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The tumor microenvironment in leukemia and solid tumors induces a shift of activated CD8+ cytotoxic T cells to an exhausted state, characterized by loss of proliferative capacity and impaired immunologic synapse formation. Efficient strategies and targets need to be identified to overcome T-cell exhaustion and further improve overall responses in the clinic. Here, we took advantage of the Eμ-TCL1 chronic lymphocytic leukemia (CLL) and B16 melanoma mouse models to assess the role of the homophilic cell-surface receptor SLAMF6 as an immune-checkpoint regulator. The transfer of SLAMF6+ Eμ-TCL1 cells into SLAMF6−/− recipients, in contrast to wild-type (WT) recipients, significantly induced expansion of a PD-1+ subpopulation among CD3+CD44+CD8+ T cells, which had impaired cytotoxic functions. Conversely, administering anti-SLAMF6 significantly reduced the leukemic burden in Eμ-TCL1 recipient WT mice concomitantly with a loss of PD-1+CD3+CD44+CD8+ T cells with significantly increased effector functions. Anti-SLAMF6 significantly reduced leukemic burden in the peritoneal cavity, a niche where antibody-dependent cellular cytotoxicity (ADCC) is impaired, possibly through activation of CD8+ T cells. Targeting of SLAMF6 affected tumor growth not only in B cell–related leukemia and lymphomas but also in nonhematopoietic tumors such as B16 melanoma, where SLAMF6 is not expressed. In vitro exhausted CD8+ T cells showed increased degranulation when anti-human SLAMF6 was added in culture. Taken together, anti-SLAMF6 both effectively corrected CD8+ T-cell dysfunction and had a direct effect on tumor progression. The outcomes of our studies suggest that targeting SLAMF6 is a potential therapeutic strategy.

Background: The World Health Organization recommends that rotavirus vaccines should be included in all national immunization programs. Some currently licensed oral rotavirus vaccines contain a buffering agent (either as part of a ready-to-use liquid formulation or added during reconstitution) to reduce possible degradation of the vaccine virus in the infant gut, which poses several programmatic challenges (the large dose volume or the reconstitution requirement) during vaccine administration. Because ROTAVAC®, a WHO prequalified vaccine, was derived from the 116E neonatal strain, we evaluated the immunogenicity and safety of ROTAVAC® without buffer and ROTAVAC® with buffer in a phase 4, multicentre, single-blind, randomized clinical trial in healthy infants in India. Methods: 900 infants, approximately 6, 10 and 14 weeks of age, were assigned to 3 groups to receive ROTAVAC® (0.5 mL dose) orally: (i) 2.5 mL of citrate-bicarbonate buffer 5 minutes prior to administration of ROTAVAC® (Group I), (ii) ROTAVAC®, alone, without any buffer (Group II), or (iii) ROTAVAC®, mixed with buffer immediately before administration (Group III). Non–inferiority was compared among the groups for differences in serological responses (detected by serum anti-rotavirus IgA) and safety. Results: Geometric mean titers post vaccination at day 84 (28 days after dose 3) were 19.6 (95%CI: 17.0, 22.7), 20.7 (95%CI: 17.9, 24) and 19.2 (95%CI: 16.8, 22.1) for groups I, II and III respectively. Further, seroconversion rates and distribution of adverse events were similar among groups. Conclusions: Administration of ROTAVAC® at a 0.5 mL dose volume without buffering agent was shown to be well tolerated and immunogenic. Given the homologous nature of the strain, it is plausible that ROTAVAC® replicates well and confers immunity even without buffer administration.

Aim The aim of this study was to determine the immunologic effects and safety of oral anti-CD3 in patients with ulcerative colitis (UC). Methods An open-label pilot study of orally delivered anti-CD3 was performed in patients with moderate-to-severe UC. The primary end points were changes in immunologic parameters and evaluation for safety. Results Six subjects received oral OKT3. Biologic effects of oral anti-CD3 included significantly increased proliferation in response to anti-CD3 and anti-inflammatory gene expression profile in peripheral blood mononuclear cells. No serious treatment-related adverse events occurred. Conclusion Orally delivered anti-CD3 resulted in immunologic changes in patients with UC., There remains an unmet need for drugs given as pills that can treat the inflammation of ulcerative colitis (UC). In addition to developing new drugs, a parallel approach is to evaluate existing drugs that have been FDA approved for other diseases (repurposing). Muromunab (OKT3) is a drug that was approved in 1985 to treat acute rejection after organ transplantation. It works by binding to CD3, a protein on some immune cells, and then prevents those cells from causing inflammation. In this pilot study, the researchers gave OKT3 to patients with active UC to assess whether it would suppress the inflammation in their colon. They also measured side effects and the impact of OKT3 on their immune cells. After 3 weeks, 3 of 6 patients had improved symptoms, and 1 of 6 had improvement in the inflammation in their colon. One mild, and one moderate, side effect were recorded. At the cell level, OKT3 led to modest changes in cell proliferation and expression of genes associated with inflammation. This small study suggests that local blockade of CD3 could be a target for treatment in UC.

Rashidian et al. show that 89Zr-PEGylated single-domain antibodies that target CD8+ T cells can be used to monitor and evaluate the response to immunotherapy as a predictive tool., Immunotherapy using checkpoint-blocking antibodies against targets such as CTLA-4 and PD-1 can cure melanoma and non–small cell lung cancer in a subset of patients. The presence of CD8 T cells in the tumor correlates with improved survival. We show that immuno–positron emission tomography (immuno-PET) can visualize tumors by detecting infiltrating lymphocytes and, through longitudinal observation of individual animals, distinguish responding tumors from those that do not respond to therapy. We used 89Zr-labeled PEGylated single-domain antibody fragments (VHHs) specific for CD8 to track the presence of intratumoral CD8+ T cells in the immunotherapy-susceptible B16 melanoma model in response to checkpoint blockade. A 89Zr-labeled PEGylated anti-CD8 VHH detected thymus and secondary lymphoid structures as well as intratumoral CD8 T cells. Animals that responded to CTLA-4 therapy showed a homogeneous distribution of the anti-CD8 PET signal throughout the tumor, whereas more heterogeneous infiltration of CD8 T cells correlated with faster tumor growth and worse responses. To support the validity of these observations, we used two different transplantable breast cancer models, yielding results that conformed with predictions based on the antimelanoma response. It may thus be possible to use immuno-PET and monitor antitumor immune responses as a prognostic tool to predict patient responses to checkpoint therapies.

Summary - Significant insights into disease pathogenesis have been gleaned from population-level genetic studies; however, many loci associated with complex genetic disease contain numerous genes, and phenotypic associations cannot be assigned unequivocally. In particular, a gene-dense locus on chromosome 11 (61.5–61.65 Mb) has been associated with inflammatory bowel disease, rheumatoid arthritis, and coronary artery disease. Here, we identify TMEM258 within this locus as a central regulator of intestinal inflammation. Strikingly, Tmem258 haploinsufficient mice exhibit severe intestinal inflammation in a model of colitis. At the mechanistic level, we demonstrate that TMEM258 is a required component of the oligosaccharyltransferase complex and is essential for N-linked protein glycosylation. Consequently, homozygous deficiency of Tmem258 in colonic organoids results in unresolved endoplasmic reticulum (ER) stress culminating in apoptosis. Collectively, our results demonstrate that TMEM258 is a central mediator of ER quality control and intestinal homeostasis., Leona M. and Harry B. Helmsley Charitable Trust (2014PG-IBD016), Crohn's and Colitis Foundation of America, National Institutes of Health (U.S.) (grant DK043351), National Institutes of Health (U.S.) (grant DK097485)

