Expression Of Effector Molecules Research Articles

Enhanced T cell activation in lymphocytes from transgenic mice expressing LAT molecules resistant to ubiquitination (69.5)

Abstract Linker for activation of T cells (LAT) is critical for the propagation of T cell signals from the plasma membrane to the cytoplasm upon T cell activation. Previous studies demonstrated that substitution of LAT lysines with arginines (2KRLAT) resulted in a decrease in LAT ubiquitination and elevated T cell signaling, indicating that LAT ubiquitination is a molecular checkpoint for attenuation of T cell signaling. To investigate the role of LAT ubiquitination in vivo, we have generated transgenic mice expressing WT and ubiquitin-defective 2KRLAT. Upon TCR stimulation, proximal T cell signaling was elevated in lymphocytes expressing 2KRLAT versus WT cells as evidenced by higher levels of phospho(p)-tyrosine proteins, p-LAT, p-phospholipase C-gamma and cytosolic calcium flux. Evaluation of various cell surface markers also revealed an enhanced memory T cell profile in 2KR lymphocytes. Moreover, upon TCR stimulation, a higher percentage of T cells expressing greater levels of effector molecules such as intracellular interferon-gamma, tumor necrosis factor-alpha and interleukin-2 was found in 2KR versus WTLAT mice. We have thus identified enhanced proximal T cell signaling, an enhanced memory T cell profile and higher expression of cytokine effector molecules in lymphocytes from 2KRLAT transgenic mice. These results suggest that 2KR mice may respond more efficiently to pathogenic challenges, an hypothesis that will be tested in various experimental models of mouse infection.

Innate memory of human Vγ2Vδ2 T cells: Distinct proliferative, migratory and tumorcidal capabilities of γδ memory subsets (52.17)

Abstract Human Vγ2Vδ2 T cells are unconventional T cells that monitor isoprenoid metabolism by using their antigen receptors to recognize foreign and self prenyl pyrophosphates. Although important for immunity to infections and for tumor immunotherapy, little is known about the development of γδ memory. Elucidation of γδ differentiation pathways will help to develop ways to boost their immunity. Here we report that there are many unique features compared with αβ T cells. Unlike αβ T cells, at birth half of Vγ2Vδ2 cells have memory phenotypes and ~25-40% express inflammatory chemokine receptors. In adults, 98.4% of Vγ2Vδ2 T cells are memory cells that can be separated into CD28+CD27+ TEarly (central); CD28+CD27- TEarly 27-; CD28-CD27+ TIntermediate (effector); and CD28-CD27- CD45RA+ TLate RA (effector) subsets. The memory subsets display distinct functional properties with differential expression of effector molecules, NK receptors, and chemokine receptors. Intermediate and late effector memory cells express higher levels of cytotoxic effector molecules, innate CXCR1, CXCR2, and CX3CR1 chemokine receptors, KIR, CD16, CD56, and CD57 NK receptors, and lower levels of the β7 integrin, and proliferate poorly compared with early memory cells that express CXCR6, CCR1, and CCR2. Thus, different inflammatory chemokines attract each Vγ2Vδ2 memory subset. The rapid conversion of Vγ2Vδ2 cells from naive to memory allows them to mount memory responses to primary bacterial and protozoan infections.

Ubc13 maintains the suppressive function of regulatory T cells and prevents their conversion into effector-like T cells

Maintenance of immune homeostasis requires regulatory T (Treg) cells. Here we show that Treg-specific ablation of Ubc13, a lysine 63-specific ubiquitin-conjugating enzyme, caused aberrant T cell activation and autoimmunity. Although Ubc13 deficiency did not affect Treg cell survival or Foxp3 expression, it impaired the in vivo suppressive function of Treg cells and rendered them sensitive for acquiring T helper (TH) 1- and TH17-like effector T cell phenotypes. This function of Ubc13 involved its downstream target, IκB kinase (IKK). The Ubc13-IKK signaling axis controlled the expression specific Treg effector molecules, including interleukin 10 (IL-10) and SOCS1. Collectively, these findings suggest that the Ubc13-IKK signaling axis regulates the molecular program that maintains Treg function and prevents Treg cells from acquiring inflammatory phenotypes.

