Victor E. Reyes, Ph.D. - US grants

The long-term objective of this project is to define the role of the invariant chain (I-i) in antigen processing and presentation. To approach this goal the following aims are proposed: (1) To define the conditions that lead to cleavage and release of I-i from class II MHC molecules; (2) To characterize the cleavage fragments generated during the proteolytic events which lead to I-i release; (3) To identify the region(s) within I-i which associate(s) with class II MHC or the antigen binding site; and, (4) To determine whether peptide binding to class II MHC molecules depends upon or is enhanced by the removal of I-i. To address Aim 1, the proposed experiments are aimed at mimicking the endosomal environment (low pH and/or the presence of specific proteases) to induce I-i release from class II MHC as detected by immunoprecipitation and SDS-PAGE analysis. As part of Aim 2, the fragments generated during cleavage and release of I-i will be characterized through Western blotting with antibodies to N-terminal (VicY1) and C-terminal (E1) epitopes in I-i as well as rabbit antisera to synthetic peptides corresponding to various regions within I-i. Partial N-terminal sequencing of the fragments will be performed on fragments isolated by either reverse phase HPLC or 2D electrophoresis and electroblotting. In Aim 3, isolated I-i fragments which result following cleavage and release of I-i from class II MHC molecules will be examined for their ability to block peptide presentation presumably as a result of binding to the antigen binding site on class II MHC molecules;. thus, identifying the I-i sequence which blocks the desetope. Having elucidated the conditions which lead to I-i removal, in Aim 4 those conditions will be reproduced in the presence of influenza peptides derivatized with a crosslinking reagent and iodinated. Their binding will then be assessed, subsequent to crosslinking induced by U.V. light exposure, through immunoprecipitation with anti-class II antibodies, electrophoresis, and autoradiography. Alternatively, binding can be examined through gel filtration to separate bound from free peptides. The answers derived from these studies will serve a dual role: (a) enhance our understanding of a fundamental question in immunology and (b) identify a region in Ii which might serve as a backbone in the synthesis of peptide-based vaccines.

The long-term objective of this project is to define the role of the invariant chain (I-i) in antigen processing and presentation. To approach this goal the following aims are proposed: (1) To define the conditions that lead to cleavage and release of I-i from class II MHC molecules; (2) To characterize the cleavage fragments generated during the proteolytic events which lead to I-i release; (3) To identify the region(s) within I-i which associate(s) with class II MHC or the antigen binding site; and, (4) To determine whether peptide binding to class II MHC molecules depends upon or is enhanced by the removal of I-i. To address Aim 1, the proposed experiments are aimed at mimicking the endosomal environment (low pH and/or the presence of specific proteases) to induce I-i release from class II MHC as detected by immunoprecipitation and SDS-PAGE analysis. As part of Aim 2, the fragments generated during cleavage and release of I-i will be characterized through Western blotting with antibodies to N-terminal (VicY1) and C-terminal (E1) epitopes in I-i as well as rabbit antisera to synthetic peptides corresponding to various regions within I-i. Partial N-terminal sequencing of the fragments will be performed on fragments isolated by either reverse phase HPLC or 2D electrophoresis and electroblotting. In Aim 3, isolated I-i fragments which result following cleavage and release of I-i from class II MHC molecules will be examined for their ability to block peptide presentation presumably as a result of binding to the antigen binding site on class II MHC molecules;. thus, identifying the I-i sequence which blocks the desetope. Having elucidated the conditions which lead to I-i removal, in Aim 4 those conditions will be reproduced in the presence of influenza peptides derivatized with a crosslinking reagent and iodinated. Their binding will then be assessed, subsequent to crosslinking induced by U.V. light exposure, through immunoprecipitation with anti-class II antibodies, electrophoresis, and autoradiography. Alternatively, binding can be examined through gel filtration to separate bound from free peptides. The answers derived from these studies will serve a dual role: (a) enhance our understanding of a fundamental question in immunology and (b) identify a region in Ii which might serve as a backbone in the synthesis of peptide-based vaccines.