// Burcu Yigit 1 , Peter J. Halibozek 1 , Shih-Shih Chen 2 , Michael S. O’Keeffe 1 , Jon Arnason 3 , David Avigan 3 , Valter Gattei 4 , Atul Bhan 5 , Osman Cen 6 , Richard Longnecker 6 , Nicholas Chiorazzi 2 , Ninghai Wang 1 , Pablo Engel 7 , Cox Terhorst 1 1 Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 2 Karches Center for Chronic Lymphocytic Leukemia Research, The Feinstein Institute for Medical Research, Manhasset, NY, USA 3 Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 4 Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, I.R.C.C.S., Aviano, Italy 5 Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 6 Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 7 Immunology Unit, Department of Cell Biology, Immunology and Neurosciences, Medical School, University of Barcelona, Barcelona, Spain Correspondence to: Burcu Yigit, email: byigit@bidmc.harvard.edu Cox Terhorst, email: cterhors@bidmc.harvard.edu Keywords: CLL, TCL1-192, SLAMF6, BCR, ibrutinib Received: January 29, 2016 Accepted: March 07, 2016 Published: March 25, 2016 ABSTRACT The signaling lymphocyte activation molecule family [SLAMF] of cell surface receptors partakes in both the development of several immunocyte lineages and innate and adaptive immune responses in humans and mice. For instance, the homophilic molecule SLAMF6 (CD352) is in part involved in natural killer T cell development, but also modulates T follicular helper cell and germinal B cell interactions. Here we report that upon transplantation of a well-defined aggressive murine B220 + CD5 + Chronic Lymphocytic Leukemia (CLL) cell clone, TCL1-192, into SCID mice one injection of a monoclonal antibody directed against SLAMF6 (αSlamf6) abrogates tumor progression in the spleen, bone marrow and blood. Similarly, progression of a murine B cell lymphoma, LMP2A/λMyc, was also eliminated by αSlamf6. But, surprisingly, αSLAMF6 neither eliminated TCL1-192 nor LMP2A/λMyc cells, which resided in the peritoneal cavity or omentum. This appeared to be dependent upon the tumor environment, which affected the frequency of sub-populations of the TCL1-192 clone or the inability of peritoneal macrophages to induce Antibody Dependent Cellular Cytotoxicity (ADCC). However, co-administering αSlamf6 with the Bruton tyrosine kinase (Btk) inhibitor, ibrutinib, synergized to efficiently eliminate the tumor cells in the spleen, bone marrow, liver and the peritoneal cavity. Because an anti-human SLAMF6 mAb efficiently killed human CLL cells in vitro and in vivo , we propose that a combination of αSlamf6 with ibrutinib should be considered as a novel therapeutic approach for CLL and other B cell tumors.

A phase III randomized double-blind placebo-controlled trial was conducted in the urban neighborhoods of Delhi to assess whether Oral Rotavirus Vaccine ROTAVAC® interferes with the immune response to childhood vaccines when coadministered. Infants aged 6 weeks were randomized to receive three doses of either ROTAVAC® or placebo along with childhood vaccines: Oral Polio Vaccine and vaccines against Diphtheria, Pertussis, Tetanus, Hepatitis B and Haemophilus influenza type b given as Pentavalent at 6, 10, 14 weeks of age. Blood specimens were collected from all infants at baseline and 4 weeks post dose 3 to assess the immune response to antigens in Oral Polio Vaccine, Pentavalent and ROTAVAC® vaccines. Non-inferiority of immune response to all vaccine components of the childhood vaccines when ROTAVAC® was administered concurrently was demonstrated. Non-inferior immune responses to childhood vaccines were evaluated based on the seroprotective levels of antibodies against polio types 1, 2, and 3, Diphtheria toxoid, Tetanus toxoid, Haemophilus influenza type b anti- polyribosyl ribitol phosphate antibodies and Hepatitis B antibodies; and the Geometric Mean Concentration for Pertussis. The proportion of infants who seroconverted (≥4 fold rise) was 38.6% in the ROTAVAC® group and 12.2% in the placebo group. The frequency and severity of immediate adverse events, adverse events and serious adverse events were similar in both groups. None of the five reported deaths were considered to be related to the ROTAVAC® and no case of intussusception meeting Brighton Diagnostic Certainty Level I criteria was reported. This study demonstrated that ROTAVAC® can be safely administered with childhood vaccines without interfering with the immune response to the antigens contained in these vaccines.

Intact interleukin-10 receptor (IL-10R) signaling on effector and T regulatory (Treg) cells are each independently required to maintain immune tolerance. Here we show that IL-10 sensing by innate immune cells, independent of its effects on T cells, was critical for regulating mucosal homeostasis. Following wild-type (WT) CD4(+) T cell transfer, Rag2(-/-)Il10rb(-/-) mice developed severe colitis in association with profound defects in generation and function of Treg cells. Moreover, loss of IL-10R signaling impaired the generation and function of anti-inflammatory intestinal and bone-marrow-derived macrophages and their ability to secrete IL-10. Importantly, transfer of WT but not Il10rb(-/-) anti-inflammatory macrophages ameliorated colitis induction by WT CD4(+) T cells in Rag2(-/-)Il10rb(-/-) mice. Similar alterations in the generation and function of anti-inflammatory macrophages were observed in IL-10R-deficient patients with very early onset inflammatory bowel disease. Collectively, our studies define innate immune IL-10R signaling as a key factor regulating mucosal immune homeostasis in mice and humans.

Tumour necrosis factor (TNF)-α secreted by macrophages and dendritic cells (DCs) plays a predominant role in arthritis. Our previous studies suggest that a small peptide, RVG-9R (29-aa peptide derived from the rabies virus glycoprotein, fused to 9R residues), can deliver small interfering RNA (siRNA) to macrophages and DCs. We therefore tested whether knockdown of TNF-α expression in macrophages and DCs by RVG-9R/bound siRNA targeting TNF-α reduces the severity of collagen antibody-induced arthritis (CAIA) in mice.Arthritis was induced in mice by injecting a combination of antibodies to collagen followed by lipopolysaccharide (LPS) treatment. Mice were also injected with TNF-α siRNA complexed with RVG-9R peptide or an irrelevant peptide RVMAT-9R on days 1, 3, 5, and 7. As a positive control, dexamethasone was injected intravenously. Paw thickness was measured every 2 days and the mice were killed on day 10 for testing synovial TNF-α levels and histological analysis of joints.In control mice, arthritis developed on day 4 and reached its peak between day 7 and day 9. Treatment with siTNF-α bound to RVG-9R, but not to RVMAT-9R, resulted in reducing paw thickness scores to the same level as dexamethasone treatment, associated with reduced TNF-α level in synovial fluid. Histological analysis of joints in the control RVMAT-9R/TNF-α siRNA-treated mice showed marked pannus formation and destruction of cartilage and subchondrial bone, as well as severe infiltration of inflammatory cells into the synovium. By contrast, the joint pathology was markedly reduced in RVG-9R/TNF-α siRNA-treated mice resembling the dexamethasone-treated mice.Suppression of TNF-α expression in macrophages and DCs by RVG-9R-mediated siRNA delivery could potentially be a clinically viable strategy for treatment of arthritis.

B cells are an important component of mucosal immune responses in normal and diseased states. Whether B cells play a regulatory or pathogenic role in inflammatory bowel disease (IBD) has not been determined. The pathogenic role of antibodies has been suggested by the frequent presence of circulating antibodies to self- and microbial antigens in IBD. Antibodies could also regulate inflammation through Fc receptors of IgG. Although most studies performed in the experimental models of IBD suggest that B cells suppress mucosal inflammation either by secreting cytokines or by interacting with T cells, a pathogenic role of B cells has been raised in a model of ileitis. An inducible B-cell subset, termed Breg, develops in gut-associated lymphoid tissues under intestinal inflammatory conditions and causes attenuation of colitis through production of a regulatory cytokine IL-10 and by regulating T-cell expansion. An IL-12p70 producing B-cell subset capable of inhibiting colitis has also been identified. Future human studies including functional characterization of mucosal lymphocytes in normal and diseased states are needed to determine whether B cells regulate human intestinal inflammation as shown in the experimental models.