Comparison of Influenza and SIV Specific CD8 T Cell Responses in Macaques

Macaques are a potentially useful non-human primate model to compare memory T-cell immunity to acute virus pathogens such as influenza virus and effector T-cell responses to chronic viral pathogens such as SIV. However, immunological reagents to study influenza CD8+ T-cell responses in the macaque model are limited. We recently developed an influenza-SIV vaccination model of pigtail macaques (Macaca nemestrina) and used this to study both influenza-specific and SIV-specific CD8+ T-cells in 39 pigtail macaques expressing the common Mane-A*10+ (Mane-A01*084) MHC-I allele. To perform comparative studies between influenza and SIV responses a common influenza nucleoprotein-specific CD8+ T-cell response was mapped to a minimal epitope (termed RA9), MHC-restricted to Mane-A*10 and an MHC tetramer developed to study this response. Influenza-specific memory CD8+ T-cell response maintained a highly functional profile in terms of multitude of effector molecule expression (CD107a, IFN-γ, TNF-α, MIP-1β and IL-2) and showed high avidity even in the setting of SIV infection. In contrast, within weeks following active SIV infection, SIV-specific CD8+ effector T-cells expressed fewer cytokines/degranulation markers and had a lower avidity compared to influenza specific CD8+ T-cells. Further, the influenza specific memory CD8 T-cell response retained stable expression of the exhaustion marker programmed death-marker-1 (PD-1) and co-stimulatory molecule CD28 following infection with SIV. This contrasted with the effector SIV-specific CD8+ T-cells following SIV infection which expressed significantly higher amounts of PD-1 and lower amounts of CD28. Our results suggest that strategies to maintain a more functional CD8+ T-cell response, profile may assist in controlling HIV disease.

AP-1 Transcription Factors, Mucin-Type Molecules and MMPs Regulate the IL-11 Mediated Invasiveness of JEG-3 and HTR-8/SVneo Trophoblastic Cells

This study examines the IL-11 mediated activation of downstream signaling and expression of effector molecules to resolve the controversies associated with the IL-11 mediated regulation of the invasiveness of two commonly used trophoblastic cell models viz. JEG-3 and HTR-8/SVneo cells. It has been reported that IL-11 increases the invasiveness of JEG-3 cells while, reduces the invasiveness of HTR-8/SVneo cells. Invasion assay performed simultaneously for both the cell lines confirmed the above findings. In addition, HTR-8/SVneo cells showed a higher basal invasiveness than JEG-3 cells. Western blot showed the IL-11 mediated activation of STAT3(tyr705) and STAT1(tyr701) in both the cell lines. However, IL-11 activated the ERK1/2 phosphorylation in JEG-3 cells but, inhibited it in HTR-8/SVneo cells. Within 10 min of IL-11 treatment, p-STAT3(tyr705) was localized inside the nucleus of both the cell lines but, there was enhanced co-localization of protein inhibitor of activated STAT1/3 (PIAS1/3) and p-STAT3(tyr705) in HTR-8/SVneo cells and not in JEG-3 cells. This could be reason for the poor responsiveness of STAT3 responsive genes like mucin 1 (MUC1) in HTR-8/SVneo cells and not in JEG-3 cells. Further, microarray analysis of the IL-11 treated cells revealed differential responsiveness of JEG-3 as compared to HTR-8/SVneo cells. Several family of genes like activator protein-1 (AP-1) transcription factors (Jun and Fos), mucin-type molecules, MMP23B etc showed enhanced expression in IL-11 treated JEG-3 cells while, there was no response or decrease in their expression in IL-11 treated HTR-8/SVneo cells. Expression of these molecules was confirmed by quantitative RT-PCR. In addition, HTR-8/SVneo cells also showed a significant decrease in the expression of MMP2, MMP3 and MMP9 upon IL-11 treatment. Hence, IL-11 mediated differential activation of signaling and expression of effector molecules is responsible for the differential invasive response of JEG-3 and HTR-8/SVneo cells.

