Robert M. Hershberg

Antigen processing and presentation by intestinal epithelial cells – polarity and complexity

The mechanisms by which gut-associated lymphoid tissue (GALT) maintains a balance between oral tolerance and active immune response in the face of exposure to high antigen concentrations remains a central question in mucosal immunity. Here, Robert Hershberg and colleagues discuss the evidence that human intestinal epithelial cells function as antigen-presenting cells (APCs) capable of regulating T-cell responses in the intestinal mucosa

Taking toll: lipid A mimetics as adjuvants and immunomodulators

Vaccine adjuvants based on the structure of lipid A, such as monophosphoryl lipid A (MLA), have proven to be safe and effective in inducing immune responses to heterologous proteins in animal and human vaccines. Recent work on the development of a recombinant vaccine for leishmaniasis has demonstrated that a clinical grade MLA formulation - MPL(R) adjuvant - is essential in the development of a protective response. Preliminary evidence suggests that MLA and a chemically distinct family of lipid A mimetics - the aminoalkyl glucosaminide 4-phosphates - act on Toll-like receptor 4 (TLR4). As TLR4 agonists, they have potent immunomodulatory effects when used both as vaccine adjuvants and as stand-alone products. Novel approaches to vaccine development could benefit from taking full advantage of the effects of these compounds on innate and adaptive responses.

Serum Immune Responses Predict Rapid Disease Progression among Children with Crohn's Disease: Immune Responses Predict Disease Progression

BACKGROUND AND AIM:Crohns disease (CD) is a heterogeneous disorder characterized by diverse clinical phenotypes. Childhood-onset CD has been described as a more aggressive phenotype. Genetic and immune factors may influence disease phenotype and clinical course. We examined the association of immune responses to microbial antigens with disease behavior and prospectively determined the influence of immune reactivity on disease progression in pediatric CD patients.METHODS:Sera were collected from 196 pediatric CD cases and tested for immune responses: anti-I2, anti-outer membrane protein C (anti-OmpC), anti-CBir1 flagellin (anti-CBir1), and anti-Saccharomyces-cerevisiae (ASCA) using ELISA. Associations between immune responses and clinical phenotype were evaluated.RESULTS:Fifty-eight patients (28%) developed internal penetrating and/or stricturing (IP/S) disease after a median follow-up of 18 months. Both anti-OmpC (p < 0.0006) and anti-I2 (p < 0.003) were associated with IP/S disease. The frequency of IP/S disease increased with increasing number of immune responses (p trend = 0.002). The odds of developing IP/S disease were highest in patients positive for all four immune responses (OR (95% CI): 11 (1.5–80.4); p= 0.03). Pediatric CD patients positive for ≥1 immune response progressed to IP/S disease sooner after diagnosis as compared to those negative for all immune responses (p < 0.03).CONCLUSIONS:The presence and magnitude of immune responses to microbial antigens are significantly associated with more aggressive disease phenotypes among children with CD. This is the first study to prospectively demonstrate that the time to develop a disease complication in children is significantly faster in the presence of immune reactivity, thereby predicting disease progression to more aggressive disease phenotypes among pediatric CD patients.

A Synthetic TLR4 Antagonist Has Anti-Inflammatory Effects in Two Murine Models of Inflammatory Bowel Disease

Current evidence indicates that the chronic inflammation observed in the intestines of patients with inflammatory bowel disease is due to an aberrant immune response to enteric flora. We have developed a lipid A-mimetic, CRX-526, which has antagonistic activity for TLR4 and can block the interaction of LPS with the immune system. CRX-526 can prevent the expression of proinflammatory genes stimulated by LPS in vitro. This antagonist activity of CRX-526 is directly related to its structure, particularly secondary fatty acyl chain length. In vivo, CRX-526 treatment blocks the ability of LPS to induce TNF-α release. Importantly, treatment with CRX-526 inhibits the development of moderate-to-severe disease in two mouse models of colonic inflammation: the dextran sodium sulfate model and multidrug resistance gene 1a-deficient mice. By blocking the interaction between enteric bacteria and the innate immune system, CRX-526 may be an effective therapeutic molecule for inflammatory bowel disease.

