth of MAC in murine peritoneal mphis was accelerated by scavengers for ROI or RNI and inhibitors of nitric oxide synthase or phospholipase A2, indicating roles of ROI, RNI, and FFA in mphi anti-MAC functions. (2) Acidified NaNO2-derived RNI, FFA (linolenic and arachidonic acids), and the H2O2-mediated halogenation system exhibited a significant anti-MAC bactericidal activity. The combination of RNI with FFA showed a synergistic effect. However, the H2O2-halogenation system in combination with either RNI or FFA showed an antagonism. When Listeria monocytogenes (Lm) was used as a target organism, the combinations of RNI + FFA and RNI + H2O2-halogenation gave a synergistic effect, whereas FFA + H2O2-halogenation showed an antagonism in exerting bactericidal activity. In addition, when ROI generated by the xanthine oxidase-acetaldehyde system was combined with RNI, anti-Lm but not anti-MAC activity was potentiated. (3) ROI production by murine peritoneal mphis was observed immediately after contact with MAC organisms (MAC stimulation) and ceased within 2 h. FFA release was seen 1-24 h after MAC stimulation. RNI production was initiated from 3 h and increased during the first 36 h and continued at least for 4 days. These findings suggest that RNI and FFA rather than ROI are important effectors of anti-MAC functions of mphis, and the collaborating action of RNI with FFA temporarily participates in mphi-mediated killing of MAC in the relatively early phase after MAC stimulation.

Animals↗

The role of CD40/CD154 interactions in the priming, differentiation, and effector function of helper and cytotoxic T cells.

This review focuses on the emerging body of literature suggesting a critical role for CD40/CD154 interactions in antigen-presenting cell (APC) activation, CD4+ and CD8+ T cell priming, and effector T cell maturation. In this context effective antigen presentation involves not only T cell expansion and long-term survival but also the ability of the APC to guide the T cell response toward the Th1 (interferon-gamma producing) or the Th2 (interleukin-4 producing) phenotype. We suggest a model to explain why CD40/CD154 interactions are critical for some helper and cytotoxic T cell responses, whereas others occur independently of this receptor/ligand pair. In addition, we will discuss the potential role for CD40/CD154 interactions in effector T cell maturation and cytokine production.

Animals↗

Chemokines: signal lamps for trafficking of T and B cells for development and effector function.

Chemokines can act as signal lamps for trafficking of lymphocytes at the important crossing points of lymphoid tissues. Lymphoid progenitors at different differentiation stages are differentially localized in primary lymphoid tissues and have differential responsiveness to thymic or bone marrow chemokines: SDF-1, CKbeta-11/MIP-3beta/ELC, SLC/6Ckine/Exodus2, MIP-1beta, and TECK. Naive T cells and B cells circulate to secondary lymphoid tissues for possible activation. Chemokines, SDF-1, SLC/6Ckine/Exodus2, CKbeta-11/MIP-3beta/ELC, BLC/BCA-1, and DC-CK1/PARC, are expressed in the specialized microenvironments of secondary lymphoid tissues and regulate the migration of naive lymphocytes. Effector lymphocytes express a different set of chemokine receptors from naive lymphocytes. T helper (Th) 0 and 1 cells predominantly express CXCR3 and CCR5, whereas Th2 cells express CCR3, CCR4, and CCR8, which, with other factors such as expression patterns of adhesion molecules, likely determine the tissue-specific infiltration of effector lymphocytes.

Animals↗

Presynaptic effectors contributing to cAMP-induced synaptic potentiation in Drosophila.

cAMP analogs and activation of adenylyl cyclase by forskolin strongly potentiate synaptic transmission at the Drosophila neuromuscular junction. These effects are generally attributed to activation of cAMP-dependent protein kinase. Recent reports on crustacean and mammalian synapses have implicated other cAMP-dependent effectors in synaptic potentiation. Drosophila neuromuscular junctions were tested for effects of two known cAMP-dependent effectors: hyperpolarization-activated, cyclic nucleotide-regulated channels (HCNCs) and guanine nucleotide exchange protein activated by cAMP (Epac). Forskolin-induced enhancement of synaptic transmission was drastically reduced by a blocker of HCNCs, but not completely eliminated. A specific agonist for Epac modestly enhanced synaptic potentials. This agonist also stabilized their amplitudes in the presence of a blocker of HCNCs. The observations implicate HCNCs and Epac in cAMP-dependent potentiation that does not require cAMP-dependent protein kinase, indicating that additional previously unexplored factors contribute to synaptic plasticity in Drosophila. Genetic and molecular techniques available for Drosophila can be used to define the underlying molecular basis for cAMP-dependent synaptic potentiation.

Animals↗