Helicobacter pylori is an important gastrointestinal pathogen which is implicated in chronic gastritis, recurrent peptic ulceration and gastric cancer. As adenocarcinomas and lymphomas are a complication of other chronic inflammatory diseases of the gastrointestinal tract, including celiac disease and ulcerative colitis, the inflammatory process itself has been implicated in the pathogenesis of tumor development. Interestingly, altered T cell function may play a role in tumor development since patients or laboratory animals with congenital T cell deficiencies develop significantly more tumors, often in association with chronic gastrointestinal inflammation. Several mechanisms may allow T cell function to control the development of cancer during H. pylori infection. First of all, T cell-mediated immune surveillance against developing gastric neoplasms could be impaired if certain T cell responses are suppressed as a consequence of H. pylori infection. Secondly, the tumor targets themselves may avoid detection of bacteria, inflammatory mediators or cytokines, decrease the expression of surface molecules that normally signal T cells to recognize the destroy transformed cells. Although few studies have addressed this aspect of the pathogenesis of gastric disease associated with H. pylori, peripheral blood mononuclear cells from patients with gastric cancer have been shown to have suppressed cytotoxic activity against tumor cells. Our own preliminary data suggest that the expression of surface antigens by gastric epithelial cells is altered during infection with H. pylori which my, in turn, lead to inappropriate T cell activation and suboptimal tumor surveillance. These observations highlight the need to define how gastric T cell responses are regulated during H. pylori infection and lead to our hypothesis that infection with H. pylori modulates the role of the gastric epithelial cell as an antigen presenting cell leading to altered T cell activation and diminished surveillance for gastric neoplasms. To test this hypothesis we will address the following specific aims; 1) Characterize athe ability of gastric epithelial cells to modulate T cell function. This aim will define the elements required for gastric epithelial cells to be considered antigen presenting cells thereby enabling them to influence T cell activation or effector function. 2) Characterize the mechanisms of peptide generation and association with class II MHC in gastric epithelial cells. This aim determine how H. pylori antigens are handled by gastric epithelial cells an will identify the intracellular site of Class II MHC-H. pylori antigen encounter. 3) Characterize H. pylori peptides which are naturally processed and selected for presentation by gastric epithelial cells. These studies will identify dominant, H. pylori-derived peptides which are bound by Class II MHC expressed by gastric epithelial cells. 4) Determine the effects of H. pylori infection on T cell function. The studies in this aim will examine the impact of H. pylori stimulated antigen presentation of T cell activation/suppression. Together, these studies will enhance our understanding of the control of cell mediated immunity in the gastric mucosa in response to H. pylori infection and identify molecular markers which are associated with the pathogenesis of gastric cancer.

DESCRIPTION (provided by applicant): Helicobacter pylori (Hp) infects the gastric mucosa of >50% of the world's population. Infection with this gram negative bacterium results in significant gastroduodenal diseases that include chronic gastritis, ulcers and cancer. Prevalence rates of Hp infection are high among underrepresented minority and lower income populations. These groups also have the highest risk of developing gastric cancer. The chronic inflammation that is a hallmark of the infection is considered a risk factor for the development of gastric cancer. Individuals infected with strains that carry the functional form of the cytotoxin-associated gene A, cagA, have an increased risk of developing gastric cancer. Hp is thus the first bacterial agent to be classified as a class I carcinogen. Since gastric cancer is the second deadliest form of cancer and delays in diagnosis contribute to poor prognosis, the detection of biomarkers that not only report the infection, but also allow detection of carcinoma development is clearly important. Since duodenal ulcer disease and gastric cancer are mutually exclusive, we hypothesize that a comparison of the proteomic profile of gastric epithelial cells of patients infected with Hp but with different diseases linked to this infection will reveal important disease-specific biomarkers of gastric cancer. To test this hypothesis we plan to address the following specific aims: Aim 1. Characterize the differences in the biomarker profile of gastric epithelial cells from