Summary Circulatory antigens transit through the small intestine via the fenestrated capillaries in the lamina propria prior to entering into the draining lymphatics. But whether or how this process controls mucosal immune responses remains unknown. Here we demonstrate that dendritic cells (DCs) of the lamina propria can sample and process both circulatory and luminal antigens. Surprisingly, antigen cross-presentation by resident CX3CR1 + DCs induced differentiation of precursor cells into CD8 + T cells that expressed interleukin-10 (IL-10), IL-13, and IL-9 and could migrate into adjacent compartments. We conclude that lamina propria CX3CR1 + DCs facilitate the surveillance of circulatory antigens and act as a conduit for the processing of self- and intestinally absorbed antigens, leading to the induction of CD8 + T cells, that partake in the control of T cell activation during mucosal immune responses.

Bacterially derived factors from the gut play a major role in the activation of inflammatory pathways in the liver and in the pathogenesis of alcoholic liver disease. The intestinal brush-border enzyme intestinal alkaline phosphatase (IAP) detoxifies a variety of bacterial pro-inflammatory factors and also functions to preserve gut barrier function. The aim of this study was to investigate whether oral IAP supplementation could protect against alcohol-induced liver disease. Mice underwent acute binge or chronic ethanol exposure to induce alcoholic liver injury and steatosis ± IAP supplementation. Liver tissue was assessed for biochemical, inflammatory, and histopathological changes. An ex vivo co-culture system was used to examine the effects of alcohol and IAP treatment in regard to the activation of hepatic stellate cells and their role in the development of alcoholic liver disease. Pretreatment with IAP resulted in significantly lower serum alanine aminotransferase compared to the ethanol alone group in the acute binge model. IAP treatment attenuated the development of alcohol-induced fatty liver, lowered hepatic pro-inflammatory cytokine and serum LPS levels, and prevented alcohol-induced gut barrier dysfunction. Finally, IAP ameliorated the activation of hepatic stellate cells and prevented their lipogenic effect on hepatocytes. IAP treatment protected mice from alcohol-induced hepatotoxicity and steatosis. Oral IAP supplementation could represent a novel therapy to prevent alcoholic-related liver disease in humans.

Although numerous polymorphisms have been associated with inflammatory bowel disease (IBD), identifying the function of these genetic factors has proved challenging. Here we identified a role for nine genes in IBD susceptibility loci in antibacterial autophagy and characterized a role for one of these genes, GPR65, in maintaining lysosome function. Mice lacking Gpr65, a proton-sensing G protein-coupled receptor, showed increased susceptibly to bacteria-induced colitis. Epithelial cells and macrophages lacking GPR65 exhibited impaired clearance of intracellular bacteria and accumulation of aberrant lysosomes. Similarly, IBD patient cells and epithelial cells expressing an IBD-associated missense variant, GPR65 I231L, displayed aberrant lysosomal pH resulting in lysosomal dysfunction, impaired bacterial restriction, and altered lipid droplet formation. The GPR65 I231L polymorphism was sufficient to confer decreased GPR65 signaling. Collectively, these data establish a role for GPR65 in IBD susceptibility and identify lysosomal dysfunction as a potentially causative element in IBD pathogenesis with effects on cellular homeostasis and defense. Gene mapping efforts have identified numerous disease-associated genes, but understanding how these genes influence disease has proved challenging. Xavier and colleagues identify a role for GPR65 in lysosomal homeostasis and demonstrate that the IBD-associated risk variant GPR65 I231L confers lysosomal dysfunction with effects on autophagy, pathogen clearance, and intestinal homeostasis.

Purpose: Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor, often metastatic at presentation, for which current chemotherapeutic regimens are largely ineffective. As its pathogenesis is still unknown, we hypothesized that deregulation of signaling pathways commonly activated in cancer may contribute to MCC tumorigenesis and may provide insights into targeted therapy approaches for this malignancy. Experimental Design: We retrospectively profiled 60 primary MCC samples using a SNaPshot-based tumor genotyping assay to screen for common mutations in 13 cancer genes. Results: We identified mutations in 9 (15%) MCC primary tumors, including mutations in TP53 (3 of 60) and activating mutations in the PIK3CA gene (6 of 60). Sanger sequencing of the primary MCC tumors detected one additional PIK3CA mutation (R19K) that had not been previously described in cancer. Merkel cell polyoma virus (MCPyV) was detected in 38 (66%) MCC cases and patients with MCPyV-positive cancers showed a trend toward better survival. With one exception, the presence of MCPyV and activating mutations in PIK3CA appeared mutually exclusive. We observed that signaling through the PI3K/pAKT pathway was active in one MCPyV-positive and in all MCPyV-negative MCC cell lines, as evidenced by AKT phosphorylation. Importantly, the presence of a PIK3CA-activating mutation was associated with sensitivity to treatment with ZST474, a specific phosphoinositide 3-kinase (PI3K) inhibitor, and to NVP-BEZ235, a dual PI3K/mTOR inhibitor, targeted agents under active clinical development. Conclusions: PI3K pathway activation may drive tumorigenesis in a subset of MCC and screening these tumors for PIK3CA mutations could help identify patients who may respond to treatment with PI3K pathway inhibitors. Clin Cancer Res; 18(5); 1227–36. ©2012 AACR.

Vaccines against important enteric pathogens such as rotavirus and poliovirus have shown lower efficacy in some populations. The application of new technologies and diverse scientific disciplines are needed to realize the promise of truly universal and effective solutions to combat those and other enteric diseases.

Background/Aims The response rates of HCV infection to interferon therapy vary depending on viral and host factors. We hypothesized that key regulators of the IFN signaling pathway are predictive of treatment outcome. Methods We measured the expression of signal transducer and activator of transcription 1 (STAT1) and suppressor of cytokine signaling 3 (SOCS3) in pretreatment liver biopsies. Staining quantitation was compared to treatment outcomes. Results Forty-nine patients with HCV and 25 patients with HCV/HIV infection treated with peginterferon/ribavirin were analyzed. Pretreatment hepatic SOCS3 expression was higher in non-responders than responders. Genotype 1 responders had similar levels of SOCS3 as genotype 2/3 responders. African Americans (AA) had higher hepatic SOCS3 than non-AA. Pretreatment hepatic SOCS3 was the most powerful independent predictor of sustained virologic response (SVR), even more so than genotype by logistic regression analysis. Failure to achieve SVR and AA race were independently associated with high hepatic SOCS3 levels. The hepatic expression of STAT-1 did not differ between responders and non-responders. Conclusions Our data indicate that hepatic SOCS3 is a stronger baseline predictor of antiviral response than viral genotype. Poor response to antiviral therapy in AA may be associated with higher hepatic SOCS3 expression.

Over 80% of the body's activated B cells are located in mucosal sites, including the intestine. The intestine contains IgM+ B cells, but these cells have not been characterized phenotypically or in terms of their developmental origins. We describe a previously unidentified and unique subset of immunoglobulin M+ B cells that present with an AA4.1−CD21−CD23− major histocompatibility complex class IIbright surface phenotype and are characterized by a low frequency of somatic hypermutation and the potential ability to produce interleukin-12p70. This B cell subset resides within the normal mucosa of the large intestine and expands in response to inflammation. Some of these intestinal B cells originate from the AA4.1+ immature B2 cell pool in the steady state and are also recruited from the recirculating naive B cell pool in the context of intestinal inflammation. They develop in an antigen-independent and BAFF-dependent manner in the absence of T cell help. Expansion of these cells can be induced in the absence of the spleen and gut-associated lymphoid tissues. These results describe the existence of an alternative pathway of B cell maturation in the periphery that gives rise to a tissue-specific B cell subset.

Background & Aims: T-cell receptor (TCR) γδ T cells are an important component of the mucosal immune system and regulate intestinal epithelial homeostasis. Interestingly, there is a significant increase in γδ T cells in the inflamed mucosa of patients with ulcerative colitis (UC). However, the role of γδ T cells in chronic colitis has not been fully identified. Methods: TCRα-deficient mice, which spontaneously develop chronic colitis with many features of human UC including an increase in γδ T-cell population, represent an excellent model to investigate the role of γδ T cells in UC-like colitis. To identify the role of γδ T cells in this colitis, we herein have generated TCRγ-deficient mice through deletion of all TCR Cγ genes (Cγ1, Cγ2, Cγ3, and Cγ4) using the Cre/ loxP site-specific recombination system and subsequently crossing these mice with TCRα-deficient mice. Results: An increase in colonic γδ T cells was associated with the development of human UC as well as UC-like disease seen in TCRα-deficient mice. Interestingly, the newly established TCRα −/− × TCRγ −/− double mutant mice developed significantly less severe colitis as compared with TCRα-deficient mice. The suppression of colitis in TCRα −/− × TCRγ −/− double mutant mice was associated with a significant reduction of proinflammatory cytokine and chemokine productions and a decrease in neutrophil infiltration. Conclusions: γδ T cells are involved in the exacerbation of UC-like chronic disease. Therefore, γδ T cells may represent a promising therapeutic target for the treatment of human UC.