Critical Role of cAMP Response Element Binding Protein Expression in Hypoxia-elicited Induction of Epithelial Tumor Necrosis Factor-α

Tissue hypoxia is intimately associated with a number of chronic inflammatory conditions of the intestine. In this study, we investigated the impact of hypoxia on the expression of a panel of inflammatory mediators by intestinal epithelia. Initial experiments revealed that epithelial (T84 cell) exposure to ambient hypoxia evoked a time-dependent induction of the proinflammatory markers tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and major histocompatibility complex (MHC) class II (37 ± 6.1-, 7 ± 0.8-, and 9 ± 0.9-fold increase over normoxia, respectively, each p < 0.01). Since the gene regulatory elements for each of these molecules contains an NF-κB binding domain, we investigated the influence of hypoxia on NF-κB activation. Cellular hypoxia induced a time-dependent increase in nuclear p65, suggesting a dominant role for NF-κB in hypoxia-elicited induction of proinflammatory gene products. Further work, however, revealed that hypoxia does not influence epithelial intercellular adhesion molecule 1 (ICAM-1) or MHC class I, the promoters of which also contain NF-κB binding domains, suggesting differential responses to hypoxia. Importantly, the genes for TNF-α, IL-8, and MHC class II, but not ICAM-1 or MHC class I, contain cyclic AMP response element (CRE) consensus motifs. Thus, we examined the role of cAMP in the hypoxia-elicited phenotype. Hypoxia diminished CRE binding protein (CREB) expression. In parallel, T84 cell cAMP was diminished by hypoxia (83 ± 13.2% decrease, p < 0.001), and pharmacologic inhibition of protein kinase A induced TNF-α and protein release (9 ± 3.9-fold increase). Addback of cAMP resulted in reversal of hypoxia-elicited TNF-α release (86 ± 3.2% inhibition with 3 mm 8-bromo-cAMP). Furthermore, overexpression of CREB but not mutated CREB by retroviral-mediated gene transfer reversed hypoxia-elicited induction of TNF-α defining a causal relationship between hypoxia-elicited CREB reduction and TNF-α induction. Such data indicate a prominent role for CREB in the hypoxia-elicited epithelial phenotype and implicate intracellular cAMP as an important second messenger in differential induction of proinflammatory mediators.

Synthetic Toll-Like Receptor 4 Agonists Stimulate Innate Resistance to Infectious Challenge

ABSTRACT A compound family of synthetic lipid A mimetics (termed the aminoalkyl glucosaminide phosphates [AGPs]) was evaluated in murine infectious disease models of protection against challenge with Listeria monocytogenes and influenza virus. For the Listeria model, intravenous administration of AGPs was followed by intravenous bacterial challenge 24 h later. Spleens were harvested 2 days postchallenge for the enumeration of CFU. For the influenza virus model, mice were challenged with virus via the intranasal/intrapulmonary route 48 h after intranasal/intrapulmonary administration of AGPs. The severity of disease was assessed daily for 3 weeks following challenge. Several types of AGPs provided strong protection against influenza virus or Listeria challenge in wild-type mice, but they were inactive in the C3H/HeJ mouse, demonstrating the dependence of the AGPs on toll-like receptor 4 (TLR4) signaling for the protective effect. Structure-activity relationship studies showed that the activation of innate immune effectors by AGPs depends primarily on the lengths of the secondary acyl chains within the three acyl-oxy-acyl residues and also on the nature of the functional group attached to the aglycon component. We conclude that the administration of synthetic TLR4 agonists provides rapid pharmacologic induction of innate resistance to infectious challenge by two different pathogen classes, that this effect is mediated via TLR4, and that structural differences between AGPs can have dramatic effects on agonist activity in vivo.