patients infected with Hp who are diagnosed with gastric cancer or duodenal ulcers. The protein profile of uninfected donors will be used as a baseline control. In this aim, gastric biopsies will be used to isolate epithelial cells, whose proteins will be solubilized for profiling by high resolution two-dimensional gel electrophoresis (2-DE). Biomarkers whose expression is consistently altered in gastric cancer patients and not in duodenal ulcer patients will be identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flight MS (MALDI-TOF- MS). Changes in expression of these proteins will be validated by western blotting with specific antibodies and confirmed by immunohistochemistry of representative tissues. Aim 2. Determine what biomarkers are induced by in vitro infection of gastric epithelial cells with Hp. In this aim, gastric epithelial cells from uninfected individuals will be infected with cagA+ strains of Hp or cagA- strains, as controls, to determine what biomarkers detected in Aim 1 are induced early in the course of infection and could thus be considered early biomarkers of cell transformation. PUBLIC HEALTH RELEVANCE: Gastric cancer is the second deadliest cancer worldwide and its linked to infection with the Helicobacter pylori bacterium. Since in its early stages gastric cancer causes no symptoms, delays in diagnosis contribute to the high mortality rate associated with this form of cancer. The objective of this project is to implement high throughput technology to identify proteins that could become candidate biomarker signatures that would aid in early diagnosis of Helicobacter pylori infection and gastric cancer development.

DESCRIPTION (provided by applicant): Ulcerative colitis (UC) and Crohn's disease (CD) are chronic inflammatory conditions globally known as inflammatory bowel diseases (IBD). IBD is considered a disruption of tolerance to intestinal microflora, leading to dysregulation of mucosal CD4+ T cell responses and chronic inflammation. Most damage in IBD involves activated CD4+ T helper (Th) cells. Naive T cells activated by antigen-presenting cells (APCs) differentiate into Th cell subsets depending on the signals received. CD and UC differ in the types of activated Th cells. CD is linked with a Th1, and perhaps a Th17, cell phenotype. UC is linked with an atypical Th2 phenotype. PD-L1 (a.k.a. CD274, B7-H1) plays a key role in peripheral T cell tolerance and is thought to control acute inflammation via suppression of ongoing activated T cell responses. Altered expression of PD-L1 on APCs in nonlymphoid peripheral tissue appears in the progression of chronic inflammatory disease associated with Th1/Th2 unbalanced responses. PD-L1 expression increases in patients with IBD and in a model of colitis. Thus, as dysfunction of immune regulation is key in the onset and development IBD, the role of PD-L1 and PD- L2, which share the PD-1 receptor on T cells, has to be considered. Despite ample information on the infiltrating immune cells in the inflamed mucosa, the phenotype of the PD-L1 overexpressing cells and their role in IBD is unknown. Our studies show that colonic subepithelial myofibroblasts (CMFs) are the most common (~70%) class II MHC+ and PD-L1+ cell phenotype in the colonic mucosa, whose PD-L1 expression increases in IBD. Together, these observations/preliminary data obtained led us to hypothesize that overexpression of PD-L1 by CMFs plays a role in the dysregulated CD4+ T cell response in the intestinal mucosa leading to IBD. The studies are significant because understanding the mechanisms by which CMFs contribute to mucosal tolerance and how an alteration in their expression of PD-1 ligands contributes to chronic gut inflammation will offer vital insights into the onset and development of IBD. We propose to address the following aims: 1. Define the mechanism(s) responsible for PD-L1 overexpression in CMFs. We will investigate how TLRs recognizing bacterial products and cytokines produced during IBD affect PD-L1 expression. 2. Determine the role that PD-L1 expression by CMFs has in the regulation of the Th1/Th2 balance. We will examine CMFs (or DC controls) in (A) co-cultures with CD4+ T cells activated under conditions polarizing to Th1 or Th2 profiles and (B) co-cultures with activated Th1/Th2 CD4+ T cells to assess inhibition of T cell proliferation. 3. Determine the contribution of PD-L1 overexpression by CMFs in a mouse model of ulcerative colitis. To determine the relative in vivo contribution of CMFs in UC, we will use the oxazolone-induced colitis model to study the expression of PD-L1 and PD-L2 by CMFs and if blocking antibodies to PD-L1/PD-L2 ameliorate colitis. Further, PD-L1-/- and PD-L1-/-/PD-L2-/- knockout mice will allow to assess the role of PD-L1 while (MK2-/-) deficient in myofibroblast development will allow us to examine the role of CMFs in colitis.