Granulomas represent a localized inflammatory reaction that is characteristically observed in many inflammatory conditions. However, the mechanisms of granuloma formation have not been fully defined. Herein we demonstrate, by using experimental models of intestinal inflammation, that a unique CD11c+ DC-like cell subset that exhibits phenotypic and functional features of immature myeloid DCs and is characterized by the expression of a macrophage marker (F4/80) produces large amounts of IL-23 and directly induces the development of granulomas under a Th1-predominant intestinal inflammatory condition. Importantly, both IL-4 and IgG contribute to the suppression of F4/80+ DC-like cell–mediated granuloma formation by regulating the function and differentiation of this cell subset. In addition, enteric flora is required for the F4/80+ DC-like cell–mediated granuloma formation. Collectively, our data provide what we believe are novel insights into the involvement of F4/80+ DC-like cells in intestinal granuloma formation and demonstrate the role of host (IL-4 and IgG) and environmental (enteric flora) factors that regulate this function.

The Wiskott-Aldrich syndrome, a primary human immunodeficiency, results from defective expression of the hematopoietic-specific cytoskeletal regulator Wiskott-Aldrich syndrome protein (WASP). Because CD4+CD25+Foxp3+ naturally occurring regulatory T (nTreg) cells control autoimmunity, we asked whether colitis in WASP knockout (WKO) mice is associated with aberrant development/function of nTreg cells. We show that WKO mice have decreased numbers of CD4+CD25+Foxp3+ nTreg cells in both the thymus and peripheral lymphoid organs. Moreover, we demonstrate that WKO nTreg cells are markedly defective in both their ability to ameliorate the colitis induced by the transfer of CD45RBhi T cells and in functional suppression assays in vitro. Compared with wild-type (WT) nTreg cells, WKO nTreg cells show significantly impaired homing to both mucosal (mesenteric) and peripheral sites upon adoptive transfer into WT recipient mice. Suppression defects may be independent of antigen receptor–mediated actin rearrangement because both WT and WKO nTreg cells remodeled their actin cytoskeleton inefficiently upon T cell receptor stimulation. Preincubation of WKO nTreg cells with exogenous interleukin (IL)-2, combined with antigen receptor–mediated activation, substantially rescues the suppression defects. WKO nTreg cells are also defective in the secretion of the immunomodulatory cytokine IL-10. Overall, our data reveal a critical role for WASP in nTreg cell function and implicate nTreg cell dysfunction in the autoimmunity associated with WASP deficiency.

Inflammatory bowel disease (IBD) is an idiopathic inflammatory disease of the intestine. CD4+ T lymphocytes play an important role in both initiating and regulating intestinal inflammatory immune responses. CD4+CD25+CD45RBlow regulatory T (T reg) cells are capable of preventing the development of colitis in a mouse model of IBD. The precise mechanism of T reg cell–mediated prevention of colitis in this model is unclear, and the role of chemokine receptors in the trafficking and function of T reg cells in this model has not been determined. We examined the role of the chemokine receptor CCR4 in in vivo trafficking and suppressive function of T reg cells in a mouse adoptive transfer model of IBD. CCR4-deficient T reg cells failed to accumulate in the mesenteric lymph nodes (MLNs) at early time points (2–5 d) after adoptive transfer, resulting in a failure to suppress the generation of pathogenic T cells and the development of colitis. Moreover, although CCR4-deficent T cells had equivalent in vitro suppressive activity and accumulated in MLNs at later time points (42–56 d), they were unable to suppress colitis. Our study demonstrates that CCR4 plays an important role in T reg cell trafficking in LNs and that this is critical for T reg cell suppressive function in vivo.

Aims To examine whether co-administration of intestinal alkaline phosphatase (IAP) with antibiotics early in life may have a preventive role against metabolic syndrome (MetS) in mice. Methods A total of 50 mice were allocated to four treatment groups after weaning. Mice were treated with azithromycin (AZT) ± IAP, or with no AZT ± IAP, for three intermittent 7-day cycles. After the last treatment course, the mice were administered a regular chow diet for 5 weeks and subsequently a high-fat diet for 5 weeks. Body weight, food intake, water intake, serum lipids, glucose levels and liver lipids were compared. 16S rRNA gene pyrosequencing was used to determine the differences in microbiome composition. Results Exposure to AZT early in life rendered mice susceptible to MetS in adulthood. Co-administration of IAP with AZT completely prevented this susceptibility by decreasing total body weight, serum lipids, glucose levels and liver lipids to the levels of control mice. These effects of IAP probably occur as a result of changes in the composition of specific bacterial taxa at the genus and species levels (e.g. members of Anaeroplasma and Parabacteroides). Conclusions Co-administration of IAP with AZT early in life prevents mice from susceptibility to the later development of MetS. This effect is associated with alterations in the composition of the gut microbiota. IAP may represent a novel treatment against MetS in humans.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, is a potential therapeutic target. MIF is markedly different from other cytokines because it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention is challenging because of the need to neutralize a ubiquitous protein. In this article, we report that MIF occurs in two redox-dependent conformational isoforms. We show that one of the two isoforms of MIF, that is, oxidized MIF (oxMIF), is specifically recognized by three mAbs directed against MIF. Surprisingly, oxMIF is selectively expressed in the plasma and on the cell surface of immune cells of patients with different inflammatory diseases. In patients with acute infections or chronic inflammation, oxMIF expression correlated with inflammatory flare-ups. In addition, anti-oxMIF mAbs alleviated disease severity in mouse models of acute and chronic enterocolitis and improved, in synergy with glucocorticoids, renal function in a rat model of crescentic glomerulonephritis. We conclude that oxMIF represents the disease-related isoform of MIF; oxMIF is therefore a new diagnostic marker for inflammation and a relevant target for anti-inflammatory therapy.

The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity. DOI: http://dx.doi.org/10.7554/eLife.05920.001, eLife digest Inflammation is used by the immune system to protect and repair tissues after an injury or infection. However, if inflammation is too strong, or goes on for too long, it can damage tissues. This is seen in autoimmune diseases such as inflammatory bowel disease and type 1 diabetes. Therefore, precise regulation of the inflammatory response is essential for maintaining human health. White blood cells known as T cells are central regulators of tissue inflammation. To achieve this goal, they develop into subtypes with specialized roles. For example, some T helper cells release chemical signals that trigger inflammation and other immune responses. Regulatory T (Treg) cells then shut down these immune responses once they are no longer needed. Many autoimmune and other inflammatory diseases are thought to arise—at least partially—because Treg cells fail to stop the inflammatory response. Boosting the number or the activity of Treg cells could therefore help to treat these diseases. However, technical difficulties have made it difficult to investigate the genes and molecular pathways that control how this subtype of white blood cells develops. Khor et al. thought that discovering new chemicals that increase the number of Treg cells without harming them could help to identify the pathways that control their development. Khor et al. screened over 3000 chemicals, many of which are drugs currently approved for use in humans, for their effect on immature T cells that were taken from mice and grown in the laboratory. This ‘unbiased chemical biology’ approach identified several chemicals that both encouraged the T cells to develop into Treg cells and reduced the numbers that became inflammation-promoting T helper cells. Khor et al. then focused on one of these chemicals, called harmine. Tests in mice showed that harmine reduces the extent of experimentally induced inflammatory reactions. Treg cells generated by treating immature T cells with harmine had the same effect. Further experiments showed that harmine exerts these effects, at least in part, by inhibiting the activity of a protein called DYRK1A. When DYRK1A was removed from maturing mouse T cells grown in the laboratory, the T cells tended to develop into anti-inflammatory Treg cells. These findings therefore identify DYRK1A as part of a pathway that suppresses the development of Treg cells. It remains to be discovered how it does this, and whether other DYRK protein family members have similar roles. DOI: http://dx.doi.org/10.7554/eLife.05920.002

