Enhanced Cytokine Production Research Articles

Accelerated Ca2+ entry by membrane hyperpolarization due to Ca2+-activated K+ channel activation in response to histamine in chondrocytes

In articular cartilage inflammation, histamine release from mast cells is a key event. It can enhance cytokine production and matrix synthesis and also promote cell proliferation by stimulating chondrocytes. In this study, the functional impact of Ca(2+)-activated K(+) (K(Ca)) channels in the regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in chondrocytes in response to histamine was examined using OUMS-27 cells, as a model of chondrocytes derived from human chondrosarcoma. Application of histamine induced a significant [Ca(2+)](i) rise and also membrane hyperpolarization, and both effects were mediated by the stimulation of H(1) receptors. The histamine-induced membrane hyperpolarization was attenuated to approximately 50% by large-conductance K(Ca) (BK) channel blockers, and further reduced by intermediate (IK) and small conductance K(Ca) (SK) channel blockers. The tonic component of histamine-induced [Ca(2+)](i) rise strongly depended on the presence of extracellular Ca(2+) ([Ca(2+)](o)) and was markedly reduced by La(3+) or Gd(3+) but not by nifedipine. It was significantly attenuated by BK channel blockers, and further blocked by the cocktail of BK, IK, and SK channel blockers. The K(Ca) blocker cocktail also significantly reduced the store-operated Ca(2+) entry (SOCE), which was induced by Ca(2+) addition after store-depletion by thapsigargin in [Ca(2+)](o) free solution. Our results demonstrate that the histamine-induced membrane hyperpolarization in chondrocytes due to K(Ca) channel activation contributes to sustained Ca(2+) entry mainly through SOCE channels in OUMS-27 cells. Thus, K(Ca) channels appear to play an important role in the positive feedback mechanism of [Ca(2+)](i) regulation in chondrocytes in the presence of articular cartilage inflammation.

A Minor Catalytic Activity of Src Family Kinases Is Sufficient for Maximal Activation of Mast Cells via the High-Affinity IgE Receptor

Src family kinases (SFK) are critical for initiating and regulating the response of mast cells activated by engagement of the high-affinity IgE receptor, FcepsilonRI. Lyn is the predominant SFK in mast cells and has been ascribed both positive and negative roles in regulating mast cell activation. We analyzed the mast cell phenotype of WeeB, a recently described mouse mutant that expresses a Lyn protein with profoundly reduced catalytic activity. Surprisingly, we found that this residual activity is sufficient for wild-type levels of cytokine production and degranulation in bone marrow-derived mast cells after low-intensity stimulation with anti-IgE. High-intensity stimulation of lyn(-/-) bone marrow-derived mast cells with highly multivalent Ag resulted in enhanced cytokine production as previously reported, and WeeB cells displayed an intermediate phenotype. Under this latter condition, SFK inhibition using PP2 increased cytokine production in wild-type and WeeB but not lyn(-/-) cells, resulting in substantially higher levels in the PP2-treated WeeB than in lyn(-/-) cells. Restoration of wild-type and WeeB lyn alleles in lyn(-/-) cells generated activation phenotypes similar to those in nontransduced wild-type and WeeB cells, respectively, whereas a kinase-dead allele resulted in a phenotype similar to that of empty-vector-transduced cells. These data indicate that inhibition of Lyn and/or SFK activity can result in higher levels of mast cell activation than simple deletion of lyn and that only near-complete inhibition of Lyn can impair its positive regulatory functions. Furthermore, the data suggest that both positive and negative regulatory functions of Lyn are predominantly carried out by its catalytic activity and not an adaptor function.

TLR3-dependent upregulation of RIG-I leads to enhanced cytokine production from cells infected with the parainfluenza virus SV5

Here we address the role of RIG-I and TLR3 in differential cytokine responses against Simian Virus 5 (SV5) and two distinct cytokine inducing SV5 mutants. IFN-beta and IL-6 secretion was induced by infection with P/V-CPI-, an SV5 mutant with P/V substitutions, and were reduced by either siRNA-mediated knockdown of RIG-I expression or by expression of a dsRNA-binding protein. TLR3 overexpression did not alter cytokine secretion induced by P/V-CPI- or by Le-(U5C, A14G), an SV5 promoter mutant. TLR3 signaling by addition of exogenously added dsRNA was not blocked by WT SV5 or either SV5 mutant. Unexpectedly, TLR3 activation in infected cells led to enhanced IL-8 secretion, which correlated with increased RIG-I expression. Dominant negative RIG-I and TRIF supported a model whereby TLR3 activation upregulates RIG-I expression and in turn hypersensitizes cells to RIG-I-mediated cytokine secretion. Implications for crosstalk between different innate immunity pathways in mounting antiviral responses to paramyxoviruses are discussed.

