Altered immunity to microbiota, B cell activation and depleted γδ/resident memory T cells in colorectal cancer.

The role of microbiota:immune system dysregulation in the etiology of colorectal cancer (CRC) is poorly understood. CRC develops in gut epithelium, accompanied by low level inflammatory signaling, intestinal microbial dysbiosis and immune dysfunction. We examined populations of intraepithelial lymphocytes in non-affected colonic mucosa of CRC and healthy donors and circulating immune memory to commensal bacterial species and yeasts. γδ T cells and resident memory T cells, populations with a regulatory CD39-expressing phenotype, were found at lower frequencies in the colonic tissue of CRC donors compared to healthy controls. Patterns of T cell proliferative responses to a panel of commensal bacteria were distinct in CRC, while B cell memory responses to several bacteria/yeast were significantly increased, accompanied by increased proportions of effector memory B cells, transitional B cells and plasmablasts in blood. IgA responses to mucosal microbes were unchanged. Our data describe a novel immune signature with similarities to and differences from that of inflammatory bowel disease. They implicate B cell dysregulation as a potential contributor to parainflammation and identify pathways of weakened barrier function and tumor surveillance in CRC-susceptible individuals.

Altered Mucosal Immune-Microbiota Interactions in Familial Adenomatous Polyposis.

INTRODUCTION: Familial adenomatous polyposis (FAP) is a condition caused by a constitutional pathogenic variant of the adenomatous polyposis coli gene that results in intestinal adenoma formation and colorectal cancer, necessitating pre-emptive colectomy. We sought to examine interaction between the mucosal immune system and commensal bacteria in FAP to test for immune dysfunction that might accelerate tumorigenesis. METHODS: Colonic biopsies were obtained from macroscopically normal mucosal tissue from 14 healthy donors and 13 patients with FAP during endoscopy or from surgical specimens. Intraepithelial and lamina propria lymphocytes were phenotyped. Intraepithelial microbes were labeled with anti-IgA/IgG and analyzed by flow cytometry. RESULTS: Proportions of resident memory CD103-expressing CD8 + and γδ T-cell receptor + intraepithelial lymphocytes were dramatically reduced in both the left and right colon of patients with FAP compared with healthy controls. In lamina propria, T cells expressed less CD103, and CD4 + CD103 + cells expressed less CD73 ectonucleotidase. IgA coating of epithelia-associated bacteria, IgA + peripheral B cells, and CD4 T-cell memory responses to commensal bacteria were increased in FAP. DISCUSSION: Loss of resident memory T cells and γδ T cells in mucosal tissue of patients with FAP accompanies intestinal microbial dysbiosis previously reported in this precancerous state and suggests impaired cellular immunity and tumor surveillance. This may lead to barrier dysfunction, possible loss of regulatory T-cell function, and excess IgA antibody secretion. Our data are the first to implicate mucosal immune dysfunction as a contributing factor in this genetically driven disease and identify potentially critical pathways in the etiology of CRC.

In Vitro Priming of Human T Cells by Dendritic Cells Provides a Screening Tool for Candidate Vaccines for Burkholderia pseudomallei.

Murine dendritic cells, when pulsed with heat-killed Burkholderia pseudomallei and used to immunise naïve mice, have previously been shown to induce protective immunity in vivo. We have now demonstrated the in vitro priming of naïve human T cells against heat-killed B. pseudomallei, by co-culture with syngeneic B. pseudomallei-pulsed dendritic cells. Additionally, we have enriched the DC fraction such that a study of the differential response induced by pulsed DCs of either myeloid or plasmacytoid lineage in syngeneic human T cells was achievable. Whilst both mDCs and pDCs were activated by pulsing, the mDCs contributed the major response to B. pseudomallei with the expression of the migration marker CCR7 and a significantly greater secretion of the proinflammatory TNFα and IL1ß. When these DC factions were combined and used to prime syngeneic T cells, a significant proliferation was observed in the CD4+ fraction. Here, we have achieved human T cell priming in vitro with unadjuvanted B. pseudomallei, the causative organism of melioidosis, for which there is currently no approved vaccine. We propose that the approach we have taken could be used to screen for the human cellular response to candidate vaccines and formulations, in order to enhance the cell-mediated immunity required to protect against this intracellular pathogen and potentially more broadly against other, difficult-to-treat intracellular pathogens. To date, the polysaccharide capsule of B. pseudomallei, fused to a standard carrier protein, e.g., Crm, looks a likely vaccine candidate. Dendritic cells (DCs), providing, as they do, the first line of defence to infection, process and present microbial products to the immune system to direct downstream immune responses. Here, we have sought to use DCs ex vivo to identify immunogenic products from heat-killed B. pseudomallei. Using practical volumes of fresh human donor blood, we show that heat-killed B. pseudomallei activated and stimulated the expression of pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 from both myeloid and plasmacytoid DCs. Furthermore, B. pseudomallei-pulsed DCs cultured with naïve syngeneic T cells ex vivo, induced the activation and proliferation of the CD4+ T-cell population, which was identified by cell surface marker staining using flow cytometry. Thus, both DC subsets are important for driving primary T helper cell responses to B. pseudomallei in healthy individuals and have the potential to be used to identify immunogenic components of B. pseudomallei for future therapies and vaccines.