Anti-Flagellin (CBir1) Phenotypic and Genetic Crohn's Disease Associations

Background Antibody reactivity to microbial antigens correlates with distinct Crohns disease (CD) phenotypes such as fistulizing or fibrostenosing disease. We examined the association between anti‐CBir1 and clinical phenotypes and NOD2 variants in a large cohort of adult CD patients. Methods Sera and genomic DNA were collected from 731 patients with CD and tested for immune responses to I2, CBir1, oligomannan, and outer membrane porin C (OmpC) and the 3 most common CD‐associated NOD2 variants. Results Anti‐CBir1 reactivity was significantly associated with fibrostenosis (FS), internal penetrating (IP) disease phenotypes, small bowel (SB) involvement, and SB surgery but negatively associated with ulcerative colitis (UC)‐like CD. Multivariate logistic regression analysis showed that anti‐CBir1 was independently associated with FS and UC‐like CD irrespective of the antibody reactivity to I2, oligomannan, or OmpC, but not with SB involvement or SB surgery. The magnitude of anti‐CBir1 reactivity, when added to the quantitative response toward the other 3 CD‐associated antigens, enhances the discrimination of FS, IP, UC‐like CD, and SB involvement, but not SB surgery. Finally, although the frequency of anti‐CBir1 was similar in patients with none versus at least 1 NOD2 variant, the quantitative response to CBir1 flagellin was significantly higher in patients with CD carrying at least 1 NOD2 variant versus those carrying no variants (median anti‐CBir1 titer 33.39 versus 28.36, respectively; P = 0.01). Conclusions Anti‐CBir1 serum reactivity in CD patients is independently associated with FS and complicated SB CD. Quantitative, but not qualitative, response to CBir1 is also significantly associated with the CD‐associated NOD2 variants. (Inflamm Bowel Dis 2007)

Delineation of a CD1d-restricted antigen presentation pathway associated with human and mouse intestinal epithelial cells

BACKGROUND & AIMS CD1d, a major histocompatibility complex (MHC) class I-related molecule that is responsible for the presentation of glycolipid antigens to subsets of natural killer T (NK-T) cells, is expressed by intestinal epithelial cells (IECs). However, CD1d-restricted antigen presentation has not yet been examined on IECs. METHODS A mouse intestinal epithelial cell line (MODE-K), a human epithelial cell line (T84), T84 cells transfected with CD1d and/or MHC class II, and freshly isolated human IECs were examined for their ability to present model glycolipid antigens to NK-T cells as defined by interleukin (IL)-2 or IL-4 secretion. RESULTS MODE-K and freshly isolated human IECs exhibited dose-dependent, CD1d-restricted presentation of the functional glycolipid antigen, alpha-galactosylceramide (alpha GalCer), to the mouse NK-T cell hybridoma, DN32.D3. The human IEC line, T84, mainly presented alpha GalCer when transfected with human CD1d. Presentation of alpha GalCer by CD1d-transfected T84 cells (T84d) to DN32.D3 cells was greater along the basal surface in comparison with the apical surface. Induction of the MHC class II antigen presentation machinery by cotransfecting T84d with the MHC class I transactivator (CIITA) did not alter this polarity of presentation. Neither MODE-K nor T84 cells transfected with CD1d, CD1d plus CIITA, or CD1d plus HLA-DR were able to present glycolipid antigens requiring intracellular processing. The MODE-K cell line could also present alpha GalCer to primary mouse NK-T cells. CONCLUSIONS CD1d is expressed functionally on IECs with a polarity of presentation (basal > apical) predicting a role in presentation of mucosal glycolipid antigens to local CD1d-restricted T cells.