Mouse Embryonic Stem Cells Express Functional Toll Like Receptor 2.

Abstract 1473Poster Board I-496Mouse embryonic stem cells (mESCs) are unique in that they give rise to every cell type of the body. Little is known about stimuli that promote mESC differentiation and proliferation. We hypothesized that TLRs are expressed and functional, and when activated by its ligand will influence survival and proliferation of mESCs in the presence of Leukemia Inhibitory Factor (LIF). This study evaluated three mESC lines, R1, CGR8, and E14 to first determine if they express Toll Like Receptors (TLRs) at mRNA and protein levels. Next, we evaluated if the TLR ligands would induce or modulate mESC proliferation and survival of the mESCs in the presence of LIF. We then assessed the E14 mESC line to determine if the mESCs expressed MyD88 an adaptor protein molecule, known to be involved in the TLR pathway and if the TLR ligands would cause inhibitor of kinase kinase alpha/ beta (IKKα/β) phosphorylation and nuclear factor-kappa beta (NF-κβ) nuclear translocation, and cytokine production. In this study we found expression of TLRs 1, 2, 3, 5 and 6 at the mRNA level, but no mRNA expression of TLRs 4, 7, 8 and 9. We also confirmed some of these results by flow analysis. Toll Like Receptor 2 (TLR-2), but not Toll Like Receptor 4 (TLR-4), is expressed on the three mESC lines. Therefore we focused our studies on TLR-2, specifically. Pam3Cys, a synthetic triacyl-lipoprotein and a TLR-2 ligand induced a significant increase in mESC numbers on Day 3 compared to controls and also at Day 4 and Day 5 when compared to controls. Pam3Cys (10ug/ml) also enhanced the survival of mESC colony forming cells subjected to serum withdrawal and then delayed addition of serum. Next we found that E14 mESCs express molecules involved in the TLR Pathway. MyD88 was expressed in mESCs and IKKα/β phosphorylation was enhanced after 15 minute by TLR-2 ligand activation. We found a significant increase of NF-κβ nuclear translocation upon activation by Pam3Cys after 30 minutes which continued for up to an hour. Densitometry analysis of the nuclear extracts from three separate experiments shows a significant 2 fold increase in NF-κβ in the nucleus compared to control mESC nuclear extracts. Since TLR activation of leukocytes enhances cytokine production, and our group has published that mESCs produce cytokines, we studied the effect of Pam3Cys on release of cytokine from our mESC line. Cytokine release in mESCs by TLR-ligand activation was assessed by ELISA, but TLR-2 ligand stimulation did not affect cytokine release of IL-6, TNF-α, or INF-β. We found that there were no significant changes in expression of mESCs markers Oct4, KLF-4, Sox 2, and SSEA1 when compared to cells not activated by Pam3Cys. Thus the mESCs remained in a pluripotent state in the presence of LIF after activation with the TLR-2 ligand. These results demonstrate that mESCs can respond to microbial products, and TLR-2 activation enhances proliferation and survival of the mESCs. This finding expands the role of TLRs and has implications for a better understanding of the responsiveness of embryonic stem cells to certain microbial agents. Disclosures:No relevant conflicts of interest to declare.