The homophilic cell surface receptors CD150 (Slamf1) and CD352 (Slamf6) are known to modulate adaptive immune responses. Although the Th17 response was enhanced in Slamf6−/− C57BL/6 mice upon oral infection with Citrobacter rodentium, the pathologic consequences are indistinguishable from an infection of wild-type C57BL/6 mice. Using a reporter-based binding assay, we show that Slamf6 can engage structures on the outer cell membrane of several Gram− bacteria. Therefore, we examined whether Slamf6, like Slamf1, is also involved in innate responses to bacteria and regulates peripheral inflammation by assessing the outcome of C. rodentium infections in Rag−/− mice. Surprisingly, the pathology and immune responses in the lamina propria of C. rodentium-infected Slamf6−/−Rag−/− mice were markedly reduced as compared with those of Rag−/− mice. Infiltration of inflammatory phagocytes into the lamina propria was consistently lower in Slamf6−/−Rag−/− mice than in Rag−/− animals. Concomitant with the reduced systemic translocation of the bacteria was an enhanced production of IL-22, suggesting that Slamf6 suppresses a mucosal protective program. Furthermore, administering a mAb (330) that inhibits bacterial interactions with Slamf6 to Rag−/− mice ameliorated the infection compared with a control antibody. We conclude that Slamf6-mediated interactions of colonic innate immune cells with specific Gram− bacteria reduce mucosal protection and enhance inflammation, contributing to lethal colitis that is caused by C. rodentium infections in Rag−/− mice.

Transplantation of wild-type (H-2k) bone marrow into tg epsilon26 mice (BM--tg epsilon26) induces colitis, characterized by T-helper cell type 1 activation in the lamina propria. Here we determined whether pathogenic T-cell clones could be derived by serial adoptive transfers into healthy tg epsilon26 recipients, starting with the population of CD4+ cells in the mesenteric lymph nodes of BM--tg epsilon26 mice.CD4+ cells purified from the mesenteric lymph nodes of colitic BM--tg epsilon26 mice were adoptively transferred into a second group of healthy tg epsilon26 recipients. Mesenteric lymph node CD4+ cells from the second group of mice were then used for consecutive transfers. Lamina propria CD4+ cells isolated from each mouse with colitis were analyzed for their cytokine profile and for their T-cell receptor Vbeta repertoire.CD4+ T cells maintained a dominant T-helper 1 phenotype after multiple transfers (or = 8) into recipient tg epsilon26 mice. A single T-cell receptor Vbeta was enriched (as much as 90%) in 8 CD4+ T-cell lines: Vbeta8S3, Vbeta8S1/2, Vbeta10S1, or Vbeta14. Sequence analyses of the T-cell receptor Vbetas showed clonality or the presence of a very restricted number of clones within each line. Adoptive transfers of the oligoclonal lines into either C3H x Rag-/- or severe combined immunodeficiency disease mice (H-2k) also induced colitis, whereas transfers into BALB/c x Rag-/- or severe combined immunodeficiency disease mice (H-2d) did not.Colitis-inducing CD4+ T-helper 1 cell clones can be obtained by enrichment through sequential adoptive transfers of CD4+ cells from mesenteric lymph nodes. Distinct dominant T-cell receptor Vbetas in each cell line responded to antigens presented by class II major histocompatibility complex.

Cadherins are integral membrane proteins expressed in tissue-restricted patterns that mediate homophilic intercellular adhesion. During development, they orchestrate tissue morphogenesis and, in the adult, they determine tissue integrity and architecture. The synovial lining is a condensation of fibroblast-like synoviocytes (FLS) and macrophages one to three cells thick. These cells are embedded within the extracellular matrix, but the structure is neither an epithelium nor an endothelium. Previously, the basis for organization of the synovium into a tissue was unknown. Here, we cloned cadherin-11 from human rheumatoid arthritis (RA)-derived FLS. We developed L cell transfectants expressing cadherin-11, cadherin-11 fusion proteins, and anti–cadherin-11 mAb. Cadherin-11 was found to be expressed mainly in the synovial lining by immunohistologic staining of human synovium. FLS adhered to cadherin-11–Fc, and transfection of cadherin-11 conferred the formation of tissue-like sheets and lining-like structures upon fibroblasts in vitro. These findings support a key role for cadherin-11 in the specific adhesion of FLS and in synovial tissue organization and behavior in health and RA.

Signal transduction from proinflammatory stimuli leading to NF-κB-dependent gene expression is mediated by the IκB kinase 2 (IKK2/IKKβ). Therefore, IKK2 has become an important drug target for treatment of inflammatory conditions. T cells, whose activation depends to a large extent on the activity of NF-κB transcription factors, play important roles in inflammation and autoimmunity. Ablation of IKK2 specifically in T cells in CD4cre/Ikk2FL mice allows their survival and activation by polyclonal stimuli in vitro, suggesting that IKK2 is dispensable for T cell activation. We report in this study that IKK2-deficient T cells expand efficiently in response to superantigen administration in vivo, but are completely deficient in recall responses, most likely due to inefficient priming. IKK2-deficient T cells provide suboptimal B cell help and fail to support germinal center reactions. Finally, IKK2 is essential for homeostatic expansion of naive T cells, reflected by the inability of IKK2-deficient T cells to induce colitis in lymphopenic hosts.

We sought to determine the proportion of rotavirus ( RV) infections among children with severe diarrhea in Bangalore, India, and to determine the role of neonatal infection with the asymptomatic RV strain I321 in protection against subsequent RV diarrhea. At 2 major hospitals, there was a >42% decrease in diarrhea-specific admissions during the study period. At 6 hospitals, asymptomatic infections were found in 25%-50% of neonates, when screening was performed randomly, and in >58% of neonates, when screening was performed daily, with the majority of infections occurring within the first 7 days of life. All the RVs found in asymptomatic neonates were strain I321. A 24-month follow-up of a cohort of 44 children who had been neonatally infected with strain I321 and 28 children who had not (control group) revealed comparable rates of RV detection but a marked decrease in the number of RV diarrhea episodes in the strain I321-infected group (2.3%), compared with the control group (39.3%) (P < .0001). This preliminary study suggests a possible association between neonatal infection with strain I321 and protection against subsequent RV illness.

Background: Histological recurrence of the hepatitis C virus (HCV) occurs in the majority of persons transplanted for cirrhosis as a result of HCV. Herein we analyze our experience with the use of both conventional and pegylated (PEG) interferon (IFN) in combination with ribavirin (RBV) in liver transplant recipients with recurrent HCV. Methods: Patients transplanted between 1992 and 2001 with post-orthotopic liver transplantation (OLT) histological recurrence of HCV, and who were treated with at least 6 months of IFN or PEG-IFN in combination with RBV were included in this analysis. A retrospective chart review was performed. Results: A total of 31 patients were included. Fifteen were treated with IFN/RBV and 16 with PEG-IFN/RBV. Of these 16, 11 had been begun on IFN/RBV and were changed to PEG-IFN/RBV because of persistent viremia. Three patients (20%) in the IFN/RBV group and six patients (37.5%) in the PEG-IFN/RBV group experienced a virologic response (VR) on therapy. Of the six patients experiencing VR in the PEG-IFN/RBV group, three (50%) were IFN/RBV non-responders. There were two sustained VRs (SVR). The 65.6% of all patients experienced a biochemical response (BR) on therapy. Seven deaths were observed. Dose modifications of IFN or PEG-IFN (87.1%) and RBV (80.6%) and the requirement for hematopoietic growth factors were frequent. Conclusions: Treatment of recurrent HCV infection with combination of IFN or PEG-IFN and RBV produced an on-therapy VR in 29% and BR in 65% of patients. Hematologic toxicity and dose modifications were frequent. Our experience with antiviral therapy for HCV post-OLT remains disappointing but PEG-IFN + RBV appears to produce VR in a sizable portion of IFN + RBV non-responders.