Bacteroides thetaiotaomicron-derived outer membrane vesicles promote regulatory dendritic cell responses in health but not in inflammatory bowel disease.

BACKGROUND: Bacteroides thetaiotaomicron (Bt) is a prominent member of the human intestinal microbiota that, like all gram-negative bacteria, naturally generates nanosized outer membrane vesicles (OMVs) which bud off from the cell surface. Importantly, OMVs can cross the intestinal epithelial barrier to mediate microbe-host cell crosstalk involving both epithelial and immune cells to help maintain intestinal homeostasis. Here, we have examined the interaction between Bt OMVs and blood or colonic mucosa-derived dendritic cells (DC) from healthy individuals and patients with Crohn's disease (CD) or ulcerative colitis (UC). RESULTS: In healthy individuals, Bt OMVs stimulated significant (p < 0.05) IL-10 expression by colonic DC, whereas in peripheral blood-derived DC they also stimulated significant (p < 0.001 and p < 0.01, respectively) expression of IL-6 and the activation marker CD80. Conversely, in UC Bt OMVs were unable to elicit IL-10 expression by colonic DC. There were also reduced numbers of CD103+ DC in the colon of both UC and CD patients compared to controls, supporting a loss of regulatory DC in both diseases. Furthermore, in CD and UC, Bt OMVs elicited a significantly lower proportion of DC which expressed IL-10 (p < 0.01 and p < 0.001, respectively) in blood compared to controls. These alterations in DC responses to Bt OMVs were seen in patients with inactive disease, and thus are indicative of intrinsic defects in immune responses to this commensal in inflammatory bowel disease (IBD). CONCLUSIONS: Overall, our findings suggest a key role for OMVs generated by the commensal gut bacterium Bt in directing a balanced immune response to constituents of the microbiota locally and systemically during health which is altered in IBD patients. Video Abstract.

Deficient Resident Memory T Cell and CD8 T Cell Response to Commensals in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: The intestinal microbiota is closely associated with resident memory lymphocytes in mucosal tissue. We sought to understand how acquired cellular and humoral immunity to the microbiota differ in health versus inflammatory bowel disease [IBD]. METHODS: Resident memory T cells [Trm] in colonic biopsies and local antibody responses to intraepithelial microbes were analysed. Systemic antigen-specific immune T and B cell memory to a panel of commensal microbes was assessed. RESULTS: Systemically, healthy blood showed CD4 and occasional CD8 memory T cell responses to selected intestinal bacteria, but few memory B cell responses. In IBD, CD8 memory T cell responses decreased although B cell responses and circulating plasmablasts increased. Possibly secondary to loss of systemic CD8 T cell responses in IBD, dramatically reduced numbers of mucosal CD8+ Trm and γδ T cells were observed. IgA responses to intraepithelial bacteria were increased. Colonic Trm expressed CD39 and CD73 ectonucleotidases, characteristic of regulatory T cells. Cytokines/factors required for Trm differentiation were identified, and in vitro-generated Trm expressed regulatory T cell function via CD39. Cognate interaction between T cells and dendritic cells induced T-bet expression in dendritic cells, a key mechanism in regulating cell-mediated mucosal responses. CONCLUSIONS: A previously unrecognised imbalance exists between cellular and humoral immunity to the microbiota in IBD, with loss of mucosal T cell-mediated barrier immunity and uncontrolled antibody responses. Regulatory function of Trm may explain their association with intestinal health. Promoting Trm and their interaction with dendritic cells, rather than immunosuppression, may reinforce tissue immunity, improve barrier function, and prevent B cell dysfunction in microbiota-associated disease and IBD aetiology.