Adoptively transferred effector cells derived from naïve rather than central memory CD8 + T cells mediate superior antitumor immunity

Effector cells derived from central memory CD8(+) T cells were reported to engraft and survive better than those derived from effector memory populations, suggesting that they are superior for use in adoptive immunotherapy studies. However, previous studies did not evaluate the relative efficacy of effector cells derived from naïve T cells. We sought to investigate the efficacy of tumor-specific effector cells derived from naïve or central memory T-cell subsets using transgenic or retrovirally transduced T cells engineered to express a tumor-specific T-cell receptor. We found that naïve, rather than central memory T cells, gave rise to an effector population that mediated superior antitumor immunity upon adoptive transfer. Effector cells developed from naïve T cells lost the expression of CD62L more rapidly than those derived from central memory T cells, but did not acquire the expression of KLRG-1, a marker for terminal differentiation and replicative senescence. Consistent with this KLRG-1(-) phenotype, naïve-derived cells were capable of a greater proliferative burst and had enhanced cytokine production after adoptive transfer. These results indicate that insertion of genes that confer antitumor specificity into naïve rather than central memory CD8(+) T cells may allow superior efficacy upon adoptive transfer.

Human blood mononuclear cell in vitro cytokine response before and after two different strenuous exercise bouts in the presence of bloodroot and Echinacea extracts

The purpose of this multidisciplinary investigation was to characterize cytokine production by human blood mononuclear cells after 2 contrasting exercise bouts (a maximal graded oxygen consumption [VO 2max] test and 90 min of cycling at 85% of ventilatory threshold [VT]) when stimulated in vitro with extracts from bloodroot ( Sanguinaria canadensis), coneflower ( Echinacea tennesseensis), or solvent vehicle controls. Blood was sampled pre- and post-exercise. Production of TNF, IL-1β, and IL-10 were measured at 24, 48, and 72 h, respectively. In the VO 2max test there was a main effect of exercise such that exercise increased cytokine synthesis and a main effect of stimulant such that bloodroot extracts significantly increased cytokine production compared to other stimulants or controls. In the 90-min bout, there was a main effect of exercise for TNF and IL-1β (but not IL-10) such that exercise decreased cytokine synthesis and a main effect of stimulant such that bloodroot extracts significantly increased cytokine production compared to other stimulants or controls, with exercise × stimulant interactions for both IL-1β and IL-10. A similar though weaker effect was seen with Echinacea extracts; subsequent biochemical analyses suggested this was related to alkamide decay during 3 years undisturbed storage at ultralow (− 80 °C) temperature. In this study, the VO 2max test was associated with enhanced cytokine production whereas the 90-min cycling at 85% VT was associated with suppressed cytokine production. Bloodroot extracts were able to increase cytokine production in both contexts. Herbal extracts purported to offset exercise-associated effects on immune activity warrant continued investigation.

TRIL, a Functional Component of the TLR4 Signaling Complex, Highly Expressed in Brain

TLR4 is the primary sensor of LPS. In this study, we describe for the first time TLR4 interactor with leucine-rich repeats (TRIL), which is a novel component of the TLR4 complex. TRIL is expressed in a number of tissues, most prominently in the brain but also in the spinal cord, lung, kidney, and ovary. TRIL is composed of a signal sequence, 13 leucine-rich repeats, a fibronectin domain, and a single transmembrane spanning region. TRIL is induced by LPS in the human astrocytoma cell line U373, in murine brain following i.p. injection, and in human PBMC. Endogenous TRIL interacts with TLR4 and this interaction is greatly enhanced following LPS stimulation. TRIL also interacts with the TLR4 ligand LPS. Furthermore, U373 cells stably overexpressing TRIL display enhanced cytokine production in response to LPS. Finally, knockdown of TRIL using small interfering RNA attenuates LPS signaling and cytokine production in cell lines, human PBMC, and primary murine mixed glial cells. These results demonstrate that TRIL is a novel component of the TLR4 complex which may have particular relevance for the functional role of TLR4 in the brain.

Yessotoxin inhibits phagocytic activity of macrophages

Yessotoxin (YTX) is a sulphated polyether compound produced by some species of dinoflagellate algae, that can be accumulated in bivalve mollusks and ingested by humans upon eating contaminated shellfish. Experiments in mice have demonstrated the lethal effect of YTX after intraperitoneal injection, whereas its oral administration has only limited acute toxicity, coupled with an alteration of plasma membrane protein turnover in the colon of the animals. In vitro studies have shown that this effect is due to the inhibition of endocytosis induced by the toxin. In this work, we investigated the effects of YTX on phagocytosis by using the J774 macrophage cell line. We found that macrophages exposed to 10 or 1 nM YTX display a reduced phagocytic activity against Candida albicans; moreover, phagosome maturation is also inhibited in these cells. Such results were confirmed with resident peritoneal macrophages from normal mice. The inhibition of both phagocytosis and phagosome maturation likely involves cytoskeletal alterations, since a striking rearrangement of the F-actin organization occurs in YTX-treated J774 macrophages. Surprisingly, YTX also enhances cytokine production (TNF-α, MIP-1α and MIP-2) by J774 macrophages. Overall, our results show that low doses of YTX significantly affect both effector and secretory functions of macrophages.