BACKGROUND AND AIMS There is a strong epidemiologic association between diabetes mellitus (DM) and hepatitis C virus (HCV) infection. However, the pathogenetic basis for this association has not been established. We sought to evaluate the association between insulin resistance (IR), beta-cell dysfunction, and HCV among orthotopic liver transplant (OLT) recipients. METHODS We performed a cross sectional analysis comparing 39 HCV(+) with 60 HCV(-) OLT recipients. IR and beta-cell function were calculated using validated measures and were correlated with clinical variables. RESULTS By multivariate analysis of the entire cohort, HCV infection and body mass index (BMI) were independent predictors of IR (P =0.04 and 0.0006, respectively). HCV infection was associated with 35% increase in IR. Because the model used to calculate IR was derived from nondiabetic subjects, we performed additional analysis of patients who did not meet criteria for diabetes at the time of their study evaluation. In this analysis, HCV(+) subjects had greater fasting insulin and homeostasis model assessment (HOMA) IR (15.3 mu U/mL and 3.8) compared with HCV(-) patients (10.7 mu U/mL and 2.5) (P =0.03, 0.03). There was no difference in beta-cell function or hepatic insulin extraction between the HCV (+) and (-) groups. HCV (P =0.0005), BMI (P

Several autoimmune disease models depend on an imbalance in the activation of aggressor T(h)1 and CD4(+)CD25(+) regulatory T (T(reg)) cells. Here we compare the requirement for signals through the co-stimulatory molecules CD28 and inducible co-stimulator (ICOS) in chronic murine colitis, a model for inflammatory bowel disease. We used a colitis model in which disease-causing CD45RB(hi) T cells alone or in combination with CD4(+)CD25(+) T cells from either CD28-deficient or wild-type donors were transferred into T cell-deficient animals, half of which were treated with ICOS-blocking reagents. Blocking ICOS on the surface of CD28-deficient T(h)1 cells abrogated development of colitis, whereas blocking CD28 or ICOS alone had little to no effect on disease induction. In contrast to T(h)1 cells, regulatory T cell functioning depended mostly on CD28 signaling with only a minor contribution for ICOS. We conclude that CD28 and ICOS collaborate to development of murine colitis by aggressor T(h)1 cells, and that CD28 is required for T(reg) cells, which should caution against the use of CD28-blocking reagents in inflammatory bowel disease.

The “Immune Regulatory Networks” meeting in Boston gathered scientists with a passion for regulatory T cells to discuss the latest information on an increasingly important subject.

The animal models of inflammatory bowel disease provide a framework to define the immunopathogenesis of intestinal inflammation. Studies in these models support the hypothesis that exaggerated immune responses to normal enteric microflora are involved in the initiation and perpetuation of chronic intestinal inflammation. A major pathway involves development of acquired immune responses by the interactions of CD4+ T-cell receptor alphabeta T cells with antigen-presenting cells (dendritic cells). Immunoregulatory cells, including Tr1 cells, Th3 cells, and CD4+ CD25+ T cells and B cells, directly or indirectly affect the T-cell receptor alphabeta T cell-induced immune responses and bridge innate and acquired immunity. The study of these complicated immune networks provides the rationale for the development of new therapeutic interventions in inflammatory bowel disease.

Seroconversion of immunoglobulin A (IgA) and immunoglobulin G (IgG) (> or =4-fold rise) to rotavirus nonstructural protein 4 (NSP4) was determined, by use of enzyme-linked immunosorbent assay with fusion proteins glutathione S-transferase (GST)-NSP4 from strains SA11 (A), 116E (B), and RRV (C), in 40 children with acute rotavirus gastroenteritis and in 30 with the same disease due to other pathogens. The IgG seroconversion rates in the rotavirus group were 67.5%, 70%, and 60% when recombinant (r) NSP4A, -B, and -C, respectively, were used as antigen in the assay, and, for rotavirus-uninfected children, rates were 10%, 13%, and 7%. IgA seroconversion occurred in 57%, 70%, and 50%, respectively, of children with rotavirus gastroenteritis; in rotavirus-uninfected children, 1 child each seroconverted to the different rNSP4s. Among 9 children infected with strain NSP4A, 7, 6, and 5 children showed IgG seroconversion, and, among 18 infected with NSP4A, -B, and -C, 16, 17, and 15, respectively, showed IgG seroconversion. Between NSP4A-infected and NSP4B-infected children, IgA responses were similar to IgG responses. In conclusion, significant NSP4-specific antibody response occurs in natural rotavirus infection, and the antibody response appears to be broad and heterotypic in nature.

Hepatitis C virus (HCV) is currently the leading indication worldwide for orthotopic liver transplantation. However, the majority of patients receiving transplant for HCV eventually develop histopathologic evidence of recurrent allograft HCV and approximately 10% die or require retransplantation within the first 5 post-operative years because of accelerated graft injury and cirrhosis. Traditional induction immunosuppressive regimens and intensive immunosuppression used to treat episodes of acute cellular rejection (ACR) are associated with enhanced viral replication and higher likelihood and severity of recurrent HCV. At our institution, therefore, we have used low-dose steroid therapy in an effort to limit HCV replication. However, this practice has been associated with frequent early presentations consistent with ACR. Here, we present three cases consistent with histologic ACR treated with conventional antirejection therapy that improved transiently, but evolved rapidly to progressive HCV. A fourth patient with a similar presentation experienced dramatic improvement in aminotransferases when treated solely with interferon and ribavirin. We propose that histologic characteristics traditionally associated with ACR may, in fact, represent early recurrent HCV as both processes share common immunopathogenetic mechanisms, or alternatively, that both ACR and recurrent HCV may be present simultaneously. We conclude that in cases suggestive of ACR, careful consideration should be given to treatment for recurrent HCV in lieu of or in concert with intensive immunosuppression.

Although CD4(+) T cells form a major subset of TCRalphabeta T cells, only a small number of TCRgammadelta T cells express CD4. Factors contributing to the down-regulation of CD4(+) TCRgammadelta T cells have not been identified. The CD5 molecule is expressed on most TCRgammadelta T cells in the spleen, whereas only a few intestinal intraepithelial TCRgammadelta T cells express this molecule in wild-type mice and TCRbeta mutant (beta(-/-)) mice. Unexpectedly, in the present studies, the lack of CD5 led to a remarkable increase of a CD4(+) TCRgammadelta T cell subset in CD5(-/-)beta(-/-) mice. The CD4(+) TCRgammadelta T cells were also detectable in MHC II(-/-)CD5(-/-)beta(-/-) triple-mutant mice. This CD4(+) TCRgammadelta T cell subset provided help in Mycobacterium-induced germinal center (GC) formation and showed a T(h)-like cytokine profile. In contrast, CD5(+) TCRgammadelta T cells suppressed the CD4(+) TCRgammadelta T cell-mediated GC formation, presumably by eliminating this CD4(+) subset. Unlike intraepithelial gammadelta T cells,30% of TCRgammadelta T cells in the colonic lamina propria (LP) expressed CD5. The lack of CD5 also led to increased numbers of CD4(+) TCRgammadelta T cells in the colonic LP and increased susceptibility to development of chronic colitis in beta(-/-) mice. Cell transfer studies suggest that CD5(+) TCRgammadelta T cells are capable of selectively eliminating CD4(+) TCRgammadelta T cells in the intestine. The CD4(+) TCRgammadelta T cells possess immune functions similar to CD4(+) TCRalphabeta T cells.