Age-related alterations in blood and colonic dendritic cell properties.

BACKGROUND: Dendritic cells (DC) determine initiation, type and location of immune responses and, in adults, show decreased Toll-like receptors and some increased cytokine levels on ageing. Few studies in children have characterised DC or explored DC-related mechanisms producing age-related immune changes. RESULTS: The pDC marker BDCA2 (but not CD123) was absent in pre-pubertal children and numbers of pDC decreased with age. Blood and colonic DC were more mature and activated in adults. Decrease in pDC numbers correlated with reduced GM-CSF levels with aging, but increasing IL-4 and IL-8 levels correlated with a more activated DC profile in blood. CXCL16 levels decreased with age. METHODS: Blood and colonic DC phenotypes were determined in healthy adults and children by flow cytometry and correlated with aging. Blood DC were divided into plasmacytoid (pDC) and myeloid (mDC) while only mDC were identified in colon. Serum cytokine levels were determined by multiplex cytokine assays and correlated with DC properties. CONCLUSIONS: In children, lack of BDCA2, a receptor mediating antigen capture and inhibiting interferon induction, may be immunologically beneficial during immune development. Conversely, reduced pDC numbers, probably secondary to decreasing GM-CSF and increasing cytokine-induced maturation of DC are likely to determine deteriorating immunity with ageing.

Chemokine (C-C Motif) Receptor 2 Mediates Dendritic Cell Recruitment to the Human Colon but Is Not Responsible for Differences Observed in Dendritic Cell Subsets, Phenotype, and Function Between the Proximal and Distal Colon.

BACKGROUND & AIMS: Most knowledge about gastrointestinal (GI)-tract dendritic cells (DC) relies on murine studies where CD103+ DC specialize in generating immune tolerance with the functionality of CD11b+/- subsets being unclear. Information about human GI-DC is scarce, especially regarding regional specifications. Here, we characterized human DC properties throughout the human colon. METHODS: Paired proximal (right/ascending) and distal (left/descending) human colonic biopsies from 95 healthy subjects were taken; DC were assessed by flow cytometry and microbiota composition assessed by 16S rRNA gene sequencing. RESULTS: Colonic DC identified were myeloid (mDC, CD11c+CD123-) and further divided based on CD103 and SIRPα (human analog of murine CD11b) expression. CD103-SIRPα+ DC were the major population and with CD103+SIRPα+ DC were CD1c+ILT3+CCR2+ (although CCR2 was not expressed on all CD103+SIRPα+ DC). CD103+SIRPα- DC constituted a minor subset that were CD141+ILT3-CCR2-. Proximal colon samples had higher total DC counts and fewer CD103+SIRPα+ cells. Proximal colon DC were more mature than distal DC with higher stimulatory capacity for CD4+CD45RA+ T-cells. However, DC and DC-invoked T-cell expression of mucosal homing markers (ß7, CCR9) was lower for proximal DC. CCR2 was expressed on circulating CD1c+, but not CD141+ mDC, and mediated DC recruitment by colonic culture supernatants in transwell assays. Proximal colon DC produced higher levels of cytokines. Mucosal microbiota profiling showed a lower microbiota load in the proximal colon, but with no differences in microbiota composition between compartments. CONCLUSIONS: Proximal colonic DC subsets differ from those in distal colon and are more mature. Targeted immunotherapy using DC in T-cell mediated GI tract inflammation may therefore need to reflect this immune compartmentalization.

T cell responses are elicited against Respiratory Syncytial Virus in the absence of signalling through TLRs, RLRs and IL-1R/IL-18R.