A Novel Agonist Anti-Human OX40L Monoclonal Antibody That Stimulates T Cell Proliferation and Enhances Cytokine Secretion

OX40L (CD252), a membrane-bound member of the tumor necrosis factor superfamily, is known to be a critical co-stimulatory molecule during immune response. Here, we describe an agonistic mouse anti-human OX40L monoclonal antibody (clone 1E7) recognizing a novel epitope of OX40L antigen. Using this antibody, we found that OX40L was transiently upregulated on CD4(+) T cells during the early stage of activation. Cross-linking of OX40L with monoclonal antibody 1E7 markedly promoted T cell proliferation and activation and enhanced cytokine production, demonstrating that OX40L transmitted a signal to T cells. Thus, OX40L may play an important role in the early phase of T cell activation and proliferation.

Systemic activation of the immune system induces aberrant BAFF and APRIL expression in B cells in patients with systemic lupus erythematosus

Elevated levels of BAFF and APRIL are characteristic of patients with systemic lupus erythematosus (SLE). The reasons for enhanced cytokine production are not well understood. This study was undertaken to identify the cells responsible for the overproduction of these cytokines. BAFF expression was analyzed on peripheral blood mononuclear cells by multiparameter flow cytometry and in tissue samples by immunofluorescence staining. The levels of BAFF and APRIL mRNA were quantified in sorted B cells. In vitro cultures were used to analyze whether B cell survival and differentiation was supported by autocrine BAFF and/or APRIL. Aberrant activation of B cells in patients with SLE was associated with a significant up-regulation of BAFF expression in naive, memory, and plasma cells. Furthermore, strong expression of BAFF and APRIL was found in plasma cells from the lymph node, bone marrow, and kidney. The levels of BAFF and APRIL mRNA in CD19+ B cells correlated both with the titer of anti-double stranded DNA antibodies and with the SLE Disease Activity Index. In vitro experiments demonstrated that B cells released functional BAFF/APRIL upon activation. Our data show that B cells contribute to the enhanced levels of circulating BAFF and APRIL. The aberrant up-regulation of these cytokines may initiate a vicious circle in which enhanced levels of BAFF and APRIL act in an autocrine manner to reinforce the systemic activation of the humoral immune system.

An Activating and Inhibitory Signal from an Inhibitory Receptor LMIR3/CLM-1: LMIR3 Augments Lipopolysaccharide Response through Association with FcRγ in Mast Cells

Leukocyte mono-Ig-like receptor 3 (LMIR3) is an inhibitory receptor mainly expressed in myeloid cells. Coengagement of Fc epsilonRI and LMIR3 impaired cytokine production in bone marrow-derived mast cells (BMMCs) induced by Fc epsilonRI crosslinking alone. Mouse LMIR3 possesses five cytoplasmic tyrosine residues (Y241, Y276, Y289, Y303, Y325), among which Y241 and Y289 (Y241/289) or Y325 fit the consensus sequence of ITIM or immunotyrosine-based switch motif (ITSM), respectively. The inhibitory effect was abolished by the replacement of Y325 in addition to Y241/289 with phenylalanine (Y241/189/325/F) in accordance with the potential of Y241/289/325 to cooperatively recruit Src homology region 2 domain-containing phosphatase 1 (SHP)-1 or SHP-2. Intriguingly, LMIR3 crosslinking alone induced cytokine production in BMMCs expressing LMIR3 (Y241/276/289/303/325F) mutant as well as LMIR3 (Y241/289/325F). Moreover, coimmunoprecipitation experiments revealed that LMIR3 associated with ITAM-containing FcRgamma. Analysis of FcRgamma-deficient BMMCs demonstrated that both Y276/303 and FcRgamma played a critical role in the activating function of this inhibitory receptor. Importantly, LMIR3 crosslinking enhanced cytokine production of BMMCs stimulated by LPS, while suppressing production stimulated by other TLR agonists or stem cell factor. Thus, an inhibitory receptor LMIR3 has a unique property to associate with FcRgamma and thereby functions as an activating receptor in concert with TLR4 stimulation.