VTX-2337 Is a Novel TLR8 Agonist That Activates NK Cells and Augments ADCC

Purpose: We aim to characterize VTX-2337, a novel Toll-like receptor (TLR) 8 agonist in clinical development, and investigate its potential to improve monoclonal antibody–based immunotherapy that includes the activation of natural killer (NK) cells. Experimental Design: HEK-TLR transfectants were used to compare the selectivity and potency of VTX-2337, imiquimod, CpG ODN2006, and CL075. The ability of VTX-2337 to induce cytokine and chemokine production from human peripheral blood mononuclear cells (PBMC) and activation of specific immune cell subsets was examined. The potential for VTX-2337 to activate NK cell activity through direct and indirect mechanisms was also investigated. Finally, we tested the potential for VTX-2337 to augment antibody-dependent cell-mediated cytotoxicity (ADCC), especially in individuals with low-affinity FcγR3A single-nucleotide polymorphism (SNP). Results: VTX-2337 selectively activates TLR8 with an EC50 of about 100 nmol/L and stimulates production of TNFα and interleukin (IL)-12 from monocytes and myeloid dendritic cells (mDC). VTX-2337 stimulates IFNγ production from NK cells and increases the cytotoxicity of NK cells against K562 and ADCC by rituximab and trastuzumab. Effects of VTX-2337 on NK cells were, in part, from direct activation as increased IFNγ production and cytotoxic activity were seen with purified NK cells. Finally, VTX-2337 augments ADCC by rituximab in PBMCs with different FcγR3A genotypes (V/V, V/F, and F/F at position 158). Conclusions: VTX-2337 is a novel small-molecule TLR8 agonist that activates monocytes, DCs, and NK cells. Through the activation of NK cells, it has the potential to augment the effectiveness of monoclonal antibody treatments where a polymorphism in FcγR3A limits clinical efficacy. Clin Cancer Res; 18(2); 499–509. ©2011 AACR.

The Ultra-Potent and Selective TLR8 Agonist VTX-294 Activates Human Newborn and Adult Leukocytes

Background Newborns display distinct immune responses that contribute to susceptibility to infection and reduced vaccine responses. Toll-like receptor (TLR) agonists may serve as vaccine adjuvants, when given individually or in combination, but responses of neonatal leukocytes to many TLR agonists are diminished. TLR8 agonists are more effective than other TLR agonists in activating human neonatal leukocytes in vitro, but little is known about whether different TLR8 agonists may distinctly activate neonatal leukocytes. We characterized the in vitro immuno-stimulatory activities of a novel benzazepine TLR8 agonist, VTX-294, in comparison to imidazoquinolines that activate TLR8 (R-848; (TLR7/8) CL075; (TLR8/7)), with respect to activation of human newborn and adult leukocytes. Effects of VTX-294 and R-848 in combination with monophosphoryl lipid A (MPLA; TLR4) were also assessed. Methods TLR agonist specificity was assessed using TLR-transfected HEK293 cells expressing a NF-κB reporter gene. TLR agonist-induced cytokine production was measured in human newborn cord and adult peripheral blood using ELISA and multiplex assays. Newborn and adult monocytes were differentiated into monocyte-derived dendritic cells (MoDCs) and TLR agonist-induced activation assessed by cytokine production (ELISA) and co-stimulatory molecule expression (flow cytometry). Results VTX-294 was ∼100x more active on TLR8- than TLR7-transfected HEK cells (EC50, ∼50 nM vs. ∼5700 nM). VTX-294-induced TNF and IL-1β production were comparable in newborn cord and adult peripheral blood, while VTX-294 was ∼ 1 log more potent in inducing TNF and IL-1β production than MPLA, R848 or CL075. Combination of VTX-294 and MPLA induced greater blood TNF and IL-1β responses than combination of R-848 and MPLA. VTX-294 also potently induced expression of cytokines and co-stimulatory molecules HLA-DR and CD86 in human newborn MoDCs. Conclusions VTX-294 is a novel ultra-potent TLR8 agonist that activates newborn and adult leukocytes and is a candidate vaccine adjuvant in both early life and adulthood.