Pattern recognition receptors (PRRs) and cytokine receptors are key players in the initiation of immune responses to infection. PRRs detecting viral RNA, such as toll like receptor (TLR)-3, -7/8, and RIG-I like receptors (RLRs; RIG-I and MDA-5), as well as cytokine receptors such as interleukin 1 receptor (IL-1R), have been implicated in responses to RNA viruses that infect the airways. The latter includes respiratory syncytial virus (RSV), a human pathogen that can cause severe lower respiratory tract infections, especially in infants. To evaluate the collective contribution of PRRs and IL-1R signalling to RSV immunity, we generated Myd88/Trif/Mavs(-/-) mice that are deficient in signalling by all TLRs, RLRs and IL-1R, as well as other cytokine receptors such as IL-18 receptor. Early production of pro-inflammatory mediators and lung infiltration by immune cells were completely abrogated in infected Myd88/Trif/Mavs(-/-) mice. However, RSV-specific CD8(+) T cells were elicited and recruited into the lungs and airways. Consistent with these findings, Myd88/Trif/Mavs(-/-) mice survived RSV infection but displayed higher viral load and weight loss. These data highlight an unappreciated level of redundancy in pathways that couple innate virus sensing to adaptive immunity, providing the host with remarkable resilience to infection.

Alveolar macrophage-derived type I interferons orchestrate innate immunity to RSV through recruitment of antiviral monocytes.

Type I interferons (IFNs) are important for host defense from viral infections, acting to restrict viral production in infected cells and to promote antiviral immune responses. However, the type I IFN system has also been associated with severe lung inflammatory disease in response to respiratory syncytial virus (RSV). Which cells produce type I IFNs upon RSV infection and how this directs immune responses to the virus, and potentially results in pathological inflammation, is unclear. Here, we show that alveolar macrophages (AMs) are the major source of type I IFNs upon RSV infection in mice. AMs detect RSV via mitochondrial antiviral signaling protein (MAVS)-coupled retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs), and loss of MAVS greatly compromises innate immune restriction of RSV. This is largely attributable to loss of type I IFN-dependent induction of monocyte chemoattractants and subsequent reduced recruitment of inflammatory monocytes (infMo) to the lungs. Notably, the latter have potent antiviral activity and are essential to control infection and lessen disease severity. Thus, infMo recruitment constitutes an important and hitherto underappreciated, cell-extrinsic mechanism of type I IFN-mediated antiviral activity. Dysregulation of this system of host antiviral defense may underlie the development of RSV-induced severe lung inflammation.

DNGR-1 is dispensable for CD8+ T-cell priming during respiratory syncytial virus infection.

During respiratory syncytial virus (RSV) infection CD8(+) T cells both assist in viral clearance and contribute to immunopathology. CD8(+) T cells recognize viral peptides presented by dendritic cells (DCs), which can directly present viral antigens when infected or, alternatively, "cross-present" antigens after endocytosis of dead or dying infected cells. Mouse CD8α(+) and CD103(+) DCs excel at cross-presentation, in part because they express the receptor DNGR-1 that detects dead cells by binding to exposed F-actin and routes internalized cell debris into the cross-presentation pathway. As RSV causes death in infected epithelial cells, we tested whether cross-presentation via DNGR-1 is necessary for CD8(+) T-cell responses to the virus. DNGR-1-deficient or wild-type mice were intranasally inoculated with RSV and the magnitude of RSV-specific CD8(+) T-cell induction was measured. We found that during live RSV infection, cross-presentation via DNGR-1 did not have a major role in the generation of RSV-specific CD8(+) T-cell responses. However, after intranasal immunization with dead cells infected with RSV, a dependence on DNGR-1 for RSV-specific CD8(+) T-cell responses was observed, confirming the ascribed role of the receptor. Thus, direct presentation by DCs may be the major pathway initiating CD8(+) T-cell responses to RSV, while DNGR-1-dependent cross-presentation has no detectable role.

Alpha/beta interferon receptor signaling amplifies early proinflammatory cytokine production in the lung during respiratory syncytial virus infection.