OPPORTUNITIES AND CHALLENGES IN THE DEVELOPMENT OF COMBINATION THERAPY FOR THE TREATMENT OF RETINAL DISEASES

Ranibizumab, a humanized monoclonal antibody fragment (fragment, antigen binding, FAB) that neutralizes all of the soluble isoforms of vascular endothelial growth factor (VEGF)-A, was a significant step forward in the control of age-related macular degeneration. However, this agent, like others with the same mechanism, has several limitations. Although ranibizumab preserves vision in almost all patients, only a fraction achieves a halving of the visual angle. By inhibiting the activities of VEGF, ranibizumab blocks the continued growth of choroidal neovascularization, but existing neovascular lesions do not regress. Further, ranibizumab addresses only the increased production of VEGF; it does not address the underlying cause of enhanced cytokine production. Although ranibizumab is well tolerated, the course of the disease is unpredictable, necessitating frequent patient monitoring and treatment. Although strategies that guide retreatment based on the reoccurrence of retinal edema can be used to reduce treatment burden, this strategy may not provide optimal patient benefit. Because of these limitations, the identification of effective adjunctive therapies is considered an urgent goal.

Characterization of cellulose production in Escherichia coli Nissle 1917 and its biological consequences

Bacterial species of the Enterobacteriaceae family produce cellulose and curli fimbriae as extracellular matrix components, and their synthesis is positively regulated by the transcriptional activator CsgD. In this group of bacteria, cellulose biosynthesis is commonly regulated by CsgD via the GGDEF domain protein AdrA, a diguanylate cyclase that produces cyclic-diguanylic acid (c-di-GMP), an allosteric activator of cellulose synthase. In the probiotic Escherichia coli strain Nissle 1917 and its recent clonal isolates, CsgD activates the production of curli fimbriae at 28 degrees C, but neither CsgD nor AdrA is required for the c-di-GMP-dependent biosynthesis of cellulose at 28 degrees C and 37 degrees C. In these strains, the GGDEF domain protein YedQ, a diguanylate cyclase that activates cellulose biosynthesis in certain E. coli strains, is not required for cellulose biosynthesis and it has in fact evolved into a novel protein. Cellulose production in Nissle 1917 is required for adhesion of bacteria to the gastrointestinal epithelial cell line HT-29, to the mouse epithelium in vivo, and for enhanced cytokine production. The role of cellulose in this strain is in contrast to the role of cellulose in the commensal strain E. coli TOB1. Consequently, the role of cellulose in bacterial-host interaction is dependent on the E. coli strain background.

The costimulatory role of TIM molecules

The T-cell immunoglobulin domain and mucin domain (TIM) family, including TIM-1, TIM-2, TIM-3, and TIM-4, is a relatively newly described group of molecules with a conserved structure and important immunological functions, including T-cell activation, induction of T-cell apoptosis and T-cell tolerance, and the clearance of apoptotic cells. TIM-1 costimulates T-cell activation and enhances cytokine production. In humans, TIM-1 also serves as a susceptibility gene for allergy and asthma. TIM-3, expressed on T cells and dendritic cells, regulates T-cell apoptosis and immune tolerance. By contrast, TIM-4, which is expressed primarily on antigen-presenting cells and which is a receptor for phosphatidylserine, regulates T-cell activation and tolerance, in part by mediating the uptake and engulfment of apoptotic cells. The TIM molecules thus have surprisingly broad activities affecting multiple aspects of immunology.

