John J. Cebra, PhD - US grants

Peyer's patches and other mucosal lymphoid follicles may play several roles in the generation of a secretory immune response manifest at a particular near or distant site: (1) 'antigen-sampling' accomplished by passage of intact macromolecules through specimized dome epithelial cells; (2) clonal expansion of their antigen-sensitive cells stimulated locally by the passaged antigen; (3) provision of a special milieu for generation of more secondary' IgA precursor cells; (4) release of IgA precursors which rapidly become non-recirculating IgA-immunoblasts and lodge in mucosal and exocrine tissue; and (5) release of long-lived, recirculating IgA precursors that can contribute to 'local' secretory immunity at a distant site upon subsequent exposure to antigen there. One overall objective of this project has been to determine how 'environmental' antigens, maternal antibodies passively acquired perinatally, deliberate infections via a mucosal route or deliberate exposure of mucosae to macromolecular, non-living antigens may influence the population of lymphoid cells in murine mucosal follicles and hence prejudice a subsequent secretory, IgA-antibody response by the mouse. A second major objective is to elucidate the molecular bases for (a) sessile-motile transitions occurring in mucosal follicles to release specific lymphoid cells into the circulation; (b) for the restriction of these cells to certain recirculation pathways and to certain blood-tissue egress sites; and (c) for the selective lodging of IgA cells in mucosal or exocrine tissue. A third general objective of our studies is to identify the B-cell/T-cell interactions relevant to the expression and control of a mucosal IgA response and to define the pertinent in vivo sites where these interactions may occur. A final main onjective is to understand, in general, how mucosal follicles come to accumulate such relatively high frequencies of IgA precursor cells of certain specificities and, in particular, the molecular mechanisms operative during the process of B-cell differentiation leading to a commitment to IgA expression.

Reovirus will be used as a probe of the possible roles of murine gut associated lymphoid tissues (GALT) in resistance to and resolution of viral infections commencing via the intestinal route. We will focus on local priming in Peyer's patches (PP) of specific T-helper (TH) and T-cytotoxic (TC) lymphocyte populations and on any special propensity these may display for selective circulation through and/or lodging in the rest of the intestines in lamina propria (LPL) or intraperitoneal (IEL) effector T cell compartments. Thus the efficacies of various virus-specific TH- and TC-populations from different tissues primed by different routes at limiting and containing reovirus (type 3 hemagglutinin) in the gut and protecting vs. ordinarily lethal brain infection will be tested. Further, the possible potentiation by enterically delivered reovirus of non-specific naturally occurring cytotoxicity in PP, LPL, and IEL cell populations will be assayed since perturbations in natural killer and/or naturally cytotoxic cell populations may also have significance in both viral infections and oncogenic processes in the gut. A library of serotypes of reovirus and their genetic reassortants that differ in tissue tropisms, resistance to intestinal digestive enzymes, and sites of replication will be utilized to try to correlate particular viral polypeptides with efficacy of specific T cell priming in PP and the generation of protective immunity. Particular variants of reovirus, for instance one with the neurotropism of type 3 and the resistance to intestinal digestion of type 1, will be chosen to produce model infections for assay of functional activity of specific T cells in vivo. Identification of particular viral polypeptides with effective priming of PP lymphocytes may eventually lead to the construction of more general and effective gut mucosal vaccines. Two of our recent main findings that lend encouragement to our proposed program are: 1) the novel finding of sharply elevated numbers of precursors for viral-specific TC-lymphocytes in PP following an enteric virus infection; and 2) the dramatic rise in PP viral-specific precursors for antibody-secreting clones and in a high incidence of these becoming committed to secretory antibody (IgA) production. Both these perturbations followed intraduodenal infection with type 1 reovirus, a serotype with a special propensity to adhere to dome epithelium of PP, and support the potency of reovirus as a gut mucosal antigenic stimulus of both T- and B-cell compartments.