UNLABELLED: Type I interferons (IFNs) are produced early upon virus infection and signal through the alpha/beta interferon (IFN-α/ß) receptor (IFNAR) to induce genes that encode proteins important for limiting viral replication and directing immune responses. To investigate the extent to which type I IFNs play a role in the local regulation of inflammation in the airways, we examined their importance in early lung responses to infection with respiratory syncytial virus (RSV). IFNAR1-deficient (IFNAR1(-/-)) mice displayed increased lung viral load and weight loss during RSV infection. As expected, expression of IFN-inducible genes was markedly reduced in the lungs of IFNAR1(-/-) mice. Surprisingly, we found that the levels of proinflammatory cytokines and chemokines in the lungs of RSV-infected mice were also greatly reduced in the absence of IFNAR signaling. Furthermore, low levels of proinflammatory cytokines were also detected in the lungs of IFNAR1(-/-) mice challenged with noninfectious innate immune stimuli such as selected Toll-like receptor (TLR) agonists. Finally, recombinant IFN-α was sufficient to potentiate the production of inflammatory mediators in the lungs of wild-type mice challenged with innate immune stimuli. Thus, in addition to its well-known role in antiviral resistance, type I IFN receptor signaling acts as a central driver of early proinflammatory responses in the lung. Inhibiting the effects of type I IFNs may therefore be useful in dampening inflammation in lung diseases characterized by enhanced inflammatory cytokine production. IMPORTANCE: The initial response to viral infection is characterized by the production of interferons (IFNs). One group of IFNs, the type I IFNs, are produced early upon virus infection and signal through the IFN-α/ß receptor (IFNAR) to induce proteins important for limiting viral replication and directing immune responses. Here we examined the importance of type I IFNs in early responses to respiratory syncytial virus (RSV). Our data suggest that type I IFN production and IFNAR receptor signaling not only induce an antiviral state but also serve to amplify proinflammatory responses in the respiratory tract. We also confirm this conclusion in another model of acute inflammation induced by noninfectious stimuli. Our findings are of relevance to human disease, as RSV is a major cause of infant bronchiolitis and polymorphisms in the IFN system are known to impact disease severity.

Regulatory T cells prevent Th2 immune responses and pulmonary eosinophilia during respiratory syncytial virus infection in mice.

During viral infection, inflammation and recovery are tightly controlled by competing proinflammatory and regulatory immune pathways. Respiratory syncytial virus (RSV) is the leading global cause of infantile bronchiolitis, which is associated with recurrent wheeze and asthma diagnosis in later life. Th2-driven disease has been well described under some conditions for RSV-infected mice. In the present studies, we used the Foxp3(DTR) mice (which allow specific conditional depletion of Foxp3(+) T cells) to investigate the functional effects of regulatory T cells (Tregs) during A2-strain RSV infection. Infected Treg-depleted mice lost significantly more weight than wild-type mice, indicating enhanced disease. This enhancement was characterized by increased cellularity in the bronchoalveolar lavage (BAL) fluid and notable lung eosinophilia not seen in control mice. This was accompanied by abundant CD4(+) and CD8(+) T cells exhibiting an activated phenotype and induction of interleukin 13 (IL-13)- and GATA3-expressing Th2-type CD4(+) T cells that remained present in the airways even 14 days after infection. Therefore, Treg cells perform vital anti-inflammatory functions during RSV infection, suppressing pathogenic T cell responses and inhibiting lung eosinophilia. These findings provide additional evidence that dysregulation of normal immune responses to viral infection may contribute to severe RSV disease.

Defective immunoregulation in RSV vaccine-augmented viral lung disease restored by selective chemoattraction of regulatory T cells.

Human trials of formaldehyde-inactivated respiratory syncytial virus (FI-RSV) vaccine in 1966-1967 caused disastrous worsening of disease and death in infants during subsequent natural respiratory syncytial virus (RSV) infection. The reasons behind vaccine-induced augmentation are only partially understood, and fear of augmentation continues to hold back vaccine development. We now show that mice vaccinated with FI-RSV show enhanced local recruitment of conventional CD4(+) T cells accompanied by a profound loss of regulatory T cells (Tregs) in the airways. This loss of Tregs was so complete that additional depletion of Tregs (in transgenic depletion of regulatory T-cell mice) produced no additional disease enhancement. Transfer of conventional CD4(+) T cells from FI-RSV-vaccinated mice into naive RSV-infected recipients also caused a reduction in airway Treg responses; boosting Tregs with IL-2 immune complexes failed to restore normal levels of Tregs or to ameliorate disease. However, delivery of chemokine ligands (CCL) 17/22 via the airway selectively recruited airway Tregs and attenuated vaccine-augmented disease, reducing weight loss and inhibiting local recruitment of pathogenic CD4(+) T cells. These findings reveal an unexpected mechanism of vaccine-induced disease augmentation and indicate that selective chemoattraction of Tregs into diseased sites may offer a novel approach to the modulation of tissue-specific inflammation.