Lack of oviduct pathology in mice infected with TLR2-signaling deficient chlamydiae is associated with earlier resolution of oviduct inflammation, and a decreased Th17 response (129.10)

Abstract TLR2 KO mice infected with wild-type C. muridarum strain Nigg and immunologically normal mice infected with plasmid-deficient C. muridarum CM3.1, that fails to activate TLR2, are protected from oviduct pathology during primary and challenge infection. Genital tracts harvested from normal mice sacrificed 7, 14, 21, and 28 days post infection with Nigg or CM3.1 were examined histologically. On day 14, neutrophil infiltrates were significantly decreased in the oviducts from mice infected with CM3.1 versus oviducts from mice infected with Nigg. On days 21 and 28, neutrophils and lymphocytes were significantly decreased in oviducts of mice infected with CM3.1 versus Nigg. Quantitative culture of oviduct homogenates thru 35 days post infection revealed equivalent bacterial burden and resolution of infection in both groups by day 19. Significant amounts of TNF, IL-1α, IL-1β, IL-6, GM-CSF, G-CSF, MIP-2, KC and IFN-γ were detected by bead assay in the oviduct homogenates of Nigg-infected mice on days 9 through 15, but were low or undetectable in mice infected with CM3.1. ELISPOT for IFN-γ and IL-17 producing CD4 T cells in iliac nodes reveal significantly increased Th17 cells on days 7 and 20 post infection in Nigg-infected mice. Chlamydial-induced TLR2 signaling promotes enhanced cytokine production, prolonged oviduct inflammation, and activation of Th17 cells that may contribute to induction of pathology.

α-1-Antitrypsin is an endogenous inhibitor of proinflammatory cytokine production in whole blood

Several observations suggest endogenous suppressors of inflammatory mediators are present in human blood. alpha-1-Antitrypsin (AAT) is the most abundant serine protease inhibitor in blood, and AAT possesses anti-inflammatory activity in vitro and in vivo. Here, we show that in vitro stimulation of whole blood from persons with a genetic AAT deficiency resulted in enhanced cytokine production compared with blood from healthy subjects. Using whole blood from healthy subjects, dilution of blood with RPMI tissue-culture medium, followed by incubation for 18 h, increased spontaneous production of IL-8, TNF-alpha, IL-1 beta, and IL-1R antagonist (IL-1Ra) significantly, compared with undiluted blood. Dilution-induced cytokine production suggested the presence of one or more circulating inhibitors of cytokine synthesis present in blood. Serially diluting blood with tissue-culture medium in the presence of cytokine stimulation with heat-killed Staphylococcus epidermidis (S. epi) resulted in 1.2- to 55-fold increases in cytokine production compared with S. epi stimulation alone. Diluting blood with autologous plasma did not increase the production of IL-8, TNF-alpha, IL-1 beta, or IL-1Ra, suggesting that the endogenous, inhibitory activity of blood resided in plasma. In whole blood, diluted and stimulated with S. epi, exogenous AAT inhibited IL-8, IL-6, TNF-alpha, and IL-1 beta significantly but did not suppress induction of the anti-inflammatory cytokines IL-1Ra and IL-10. These ex vivo and in vitro observations suggest that endogenous AAT in blood contributes to the suppression of proinflammatory cytokine synthesis.

L-carnitine, a diet component and organic cation transporter OCTN ligand, displays immunosuppressive properties and abrogates intestinal inflammation

Allele variants in the L-carnitine (LCAR) transporters OCTN1 (SLC22A4, 1672 C --> T) and OCTN2 (SLC22A5, -207 G --> C) have been implicated in susceptibility to Crohn's disease (CD). LCAR is consumed in the diet and transported actively from the intestinal lumen via the organic cation transporter OCTN2. While recognized mainly for its role in fatty acid metabolism, several lines of evidence suggest that LCAR may also display immunosuppressive properties. This study sought to investigate the immunomodulatory capacity of LCAR on antigen-presenting cell (APC) and CD4+ T cell function by examining cytokine production and the expression of activation markers in LCAR-supplemented and deficient cell culture systems. The therapeutic efficacy of its systemic administration was then evaluated during the establishment of colonic inflammation in vivo. LCAR treatment significantly inhibited both APC and CD4+ T cell function, as assessed by the expression of classical activation markers, proliferation and cytokine production. Carnitine deficiency resulted in the hyperactivation of CD4+ T cells and enhanced cytokine production. In vivo, protection from trinitrobenzene sulphonic acid colitis was observed in LCAR-treated mice and was attributed to the abrogation of both innate [interleukin (IL)-1beta and IL-6 production] and adaptive (T cell proliferation in draining lymph nodes) immune responses. LCAR therapy may therefore represent a novel alternative therapeutic strategy and highlights the role of diet in CD.

Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1–infected individuals to a mean of 49.4 ± SD 12.9% of CD8+ T cells expressing Tim-3 in HIV-1–infected chronic progressors versus 28.5 ± 6.8% in HIV-1–uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1–infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4+ T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1–specific CD8+ T cells. Tim-3–expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1–specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1–associated T cell dysfunction.

Immunostimulatory Characteristics Induced by Linear Polyethyleneimine–Plasmid DNA Complexes in Cultured Macrophages

Intravenously injected plasmid DNA (pDNA) complexed with cationic liposome (lipoplexes) caused NF-kappaB-mediated cytokine production from macrophages, induced by CpG sequence in the pDNA. We have reported that cytokine production caused by linear polyethyleneimine (PEI)-pDNA complexes (PEI polyplexes) was much lower than that caused by lipoplexes (Kawakami, S., Ito, Y., Charoensit, P., Yamashita, F., and Hashida, M. [2006]. J. Pharmacol. Exp. Ther. 317, 1382-1390). As Toll-like receptor-9 recognizing CpG sequence is expressed in the endosomal compartment, we hypothesized that the buffering capacity of PEI enhanced the escape of PEI polyplexes from endosomes, and that consequently cytokine production was decreased. In this study, the mechanism of lower cytokine production induced by PEI polyplexes, compared with lipoplexes, was investigated using the murine macrophage-like cell line RAW 264.7. Although transfection efficacy and cellular association were similar for PEI polyplexes and lipoplexes, tumor necrosis factor-alpha and interleukin-6 production and NF-kappaB activation caused by polyplexes were significantly lower than with lipoplexes. As for intracellular distribution, PEI polyplexes spread into cytosol whereas lipoplexes accumulated in vesicles, suggesting enhancement of escape from endosomes by PEI. Bafilomycin A1, an inhibitor of early endosomes, enhanced cytokine production and NF-kappaB activation by PEI polyplexes but not by lipoplexes; however, chloroquine, an inhibitor of late endosomes, inhibited PEI polyplex-induced cytokine production and NF-kappaB activation, suggesting that the buffering effect of PEI on early endosomes decreases NF-kappaB-mediated cytokine production. In conclusion, we demonstrate that cytokine production and NF-kappaB activation induced by PEI polyplexes are significantly lower than with lipoplexes in cultured macrophages. The significantly low cytokine response of PEI polyplexes may be due to effective transition of PEI polyplexes from endosomes to cytosol.

Antigen-presenting cells containing multiple costimulatory molecules promote activation and expansion of human antigen-specific memory CD8+ T cells

We have previously demonstrated that multiple immunizations with vector-based vaccines containing transgenes for tumor Ags and a triad of costimulatory molecules (TRICOM) enhance the expansion and functional avidity of Ag-specific memory CD8(+) T cells in a mouse model. However, the effect of enhanced costimulation on human memory CD8(+) T cells is still unclear. The study reported here was an in vitro investigation of the proliferation and function of CEA-specific human memory CD8(+) T cells following enhanced costimulation. Our results demonstrated that TRICOM costimulation enhanced production of multiple cytokines and expansion of CEA-specific memory CD8(+) T cells. The lytic capacity of memory CTLs toward CEA(+) tumors was also significantly enhanced. IL-2R alpha (CD25) was upregulated dramatically following APC-TRICOM stimulation, suggesting that the enhanced expansion of memory CD8(+) T cells may be mediated by increased expression of IL-2R on memory T cells. The enhanced cytokine production and proliferation following TRICOM signaling was completely blocked by the combination of neutralizing Abs against B7-1, ICAM-1, and LFA-3, the costimulatory molecules comprising TRICOM. No difference in T-cell apoptosis was observed between APC-TRICOM and APC-wild-type groups, as determined by annexin V, Bcl-2, and active caspase-3 staining. Results indicated that enhanced costimulation greatly expanded human CEA-specific CD8(+) T cells and enhanced T-cell function, without inducing increased apoptosis of CEA-specific memory CD8(+) T cells.