DESCRIPTION (provided by applicant): Our goal is to define how Helicobacter pylori (Hp) impairs the host's immune response to establish a lifelong infection. Hp infects the gastric mucosa of >50% of the world's population. This bacterium is a causal agent for chronic gastritis, duodenal and gastric ulcers. Hp is also linked to gastric cancer. The mechanisms that allow persistent infection for decades and cause disease are not known. The inability of the host response to clear Hp infection suggests inhibition of the immune response. The CD4+ lymphocytes in the Hp-infected gastric mucosa are hyporesponsive and the mechanisms responsible are unclear. Our preliminary studies showed that Hp induces gastric epithelial expression of novel co-inhibitory receptors and cytokines that may inhibit antigen specific CD4+ T cells as well as the induction of T regulatory cells that suppress effector T cells. These data led us to hypothesize that Hp impairs protective T cell immunity using the epithelium as a mediator. To test the hypothesis we will: (1). Determine the mechanism(s) used by Hp to induce on gastric epithelial cells (GECs) the expression of B7 family ligands that inhibit CD4+T cells. We will investigate mechanisms behind the Hp induction of the expression by GECs of B7 family co-inhibitors of T cells. The expression of these ligands by GEC will be assessed by independent methods that include RT-PCR, western blot analysis, flow cytometry and functional analysis of suppression of T cells. (2). Determine the contact-independent mechanisms involved in inhibition CD4+ T cell function by gastric epithelial cells during Hp infection. We will examine the production by GECs of a cytokine (IL-16) that specifically acts on cells expressing CD4+ and inhibits their proliferation and function. Evaluation will be by ELISA, intracytoplasmic staining and specific function. Also, Hp-infected GECs will be examined for their release of exosomes that will be characterized for the expression of B7 family proteins and will be assessed for their inhibitory activity on T cells. (3). Determine the mechanisms whereby Hp-infected gastric epithelial cells influence the balance of T regulatory cells and Th17 cells. We will investigate how Hp-infected GECs support the induction of T reg cells from naove T cells populations. As Th17 are developmentally linked to T regs and are found in the Hp-infected gastric mucosa, the effect of Hp-infected GECs on Th17 induction will also be examined. The T cells that develop will be phenotypically (flow cytometry) and functionally (cytokine and bioassay) characterized. (4). Determine whether the mechanisms of Hp immune avoidance characterized in vitro are also involved in vivo in a mouse model of Hp infection. The role of B7 family members in T cell inhibition and/or T reg or Th17 expansion will be assessed in vivo by the infection of B7-H1-/- or B7-H1-/-/B7-DC-/- knockout mice. Also, antibody blocking of IL-16 in vivo will be investigated to determine its role in T cell hyporesponsiveness. The results from these studies will provide important insights into underlying causes that prevent the host to clear Hp infection, allowing the infection to remain chronic for decades and contribute to immune evasion by tumors. PUBLIC HEALTH RELEVANCE: Helicobacter pylori is a common human pathogen and infection with this bacterium causes gastritis and may lead to stomach ulcer and cancer. The objective of these studies is to define how H. pylori avoids clearance by the human immune response and becomes established as a chronic infection. Specifically, we will examine how H. pylori induces the expression by stomach epithelial cells of mediators that have immunoinhibitory effects, which could represent potential therapeutic targets for the important diseases associated with this infection.