Unconventional CD8+ T cells (CD4dimCD8bright cells) in anti-HIV immunity and neuropathogenesis

August 20-22, 2012 C paradigm of T cell biology tells us that expression of the CD4 and CD8 molecules on mature T cells is mutually exclusive. However, my group and others have identified a unique subset of CD8+ T cells that co-express CD4 dimly on their surface. This subset is termed CD4dimCD8bright T cells or double positive (DP) T cells. DP cells represent a genuine phenotype of CD8+ T cells. They express αβTCR and αβCD8 and are not prematurely released from the thymus. Post TCR or superantigen stimulation, 15-60% of purified CD8+ T cells induce stable CD4 expression on their surface. DP cells constitute 3-5% of peripheral CD8+ T cells in healthy donors and are expanded to up to 15% among HIV long-term nonprogressors. DP cells are highly enriched in anti-viral (CMV and HIV) responses. In comparison to their CD8 single-positive T cell counterparts, DP cells constitute greater than 55% of anti-HIV and -CMV responses, as evaluated by MHC-I tetramer analysis, polyfunctional cytokine and effector molecule expression, and antigen-specific proliferation. DP cells are also highly enriched in β-catenin expression, a pro-survival transcriptional co-activator that drives CD4 induction on CD8+ T cells and may protect them from activation-induced cell death. DP cells are found in the CNS of HIV-infected NOD/SCID/IL-2rcγ-/mice reconstituted with human peripheral blood lymphocytes and while they are susceptible to HIV infection, they are not depleted to the same extent as CD4+ T cells. Astrocyte-conditioned media also induces CD4 expression on CD8+ T cells. Our findings identify DP cells as a potent anti-viral T cell subset; however, their contributing role to HIV-associated neurocognitive impairment, whether protective or pathogenic, remains to be elucidated.

Role of CD44 in lymphokine-activated killer cell-mediated killing of melanoma.

In the current study, we examined the potential significance of CD44 expression on lymphokine-activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells, and NKT cells. Treatment of melanoma-bearing CD44 WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma-bearing CD44 KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2-treated CD44 KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2-treated CD44 WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred CD44 WT LAK cells were significantly more effective than CD44 KO LAK cells at controlling tumor growth in vivo. Next, the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44(lo)) or elevated (CD44(hi)) expression of CD44 are generated and that the CD44(hi) cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44 KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells.

Interferon Regulatory Factor-1 (IRF-1) Shapes Both Innate and CD8+ T Cell Immune Responses against West Nile Virus Infection

Interferon regulatory factor (IRF)-1 is an immunomodulatory transcription factor that functions downstream of pathogen recognition receptor signaling and has been implicated as a regulator of type I interferon (IFN)-αβ expression and the immune response to virus infections. However, this role for IRF-1 remains controversial because altered type I IFN responses have not been systemically observed in IRF-1 -/- mice. To evaluate the relationship of IRF-1 and immune regulation, we assessed West Nile virus (WNV) infectivity and the host response in IRF-1 -/- cells and mice. IRF-1 -/- mice were highly vulnerable to WNV infection with enhanced viral replication in peripheral tissues and rapid dissemination into the central nervous system. Ex vivo analysis revealed a cell-type specific antiviral role as IRF-1 -/- macrophages supported enhanced WNV replication but infection was unaltered in IRF-1 -/- fibroblasts. IRF-1 also had an independent and paradoxical effect on CD8+ T cell expansion. Although markedly fewer CD8+ T cells were observed in naïve animals as described previously, remarkably, IRF-1 -/- mice rapidly expanded their pool of WNV-specific cytolytic CD8+ T cells. Adoptive transfer and in vitro proliferation experiments established both cell-intrinsic and cell-extrinsic effects of IRF-1 on the expansion of CD8+ T cells. Thus, IRF-1 restricts WNV infection by modulating the expression of innate antiviral effector molecules while shaping the antigen-specific CD8+ T cell response.