IL-10 regulates viral lung immunopathology during acute respiratory syncytial virus infection in mice.

Interleukin (IL-) 10 is a pleiotropic cytokine with broad immunosuppressive functions, particularly at mucosal sites such as the intestine and lung. Here we demonstrate that infection of BALB/c mice with respiratory syncytial virus (RSV) induced IL-10 production by CD4(+) and CD8(+) T cells in the airways at later time points (e.g. day 8); a proportion of these cells also co-produced IFN-γ. Furthermore, RSV infection of IL-10(-/-) mice resulted in more severe disease with enhanced weight loss, delayed recovery and greater cell infiltration of the respiratory tract without affecting viral load. In addition, IL-10(-/-) mice had a pronounced airway neutrophilia and heightened levels of pro-inflammatory cytokines and chemokines in the bronchoalveolar lavage fluid. Notably, the proportion of lung T cells producing IFN-γ was enhanced, suggesting that IL-10 may act in an autocrine manner to dampen effector T cell responses. Similar findings were made in mice treated with anti-IL-10R antibody and infected with RSV. Therefore, IL-10 inhibits disease and inflammation in mice infected with RSV, especially during recovery from infection.

Diverse targets of the transcription factor STAT3 contribute to T cell pathogenicity and homeostasis.

STAT3, an essential transcription factor with pleiotropic functions, plays critical roles in the pathogenesis of autoimmunity. Despite recent data linking STAT3 with inflammatory bowel disease, exactly how it contributes to chronic intestinal inflammation is not known. Using a T cell transfer model of colitis, we found that STAT3 expression in T cells was essential for the induction of both colitis and systemic inflammation. STAT3 was critical in modulating the balance of T helper 17 (Th17) and regulatory T (Treg) cells, as well as in promoting CD4(+) T cell proliferation. We used chromatin immunoprecipitation and massive parallel sequencing (ChIP-Seq) to define the genome-wide targets of STAT3 in CD4(+) T cells. We found that STAT3 bound to multiple genes involved in Th17 cell differentiation, cell activation, proliferation, and survival, regulating both expression and epigenetic modifications. Thus, STAT3 orchestrates multiple critical aspects of T cell function in inflammation and homeostasis.

Tpl2 kinase regulates T cell interferon-gamma production and host resistance to Toxoplasma gondii.

Tpl2 (Tumor progression locus 2), also known as Cot/MAP3K8, is a hematopoietically expressed serine-threonine kinase. Tpl2 is known to have critical functions in innate immunity in regulating tumor necrosis factor-alpha, Toll-like receptor, and G protein-coupled receptor signaling; however, our understanding of its physiological role in T cells is limited. We investigated the potential roles of Tpl2 in T cells and found that it was induced by interleukin-12 in human and mouse T cells in a Stat4-dependent manner. Deficiency of Tpl2 was associated with impaired interferon (IFN)-gamma production. Accordingly, Tpl2(-/-) mice had impaired host defense against Toxoplasma gondii with reduced parasite clearance and decreased IFN-gamma production. Furthermore, reconstitution of Rag2(-/-) mice with Tpl2-deficient T cells followed by T. gondii infection recapitulated the IFN-gamma defect seen in the Tpl2-deficient mice, confirming a T cell-intrinsic defect. CD4(+) T cells isolated from Tpl2(-/-) mice showed poor induction of T-bet and failure to up-regulate Stat4 protein, which is associated with impaired TCR-dependent extracellular signal-regulated kinase activation. These data underscore the role of Tpl2 as a regulator of T helper cell lineage decisions and demonstrate that Tpl2 has an important functional role in the regulation of Th1 responses.

Nonredundant roles for Stat5a/b in directly regulating Foxp3.