Helicobacter pylori infection has been reported to increase the risk of cholera. This nested case-control study was conducted to determine whether H. pylori infection is associated with occurrence of typhoid fever. Eighty-three case subjects of culture-proven typhoid fever were identified through a 1-year surveillance of subjects aged 0-40 years in an urban slum. Two age- and sex-matched neighborhood control subjects were concurrently selected for each case subject. Serum anti-H. pylori immunoglobulin G antibodies were measured in case and neighborhood control subjects. For determining other risk factors, 2 additional community control subjects per case were selected. There was a significant association between the presence of serum anti-H. pylori immunoglobulin G antibodies and typhoid fever (adjusted odds ratio, 2.03; 95% confidence interval, 1.02-4.01). Illiteracy, being part of a nuclear family, nonuse of soap, and consumption of ice cream were also associated with a significantly greater risk of typhoid fever. This study provides the first empiric evidence that H. pylori infection is associated with an increased risk of typhoid fever.

To define the role of TRAF proteins in CD40-dependent isotype switching in B cells, we introduced wild-type (WT) and mutant CD40 transgenes that lacked the binding motifs for TRAF6 (CD40deltaTRAF6), TRAF2 and TRAF3 (CD40deltaTRAF2/3), or both (CD40deltaTRAFs) into B cells of CD40(-/-) mice. The in vivo isotype switch defect in CD40(-/-) mice was fully corrected by WT and CD40deltaTRAF6, partially by CD40deltaTRAF2/3, and not at all by CD40deltaTRAFs transgenes. CD40-mediated isotype switching, proliferation, and activation of p38, JNK, and NFkappaB in B cells were normal in WT and CD40deltaTRAF6 mice, severely impaired in CD40deltaTRAF2/3, and absent in CD40deltaTRAFs mice. These results suggest that binding to TRAF2 and/or TRAF3 but not TRAF6 is essential for CD40 isotype switching and activation in B cells.

The balance between pro and antiinflammatory cytokines secreted by T cells regulates both the initiation and perpetuation of inflammatory bowel diseases (IBD). In particular, the balance between interferon (IFN)-gamma/interleukin (IL)-4 and transforming growth factor (TGF)-beta activity controls chronic intestinal inflammation. However, the molecular pathways that evoke these responses are not well understood. Here, we describe a critical role for the transcription factor T-bet in controlling the mucosal cytokine balance and clinical disease. We studied the expression and function of T-bet in patients with IBD and in mucosal T cells in various T helper (Th)1- and Th2-mediated animal models of chronic intestinal inflammation by taking advantage of mice that lack T-bet and retroviral transduction techniques, respectively. Whereas retroviral transduction of T-bet in CD62L(+) CD4(+) T cells exacerbated colitis in reconstituted SCID mice, T-bet-deficient T cells failed to induce colitis in adoptive transfer experiments suggesting that overexpression of T-bet is essential and sufficient to promote Th1-mediated colitis in vivo. Furthermore, T-bet-deficient CD62L(-) CD4(+) T cells showed enhanced protective functions in Th1-mediated colitis and exhibited increased TGF-beta signaling suggesting that a T-bet driven pathway of T cell activation controls the intestinal balance between IFN-gamma/IL-4 and TGF-beta responses and the development of chronic intestinal inflammation in T cell-mediated colitis. Furthermore, TGF-beta was found to suppress T-bet expression suggesting a reciprocal relationship between TGF-beta and T-bet in mucosal T cells. In summary, our data suggest a key regulatory role of T-bet in the pathogenesis of T cell-mediated colitis. Specific targeting of this pathway may be a promising novel approach for the treatment of patients with Crohn's disease and other autoimmune diseases mediated by Th1 T lymphocytes.

Objective: Vitamin A supplementation to mothers in the postpartum period and to their infants at routine immunization contacts is being considered to reduce vitamin A deficiency in infancy. This study was conducted to determine the impact of maternal and infant vitamin A supplementation on antibody response to oral polio vaccine (OPV). Design: Randomized, double blind, placebo-controlled trial. Interventions: Mothers in the intervention group received 60 mg retinol equivalent (RE) vitamin A 3–4 weeks after delivery and their infants 7.5 mg RE with each OPV dose at 6, 10 and 14 weeks of age. The control group mothers and their infants received a placebo at each of these contacts. Main outcomes: Geometric mean (GM) titer of neutralizing antibodies and proportion of children with protective titer to the three polioviruses at 26 weeks of age. Results: Vitamin A supplementation increased the proportion of infants with protective antibody titer against poliovirus type 1 (relative risk (RR) 1.15, 95% confidence interval (CI) 1.03–1.28) and the GM antibody titer (ratio of GM 1.55, 95% CI 1.03–2.31) following immunization. The proportion of infants with protective antibody titer against poliovirus type 2 (RR 0.99, 95% CI 0.94–1.05) or type 3 (RR 1.05, 95% CI 0.96–1.15) was not significantly different in vitamin A and placebo groups. The GM antibody titer for poliovirus type 2 (ratio of GM 0.99, 95% CI 0.64–1.54) or poliovirus type 3 (ratio of GM 1.10, 95% CI 0.69–1.75) also did not differ across groups. Conclusions: Vitamin A given to the mothers in the postpartum period and their infants with OPV did not interfere with the antibody response to any of the three polioviruses and enhanced the response to poliovirus type 1. Sponsorship: Division of Child Health and Development, WHO, Geneva, Norwegian Universities Committee for Development and Research and Indian Council of Medical Research, Panacea Biotec Ltd, India (provided the oral polio vaccine).

The development of monoclonal antibodies combined with flow cytometry has revolutionized the analysis of lymphocyte subsets. These newer methods using the Q-prep leucocyte preparation system require only 1–2 ml of blood as compared to 10 ml required traditionally. One of the main impediments in the use of this superior technology in Indian laboratories has been the high cost of reagents. This study evaluated methods to reduce the cost of assays. In the first experiment from 26 healthy subjects, 2ml venous blood samples in EDTA (ethylenediamine tetra-acetate) were obtained. Each sample was divided into two equal portions, one portion was stained using diluted monoclonal antibody, whereas the other portion was stained using standard concentrations of antibodies. In the second experiment, blood samples from 12 subjects were again divided into 2 portions; one portion of each pair was processed using commercial Q-prep reagents while the other portion was processed using our own reagents. In the first experiment, which evaluated use of a diluted antibody against the standard recommended concentrations, a 5-tube panel that estimated CD3, CD4, CD8, CD20 was used. In the second experiment CD3, CD4 and CD8 were estimated. The total cost per sample for a 5-panel estimation was however reduced from $39.11 to $1.10. Given the proven advantages of using a whole blood stain-lyse method for T cell subset estimations, its use should be encouraged in developing country settings. With the suggested methods the whole blood Q-prep could be performed at appreciably reduced costs, without loss in precision.

The aim of the present study was to explore whether mice fed a diet low in Zn (2·0 mg Zn/kg diet) for a relatively short period of time were more prone to severeStreptococcus pneumoniaeinfection than mice fed a normal diet (25 mg elemental Zn/kg). The Zn-deficient mice were compared with mice in two Zn-adequate control groups; one pair-fed and another with free access to the diet. After 2 weeks feeding, the mice were infected intranasally under anaesthesia with a suspension containing about 107pneumococci. Clinical status was observed every day and blood samples were examined forS. pneumoniaeevery second day for a week. All infected mice examined carried the infecting strain intranasally. The survival time and time before positive blood culture were significantly shorter in the Zn-depleted group than in the pair-fed Zn-adequate group (hazard ratios 15·6 and 3·2, P

SummaryWe have prepared human recombinant antibody molecules against the glycoprotein antigen of the rabies virus (GPRV) based on the single chain variable fragment (scFv) format. Anti-GPRV scFvs were selected from a human synthetic scFv phage display library with a repertoire of approximately 109 specificities. After three rounds of selection against the PV11 strain of the virus, 40% of the clones tested recognized the rabies antigen. Of the 20 positive clones that were sequenced, five distinct sequences were identified. These distinct scFvs were cloned into a mammalian expression vector carrying the human IgG1 Fc region. The specificity of the resulting scFv-Fc molecules for GPRV was established by ELISA, dot blot and western blot analyses and membrane immunofluorescence. Two of the scFv-Fc fusion proteins neutralized the PV11 strain in a standard in vivo neutralization assay where the virus was incubated with the scFv-Fc molecules before intracranial inoculation in mice. These anti-GPRV scFv-Fc molecules have the potential to be used as an alternative to the presently available HRIG, for use in post-exposure preventive treatment.