Differentiation-dependent functional and epigenetic landscapes for cytokine genes in virus-specific CD8+T cells

Although the simultaneous engagement of multiple effector mechanisms is thought to characterize optimal CD8(+) T-cell immunity and facilitate pathogen clearance, the differentiation pathways leading to the acquisition and maintenance of such polyfunctional activity are not well understood. Division-dependent profiles of effector molecule expression for virus-specific T cells are analyzed here by using a combination of carboxyfluorescein succinimidyl ester dilution and intracellular cytokine staining subsequent to T-cell receptor ligation. The experiments show that, although the majority of naive CD8(+) T-cell precursors are preprogrammed to produce TNF-α soon after stimulation and a proportion make both TNF-α and IL-2, the progressive acquisition of IFN-γ expression depends on continued lymphocyte proliferation. Furthermore, the extensive division characteristic of differentiation to peak effector activity is associated with the progressive dominance of IFN-γ and the concomitant loss of polyfunctional cytokine production, although this is not apparent for long-term CD8(+) T-cell memory. Such proliferation-dependent variation in cytokine production appears tied to the epigenetic signatures within the ifnG and tnfA proximal promoters. Specifically, those cytokine gene loci that are rapidly expressed following antigen stimulation at different stages of T-cell differentiation can be shown (by ChIP) to have permissive epigenetic and RNA polymerase II docking signatures. Thus, the dynamic changes in cytokine profiles for naive, effector, and memory T cells are underpinned by specific epigenetic landscapes that regulate responsiveness following T-cell receptor ligation.

CD4+CD25+Foxp3+ Regulatory T Cells Are Dispensable for Controlling CD8+ T Cell-Mediated Lung Inflammation

Every person harbors a population of potentially self-reactive lymphocytes controlled by tightly balanced tolerance mechanisms. Failures in this balance evoke immune activation and autoimmunity. In this study, we investigated the contribution of self-reactive CD8(+) T lymphocytes to chronic pulmonary inflammation and a possible role for naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (nTregs) in counterbalancing this process. Using a transgenic murine model for autoimmune-mediated lung disease, we demonstrated that despite pulmonary inflammation, lung-specific CD8(+) T cells can reside quiescently in close proximity to self-antigen. Whereas self-reactive CD8(+) T cells in the inflamed lung and lung-draining lymph nodes downregulated the expression of effector molecules, those located in the spleen appeared to be partly Ag-experienced and displayed a memory-like phenotype. Because ex vivo-reisolated self-reactive CD8(+) T cells were very well capable of responding to the Ag in vitro, we investigated a possible contribution of nTregs to the immune control over autoaggressive CD8(+) T cells in the lung. Notably, CD8(+) T cell tolerance established in the lung depends only partially on the function of nTregs, because self-reactive CD8(+) T cells underwent only biased activation and did not acquire effector function after nTreg depletion. However, although transient ablation of nTregs did not expand the population of self-reactive CD8(+) T cells or exacerbate the disease, it provoked rapid accumulation of activated CD103(+)CD62L(lo) Tregs in bronchial lymph nodes, a finding suggesting an adaptive phenotypic switch in the nTreg population that acts in concert with other yet-undefined mechanisms to prevent the detrimental activation of self-reactive CD8(+) T cells.