Stats (signal transducers and activators of transcription) regulate multiple aspects of T-cell fate. T regulatory (Treg) cells are a critical subset that limits immune responses, but the relative importance of Stat5a/b versus Stat3 for Treg cell development has been contentious. We observed that peripheral CD25(+)CD4(+) T cells were reduced in Stat5(DeltaN) mice; however, the levels of Foxp3, a transcription factor that is critical for Treg cells, were normal in splenic CD4(+) T cells even though they were reduced in the thymus. In contrast, complete deletion of Stat5a/b (Stat5(-/-)) resulted in dramatic reduction in CD25- or Foxp3-expressing CD4(+) T cells. An intrinsic requirement was demonstrated by reduction of Stat5a/b in CD4-expressing cells and by stem cell transplantation using Stat5(-/-) fetal liver cells. Stat5a/b were also required for optimal induction of Foxp3 in vitro and bound directly to the Foxp3 gene. Reduction of Stat3 in T cells did not reduce the numbers of Treg cells in the thymus or spleen; however, Stat3 was required for IL-6-dependent down-regulation of Foxp3. Therefore, we conclude that Stat5a/b have an essential, nonredundant role in regulating Treg cells, and that Stat3 and Stat5a/b appear to have opposing roles in the regulation of Foxp3.

Role of glucocorticoid receptor in acclimation of killifish (Fundulus heteroclitus) to seawater and effects of arsenic.

Killifish are euryhaline teleosts that adapt to rapid changes in the salinity of the seawater. It is generally accepted that acclimation to seawater is mediated by cortisol activation of the glucocorticoid receptor (GR), which stimulates CFTR mRNA expression and CFTR-mediated Cl- secretion by the gill. Because there is no direct evidence in killifish that the GR stimulates CFTR gene expression, quantitative PCR studies were conducted to test the hypothesis that cortisol activation of GR upregulates CFTR mRNA expression and that this response is required for acclimation to seawater. Inhibition of the GR by RU-486 prevented killifish from acclimating to increased salinity and blocked the increase in CFTR mRNA. In contrast, inhibition of the mineralocorticoid receptor by spironolactone had no effect on acclimation to seawater. Thus acclimation to increased salinity in killifish requires signaling via the GR and includes an increase in CFTR gene expression. Because arsenic, a toxic metalloid that naturally occurs in the aquatic environment, has been shown to disrupt GR transcriptional regulation in avian and mammalian systems, studies were also conducted to determine whether arsenic disrupts cortisol-mediated activation of CFTR gene expression in this in vivo fish model and thereby blocks the ability of killifish to acclimate to increased salinity. Arsenic prevented acclimation to seawater and decreased CFTR protein abundance. However, arsenic did not disrupt the GR-induced increase in CFTR mRNA. Thus arsenic blocks acclimation to seawater in killifish by a mechanism that does not disrupt GR-mediated induction of CFTR gene expression.

Cytohesin binder and regulator (cybr) is not essential for T- and dendritic-cell activation and differentiation.

Cybr (also known as Cytip, CASP, and PSCDBP) is an interleukin-12-induced gene expressed exclusively in hematopoietic cells and tissues that associates with Arf guanine nucleotide exchange factors known as cytohesins. Cybr levels are dynamically regulated during T-cell development in the thymus and upon activation of peripheral T cells. In addition, Cybr is induced in activated dendritic cells and has been reported to regulate dendritic cell (DC)-T-cell adhesion. Here we report the generation and characterization of Cybr-deficient mice. Despite the selective expression in hematopoietic cells, there was no intrinsic defect in T- or B-cell development or function in Cybr-deficient mice. The adoptive transfer of Cybr-deficient DCs showed that they migrated efficiently and stimulated proliferation and cytokine production by T cells in vivo. However, competitive stem cell repopulation experiments showed a defect in the abilities of Cybr-deficient T cells to develop in the presence of wild-type precursors. These data suggest that Cybr is not absolutely required for hematopoietic cell development or function, but stem cells lacking Cybr are at a developmental disadvantage compared to wild-type cells. Collectively, these data demonstrate that despite its selective expression in hematopoietic cells, the role of Cybr is limited or largely redundant. Previous in vitro studies using overexpression or short interfering RNA inhibition of the levels of Cybr protein appear to have overestimated its immunological role.