Background: We recently described a murine model of atopic dermatitis (AD) elicited by epicutaneous sensitization with ovalbumin (OVA). The skin lesions in these mice were characterized by a dermal infiltrate consisting of eosinophils and T cells and by increased expression of the T H 2 cytokines IL-4 and IL-5. Epicutaneous sensitization induces a rise in the levels of serum total IgE and OVA-specific antibodies, further indicating that it elicits a predominantly T H 2 response. Objective: This study was undertaken to assess the roles of T cells, B cells, and CD40L-CD40 interactions in AD. Methods: Mice with targeted gene deletions were sensitized with OVA. Histologic and immunohistochemical examinations, as well as measurements of IL-4 mRNA, were performed on OVA-sensitized skin. Total and antigen-specific serum IgE levels were determined. Results: RAG2 –/– mice, which lack both T and B cells, did not exhibit cellular infiltration, induction of dermal IL-4 mRNA, or elevation of serum IgE after OVA sensitization; all of these features were present in B-cell–deficient IgH –/– mice. T-cell receptor α –/– mice did not display cellular infiltration, IL-4 mRNA expression, or increased IgE levels after OVA sensitization, but these responses were elicited in T-cell receptor δ –/– mice after sensitization. Absence of CD40 had no effect on these responses. Conclusion: These results suggest that αβ T cells, but not γδ T cells, B cells, or CD40L-CD40 interactions, are critical for skin inflammation and the T H 2 response in AD. (J Allergy Clin Immunol 2001;107:359-66.)

Background & Aims: T-cell receptor α mutant (TCRα −/− ) mice spontaneously develop chronic colitis mediated by CD4 + TCRα − β + T cells. The aim of this study was to analyze the mechanisms of expansion of these cells by characterization of the TCRβ repertoire. Methods: TCRβ repertoire was analyzed by reverse-transcription polymerase chain reaction/Southern blot and DNA sequencing. Clonality of T cells was examined in the lymphoid tissues and colons of TCRα −/− mice and interleukin 4–deficient TCRα −/− mice. In addition, an in vitro culture system using syngeneic colonic epithelial cells as antigens was used. Results: The clonal expansion of a restricted subset of Vβ8.2 + T cells was characterized by conservation of a single negatively charged amino acid residue in the second position of the complementarity-determining region 3 (CDR3). These T cells were observed in the diseased colon and appendix (cecal patch) of TCRα −/− mice, but not germfree TCRα −/− mice. Culture of polyclonal T cells from young TCRα −/− mice with colonic epithelial cells under T helper 2 conditions resulted in the survival of Vβ8.2 + T cells characterized by the same CDR3 pattern. In addition, the transfer of the cultivated T cells induced mild colitis in recombination-activating gene 1 mutant mice. Conclusions: In the TCRα −/− mice, the development of colitis is associated with the presence of a restricted diversity of Vβ8.2 + T-cell subsets characterized by a specific TCR motif. The limited diversity of lamina propria T cells that are derived from naive T cells expanded by reacting with luminal bacterial antigens is likely caused by the survival of these T cells after stimulation with self-antigens in the presence of a T helper 2 environment. GASTROENTEROLOGY 2000;119:983-995

Background & Aims: Experimental colitis in most animal models is caused by dysregulation of T lymphocytes that display a T helper 1 (Th1) phenotype. CD154 (CD40L/gp39), a member of the tumor necrosis factor (TNF) family, is up-regulated on T cells on activation and has been shown to play a key role in the induction of a Th1 response. We investigated whether chronic experimental colitis is dependent on the CD154/CD40 pathway and whether disease can be prevented by anti-CD154 antibody treatment. Methods: Two models of chronic colitis were used: CD45Rb hi cell transfer into recombination activation gene–deficient (Rag −/− ) mice and bone marrow transplant of tgϵ26 animals. In both models, mice were reconstituted with cells from CD154-deficient animals. In another series of experiments, wild-type CD45Rb hi T cell–reconstituted recipients were treated with anti-CD154, either from the start of the experiment or after onset of disease. Results: T cells deficient in CD154 induced a milder clinical disease, less weight loss, and fewer histologic signs of colitis than wild-type cells. The level of interleukin 12 in the serum of CD154-deficient T-cell recipients was 5-fold less than that of wild-type cell recipients. Nevertheless, no signs of deviation from a Th1 phenotype were observed. Treatment with anti-CD154 antibodies substantially impaired disease development, even when started after the onset of colitis. Conclusions: The CD154/CD40 pathway plays a critical role in Th1-induced chronic experimental colitis. Blocking CD154, even after the onset of disease, ameliorates colitis but does not induce a T helper 2 (Th2) phenotype. GASTROENTEROLOGY 2000;119:715-723

The spontaneous chronic colitis in TCR alpha mutant (TCRalpha(-/-)) mice mediated by CD4(+) TCRalpha(-)beta(+) T cells is more severe in the absence of mature B cells, suggesting a suppressive role of B cells and Ig in the development of chronic colitis. To investigate the direct role of B cells in the suppression of this colitis, cell transfer studies were performed in TCRalpha(-/-) x Igmu(-/-) (alphamu(-/-)) double-knockout mice. The chronic colitis was markedly attenuated in alphamu(-/-) mice after the adoptive transfer of peripheral B cells from TCRalpha(-/-) mice into 3- to 4-week-old alphamu(-/-) mice prior to the development of colitis. Furthermore, transfer of mature B cells from TCRalpha(-/-) mice markedly decreased the number of pathogenic colonic CD4(+) TCRalpha(-)beta(+) T cells in alphamu(-/-) mice with established colitis. This B cell effect required the presence of functional co-stimulatory molecules CD40 and B7-2 (CD86) but not B7-1 (CD80). These results indicate that mature B cells play an important role in the development of chronic colitis in TCRalpha(-/-) mice by directly regulating the pathogenic T cells (CD4(+) TCRalpha(-)beta(+) T cells).

Mice with targeted disruption of the T cell receptor alpha gene (TCR alpha-/-) spontaneously develop chronic colitis. Colonic inflammation begins at 6-8 weeks of age and chronic colitis is established in about 60% of mice by 16-20 weeks of age. The disease is also associated with autoantibodies (anti-tropomyosin antibodies, anti-neutrophil cytoplasmic antibodies) and an oligoclonal immune response to luminal bacterial antigens. Although T cells, but not B cells or autoantibodies, are essential for the development of colitis, B cells and/or autoantibodies may have a regulatory role in the pathogenesis of this colitis because the colitis is more severe in B cell deficient TCR alpha-/- mice. Cytokines, specifically IL-4 and IL-1, also play an important role in the development of colitis in TCR alpha-/- mice. Enteric bacteria located in the large intestine are an important factor in the pathogenesis of this colitis because germ-free TCR alpha-/- mice do not develop colitis and appendectomy at an early age delays the onset of this colitis. The colitis in TCR alpha-/- mice resembles human ulcerative colitis and provides a useful model to study the pathogenesis of human inflammatory bowel disease.

Human small intestinal intraepithelial lymphocytes (iIEL) are a unique population of CD8αβ+ TCR-αβ+ but CD28− T lymphocytes that may function in intestinal epithelial cell immunosurveillance. In an attempt to define novel cell surface molecules involved in iIEL function, we raised several mAbs against activated iIELs derived from the small intestine that recognized an Ag on activated, but not resting, iIELs. Using expression cloning and binding studies with Fc fusion proteins and transfectants, the cognate Ag of these mAbs was identified as the N domain of biliary glycoprotein (CD66a), a carcinoembryonic Ag-related molecule that contains an immune receptor tyrosine-based inhibitory motif. Functionally, these mAbs inhibited the anti-CD3-directed and lymphokine-activated killer activity of the P815 cell line by iIELs derived from the human small intestine. These studies indicate that the expression of biliary glycoprotein on activated human iIELs and, potentially, other mucosal T lymphocytes is involved in the down-regulation of cytolytic function.