Autoreactive Cytotoxic T Lymphocytes Acquire Higher Expression of Cytotoxic Effector Markers in the Islets of NOD Mice after Priming in Pancreatic Lymph Nodes

Cytotoxic T lymphocytes (CTLs) that cause type 1 diabetes are activated in draining lymph nodes and become concentrated as fully active CTLs in inflamed pancreatic islets. It is unclear whether CTL function is driven by signals received in the lymph node or also in the inflamed tissue. We studied whether the development of cytotoxicity requires further activation in islets. Autoreactive CTLs found in the islets of diabetes-prone NOD mice had acquired much higher expression of the cytotoxic effector markers granzyme B, interferon γ, and CD107a than had those in the pancreatic lymph node (PLN). Increased expression seemed to result from stimulation in the islet itself. T cells held up from migrating from the PLN by administration of the sphingosine-1-phosphate agonist FTY720 did not increase expression of cytotoxic molecules in the PLN. Stimulation did not require antigen presentation or cytokine secretion by the target β cells because it was not affected by the absence of class I major histocompatibility complex expression or by the overexpression of suppressor of cytokine signaling-1. Activation of CD40-expressing cells stimulated increased CTL function and β-cell destruction, suggesting that signals derived from CD40-expressing cells promote the acquisition of cytotoxicity in the islet environment. These data provide in vivo evidence that stimulation of cytotoxic effector molecule expression occurs in inflamed islets and is independent of β cells.

In vitro interactions of Candida parapsilosis wild type and lipase deficient mutants with human monocyte derived dendritic cells

BackgroundCandida parapsilosis typically is a commensal of human skin. However, when host immune defense is compromised or the normal microflora balance is disrupted, C. parapsilosis transforms itself into an opportunistic pathogen. Candida-derived lipase has been identified as potential virulence factor. Even though cellular components of the innate immune response, such as dendritic cells, represent the first line of defense against invading pathogens, little is known about the interaction of these cells with invading C. parapsilosis. Thus, the aim of our study was to assess the function of dendritic cells in fighting C. parapsilosis and to determine the role that C. parapsilosis-derived lipase plays in the interaction with dendritic cells.ResultsMonocyte-derived immature and mature dendritic cells (iDCs and mDCs, respectively) co-cultured with live wild type or lipase deficient C. parapsilosis strains were studied to determine the phagocytic capacity and killing efficiency of host cells. We determined that both iDCs and mDCs efficiently phagocytosed and killed C. parapsilosis, furthermore our results show that the phagocytic and fungicidal activities of both iDCs and mDCs are more potent for lipase deficient compared to wild type yeast cells. In addition, the lipase deficient C. parapsilosis cells induce higher gene expression and protein secretion of proinflammatory cytokines and chemokines in both DC types relative to the effect of co-culture with wild type yeast cells.ConclusionsOur results show that DCs are activated by exposure to C. parapsilosis, as shown by increased phagocytosis, killing and proinflammatory protein secretion. Moreover, these data strongly suggest that C. parapsilosis derived lipase has a protective role during yeast:DC interactions, since lipase production in wt yeast cells decreased the phagocytic capacity and killing efficiency of host cells and downregulated the expression of host effector molecules.

IFN-λ determines the intestinal epithelial antiviral host defense

Type I and type III IFNs bind to different cell-surface receptors but induce identical signal transduction pathways, leading to the expression of antiviral host effector molecules. Despite the fact that type III IFN (IFN-λ) has been shown to predominantly act on mucosal organs, in vivo infection studies have failed to attribute a specific, nonredundant function. Instead, a predominant role of type I IFN was observed, which was explained by the ubiquitous expression of the type I IFN receptor. Here we comparatively analyzed the role of functional IFN-λ and type I IFN receptor signaling in the innate immune response to intestinal rotavirus infection in vivo, and determined viral replication and antiviral gene expression on the cellular level. We observed that both suckling and adult mice lacking functional receptors for IFN-λ were impaired in the control of oral rotavirus infection, whereas animals lacking functional receptors for type I IFN were similar to wild-type mice. Using Mx1 protein accumulation as marker for IFN responsiveness of individual cells, we demonstrate that intestinal epithelial cells, which are the prime target cells of rotavirus, strongly responded to IFN-λ but only marginally to type I IFN in vivo. Systemic treatment of suckling mice with IFN-λ repressed rotavirus replication in the gut, whereas treatment with type I IFN was not effective. These results are unique in identifying a critical role of IFN-λ in the epithelial antiviral host defense.