The need for efficacious mucosal vaccines has received considerably increased attention due to: 1) the rise in antibiotic resistant enteric pathogens and the threat of gram-negative bacterial sepsis; 2) the unexpected cholera epidemics in South America; 3) the prominence of bacterial diarrhea as a killer of infants; 4) the increased incidence of tuberculosis caused by antibiotic-resistant variants; and 5) the continued spread of HIV infections. Because of our still incomplete understanding of certain major features of the mucosal immune response, attempts to develop new mucosal vaccines are still largely empiric. We plan to continue our productive program to understand the mechanisms leading to a mucosal immune response by: 1) trying to elucidate, at cellular and molecular genetic levels, the role of Peyer's patch (PP) (aggregates of lymphoid follicles in the intestinal walls) germinal centers in the development of a humoral mucosal immune response; 2) defining the cell/cell and cell/cytokine exchanges that account for the effectiveness of our B- and T-lymphocyte plus dendritic cell cultures at generating an lgA antibody response; 3) evaluating the contributions of the B2 (follicular) vs. systemic B1 subsets of B cells to protective mucosal immune responses; and 4) analyzing mechanisms that regulate the host's mucosal immune response to enteric gram-negative bacteria and facultative, intracellular gram-positive pathogens, such as Listeria, infecting via the mucosal route. We plan to use germ-free and antigen (Ag)-free mice, which have been very informative for analyzing acute, de novo gut mucosal responses, and a new generation of in vitro functional assays which we have recently developed: 1) the PP and lamina propria tissue fragment assay, which reports the immune status of the gut at the time of tissue sampling; and 2) the single/clonal B cell microculture, which reveals the lg isotype potential, frequency, and physiologic state of Ag-specific B cells. These should permit us to address our aims, for instance by permitting us to quantitate the temporal fluctuations and whereabouts of Ag-specific lgA-memory cells, IgA-pre-plasmablasts, and lgA plasma cells following acute oral stimulation with enteric viruses, bacteria, or toxins. Cross regulation and stimulation of systemic vs. mucosal compartments can also be examined in our system. Finally, we plan to probe how a mucosal response to a novel Ag, given in the context of copious amounts of a wide array of environmental Ags, may differ from the better understood systemic immune response.

We propose to continue our program concerned with the development and acquisition of competence of the gut mucosal immune system beginning in neonatal life. Our hypothesis is that gut commensal bacteria and enteric viruses drive the normal development of the gut mucosal immune system- humoral and cellular, specific and 'natural'-during neonatal life and act to maintain its 'physiologically normal' state of activation/inflammation. Despite our present appreciation of the roles and protective efficacy of some highly specific mucosal IgA antibodies and mucosal T cells, we know far less about the possible roles of the voluminous amounts of natural' IgA and the abundant 'naturally activated' T lymphocytes in the various compartments of gut-associated lymphoid tissues. Because germ-free adult mice and conventionally-reared neonatal mice share the characteristic of having a markedly underdeveloped gut mucosal immune system, we intend to compare these under controlled conditions of selective colonization with known gut bacteria of enteric viruses (gnotobiotic conditions). Thus we will rely heavily on the use of our now rather rare facility for breeding and maintaining germ-free and gnotobiotic mice. Adult germ-free mice, deliberately colonized with know microbes, provide a more tractable model for subsequent analyses of the development of the gut mucosal immune system in neonatal mice as they develop normally under either conventional or gnotobiotic conditions. We plan to use selected commensal microbes-Morganella, Ochrobactrum, Arthromitis, Helicobater, and Listeria species or mutants,, both facultative and obligate anerobes and both obligate extracellular and facultative intracellular bacteria-to colonize and perturb the 'specific' and 'natural' elements of the mucosal immune system. We plan to analyze, at a cellular and molecular level, how these organisms may drive the development of the mucosal immune system. The practical extension of these studies, which we will pursue, include: 1) mechanisms for 'colonization resistance'; 2) the cellular rationale for long-term secretory IgA mucosal immunity; 3) the bacteria/host gut epithelial interactions that may activate mucosal immunity; 4) the various mechanisms that may result in dissemination of gut bacteria to distant tissues and result in disease or to systemic immune response; 5) the role of gut bacteria in initiating or exacerbating inflammatory bowel disease; and 6) the potential enteric virus/gut bacterial interaction, via the host's gut mucosal immune system, that may affect the outcome of either the viral or the bacterial infection.