Abstract 779: Role of CD44 in LAK cell-mediated killing of melanoma

Abstract Lymphokine-activated killer cells play an important role in various immunologically-based treatments of cancer, including in the use of IL-2 therapy for the treatment of malignant melanoma. In previous studies, we demonstrated that the expression of CD44 on splenocytes is significantly elevated following exposure to IL-2. Therefore, in the current study we examined the potential significance of CD44 expression on lymphokine activated killer (LAK) cells in their interaction and killing of melanoma cells. Stimulation of splenocytes with IL-2 led to a significant increase in the expression of CD44 on T cells, NK cells and NKT cells. Treatment of melanoma bearing CD44WT mice with IL-2 led to a significant reduction in the local tumor growth while treatment of melanoma bearing CD44KO mice with IL-2 was ineffective at controlling tumor growth. Furthermore, the ability of splenocytes from IL-2 treated CD44KO mice to kill melanoma tumor targets was significantly reduced when compared to the anti-tumor activity of splenocytes from IL-2 treated CD44WT mice. The importance of CD44 expression on the LAK cells was further confirmed by the observation that adoptively transferred IL-2-activated splenocytes from CD44KO mice were significantly less effective at controlling tumor growth in vivo. Next the significance of the increased expression of CD44 in tumor killing was examined and showed that following stimulation with IL-2, distinct populations of cells with low (CD44lo) or elevated (CD44hi) expression of CD44 are generated and that the CD44hi cells are responsible for killing of the melanoma cells. The reduced killing activity of the CD44KO LAK cells did not result from reduced activation or expression of effector molecules but was due, at least in part, to a reduced ability to adhere to B16F10 tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 779. doi:10.1158/1538-7445.AM2011-779

Abstract LB-164: B lymphocytes inhibit anti-tumor response in a murine EMT6 tumor model by facilitating expansion of T regulatory cells

Abstract B lymphocytes play a central role in inhibiting the immune response against certain tumors, but the underlying mechanisms by which B cells facilitate tumor growth are still poorly understood. Murine EMT6 mammary tumors grow readily in immune competent mice (ICM) but poorly in B-cell deficient μ−/− BALB/c mice (BCDM). Both decreased T regulatory cell (Treg) expansion and impaired Treg function were observed in BCDM compared to ICM. In this study, we investigated how the presence or absence of B cells affects expansion of Tregs in response to tumor implantation. We compared tumor growth, and the number of Treg cells, T cell function, number and character of tumor infiltrating cells, immune effector molecule, chemokine and cytokine expression in tumor tissue in ICM, compared to BCDM and B cell reconstituted BCDM (BCDM+B). Partial reconstitution of BCDM with adoptively transferred B cells restored tumor growth. Transfer of both normal and IL-10−/− B cells restored tumor growth in BCDM. Restored tumor growth correlated with an increase in CD4+FoxP3+ Treg cells in the spleen and draining lymph nodes and a decrease in interferon-γ secretion and cytotoxic T cell activity against EMT6 targets, in BCDM+B to BCDM. Tumor growth was completely inhibited in BCDM+B by depletion of Treg using PC61 antibody. Markedly increased levels of NK and CD8+ T cell infiltration into the tumor bed were seen in BCDM or BCDM+B treated with Treg depletion, compared to levels of infiltration seen in ICM or BCDM+B mice. PCR array analysis of mRNA expression in tumor tissue in BCDM, BCDM+B treated with PC61 antibody, or ICM following PC61 treatment, revealed down regulation of IL-17A, IL-17F, TGF-β and IL-23, CD73, VEGF-C, Rorγt and CCR4, when compared to either BCDM+B or BALB/c control mice. We observed similar changes in immune effector molecules/cytokines mRNA in all scenarios in which Treg levels were decreased. In conclusion, our studies indicate that the T cell anti-tumor response is enhanced in the absence of B cells, in part due to effects on Treg cell expansion and changes in immune effector molecule and cytokine profile in the tumor micro-environment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-164. doi:10.1158/1538-7445.AM2011-LB-164

Increased apoptosis, curtailed expansion and incomplete differentiation of CD8+ T cells combine to decrease clearance of L. monocytogenes in old mice

Aging is accompanied by altered immunity, resulting in a variable state of poorly understood immunodeficiency. While both the numbers and the functionality of naïve T cells are decreased by aging, the impact of these changes upon immune defense against bacterial pathogens in vivo remains understudied. Using a model of Listeria monocytogenes (Lm), where the primary CD8(+) T-cell response is critically important for immune defense, we show that C57BL/6 (B6) mice exhibit an age-dependent reduction in survival, with delayed bacterial clearance in old animals. Kinetic analysis of antigen-specific CD8(+) T-cell expansion showed that CD8(+) effectors begin dividing at the same time in old and adult mice, but that the proliferative burst remained incomplete during discrete windows of time and was coupled with increased effector apoptosis in old mice. Further, antilisterial CD8(+) T cells in old mice showed altered expression of key phenotypic and effector molecules and diminished polyfunctionality, measured by the ability to simultaneously produce multiple effector molecules. These results suggest that defects in functional maturation of CD8(+) cells in aged mice, compounded by (or perhaps coupled to) their reduced expansion in response to infection, yield effector CD8(+) T-cell populations insufficient in size and capability to effectively clear newly encountered intracellular pathogens.

879 BLADDER EXPRESSION OF CYTOTOXIC EFFECTOR MOLECULES IN RESPONSE TO INTRAVESICAL MYCOBACTERIUM BOVIS BACILLUS CALMETTE-GUÉRIN (BCG)

You have accessJournal of UrologyBladder Cancer: Basic Research1 Apr 2011879 BLADDER EXPRESSION OF CYTOTOXIC EFFECTOR MOLECULES IN RESPONSE TO INTRAVESICAL MYCOBACTERIUM BOVIS BACILLUS CALMETTE-GUÉRIN (BCG) Matthew Knudson, Jonathan Henning, Michael O'Donnell, and Yi Luo Matthew KnudsonMatthew Knudson Iowa City, IA More articles by this author , Jonathan HenningJonathan Henning Iowa City, IA More articles by this author , Michael O'DonnellMichael O'Donnell Iowa City, IA More articles by this author , and Yi LuoYi Luo Iowa City, IA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.703AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder expression of cytokines and chemokines in response to intravesical (i.b.) BCG has been well documented. However, studies on BCG induction of bladder expression of cytotoxic effector molecules are underdeveloped. We investigated a panel of cytotoxic effector molecules, including IFN-γ, TNF-α, perforin, granzyme B, Fas ligand (FasL), and tumor necrosis factor related apoptosis-inducing ligand (TRAIL), in the BCG-treated bladders to determine whether expression of these molecules was correlated with the effect of BCG in treating bladder cancer. METHODS C57BL/6 female mice were treated i.b. with Pasteur strain BCG (0.1 OD/dose) twice weekly for a total of 7 treatments. Mice treated with PBS served as a control. Mice were sacrificed for bladder RNA extraction after treatments 2, 4, 6 and 7. RT-PCR was used to analyze mRNA levels of IFN-γ TNF-α, perforin, granzyme B, FasL and TRAIL. IP-10 (a Th1 chemokine) and TGF-β (an inhibitory cytokine) were included. GAPDH was used as an internal control. ImageJ software was used to quantify PCR products on agarose gels. In a parallel experiment C57BL/6 female mice were implanted i.b. with 5 X 105 syngeneic bladder cancer MB49 cells that expressed luciferase (MB49-Luc) at day 0 and treated i.b. with BCG (0.1 OD/dose) twice weekly starting at day 1 for a total of 6 treatments. Control mice were treated with PBS. Bioluminescence was measured once weekly with Xenogen IVIS. At day 23 mice were euthanized and bladder weights measured. Statistics was performed by one-way analysis of variance (ANOVA) of natural log transformed bladder weights followed by a post hoc Tukey's test. RESULTS I.b. BCG treatment led to an increase in bladder mRNAs of IFN-γ,TNF-α, IP-10 and perforin and a decrease in bladder mRNAs of TGF-β. There were no changes in bladder mRNAs of granzyme B, FasL and TRAIL. In MB49-Luc orthotopic tumor model, i.b. BCG treatment reduced bladder weight by 53% (p = 0.0503 vs. PBS-treated group). In addition, i.b. BCG treatment also led to a delay in tumor growth by bioluminescence compared to PBS-treated group. CONCLUSIONS Intravesical administration of BCG induced bladder expression of cytotoxic effector molecules IFN-γ, TNF-α and perforin, which was correlated with effective BCG treatment of bladder cancer. Increased expression of IP-10 and decreased expression of TGF-β, together with increased IFN-γ and TNF-α, support the role of BCG in the induction of Th1 immune responses in the bladders. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e352 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Matthew Knudson Iowa City, IA More articles by this author Jonathan Henning Iowa City, IA More articles by this author Michael O'Donnell Iowa City, IA More articles by this author Yi Luo Iowa City, IA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Class effects of tyrosine kinase inhibitors on osteoclastogenesis in vitro

Background/objectivesBone destruction in rheumatoid arthritis (RA) is mediated by osteoclasts (OCs). Imatinib, a tyrosine kinase inhibitor (TKI), has been shown to exert an inhibitory effect on osteoclastogenesis. The aim of...

Protein Kinase B Controls Transcriptional Programs that Direct Cytotoxic T Cell Fate but Is Dispensable for T Cell Metabolism

SummaryIn cytotoxic T cells (CTL), Akt, also known as protein kinase B, is activated by the T cell antigen receptor (TCR) and the cytokine interleukin 2 (IL-2). Akt can control cell metabolism in many cell types but whether this role is important for CTL function has not been determined. Here we have shown that Akt does not mediate IL-2- or TCR-induced cell metabolic responses; rather, this role is assumed by other Akt-related kinases. There is, however, a nonredundant role for sustained and strong activation of Akt in CTL to coordinate the TCR- and IL-2-induced transcriptional programs that control expression of key cytolytic effector molecules, adhesion molecules, and cytokine and chemokine receptors that distinguish effector versus memory and naive T cells. Akt is thus dispensable for metabolism, but the strength and duration of Akt activity dictates the CTL transcriptional program and determines CTL fate.

Evaluating the role of Toll-like receptors in diseases of the central nervous system

A key part of the innate immune system is a network of pattern recognition receptors (PRRs) and their associated intracellular signalling pathways. Toll-like receptors (TLRs) are one such group of PRRs that detect pathogen associated molecular patterns (PAMPs). Activation of the TLRs with their respective agonists results in the activation of intracellular signalling pathways leading to the expression of proinflammatory mediators and anti-microbial effector molecules. Activation of the innate immune system through TLRs also triggers the adaptive immune response, resulting in a comprehensive immune program to eradicate invading pathogens. It is now known that immune surveillance and inflammatory responses occur in the central nervous system (CNS). Furthermore it is becoming increasingly clear that TLRs have a role in such CNS responses and are also implicated in the pathogenesis of a number of conditions in the CNS, such as Alzheimer's, stroke and multiple sclerosis. This is likely due to the generation of endogenous TLR agonists in these conditions which amplifies a detrimental neurotoxic inflammatory response. However TLRs in some situations can be neuroprotective, if triggered in a favourable context. This review aims to examine the recent literature on TLRs in the CNS thus demonstrating their importance in a range of infectious and non-infectious diseases of the brain.