mucosal immunity; microorganism immunology; bacterial disease; enteric bacteria; inflammatory bowel diseases; bacterial antigens; antibacterial antibody; interleukin 2; bactericidal immunity; germ free condition; pathologic process; immunoglobulin A; cellular immunity; humoral immunity; antigen antibody reaction; SCID mouse; athymic mouse; flow cytometry;

This ongoing research program, in its eleventh year, has utilized respiratory/enteric orphan (REO) viruses as model probes of the mucosal immune system. Thusfar we have confined our studies to the gut and liver of various mouse hosts: immunocompetent or severe-combined immunodeficient neonates and adults. We have made a number of original fundamental findings concerning the host's mucosal immune response in the gut: especially ato elucidate the role of the Peyer's patch microenvironment in mediating preferred isotype switching of B cells to IgA (SecretaryAb) expression and in generating cytotoxic T cells (CTLs) that accumulate in intraepithelial spaces to contain enteric virus infections in the gut. Our studies have provided the functional bases for a 'cellular mucosal immune system' including both natural killer cells and cytotoxic T cells. We now propose to use reoviruses - which also display a tropism for respiratory epithelial cells - to address certain major, unresolved, fundamental questions concerning the 'mucosal' component of the immune system which functions in the respiratory tract (RALT). We plan to use assays which we have originally developed - the T dependent, clonal B cell microculture and organ fragment cultures for mucosal tissues - and the limiting dilution analyses for CTLs plus MAbs vs. mucosal 'homing' receptors, in these attempts: l) to determine whether RALT contains inductive sites analogous to those in Peyer's patches where preferred isotype switching to IgA occurs; 2) to assess whether CTLs with 'mucosal' homing propensities can be generated in the gut or RALT, and whether these can contribute effective protection to the respiratory tract; and 3) whether effective 'cross-priming' between gut and RALT can occur. The results of these analyses should be extremely relevant for the development of efficacious vaccines vs. respiratory pathogens.

Project 1: Use of Severe Combined Immunodeficient and Immunocompetent Mice to Analyze the Pathogenesis of Oral Listeriosis (and CNS-Disease) We propose to use Severe Combined Immunodeficient (scid) and Immunocompetent (imcomp) mice, either conventionally reared (CNV) or with no gut microbial flora (GF, germ free) as models to analyze the pathogenesis or oral infection by L. monocytogenes and the host response to such gut mucosal challenge. L. monocytogenes is an opportunistic, sometimes fatal, pathogen of humans and a more common pathogen of domestic food animals. We have developed the first laboratory animal model for Central Nervous System (CNS)-listeriosis following oral infection. Oral infection of CNV scid mice with 'wild-type' L. monocytogenes results in CNS-infection and symptoms--classically 'circling disease' and death within 14-21 days. We plan to use 'wild-type' virulent L. monocytogenes, and four virulence- factor negative ('knock-out') strains of L.monocytogenes to probe the mechanisms of pathogenesis via the gut mucosal route and the role the elements of the host's mucosal immune system may play to prevent, contain, and resolve gut mucosal infections. We believe our finds may be relevant to understanding and immunizing versus gut mucosal infections. We believe our findings may be relevant to understand and immunizing versus gut mucosal infections by many facultative, intracellular bacterial pathogens. We aim to: 1) analyze how oral/gut mucosal L. monocytogenes can translocate the gut, disseminate and transit the blood-brain barrier; 2) evaluate which elements of the host's mucosal immune system may act to prevent and limit infection; 3) determine whether 'innate' or 'natural' immune mechanisms may be effecting at limiting infection by the gut mucosal route; 4) use L. monocytogenes mucosal infections as a model for evaluating whether secretory IgA Abs can effectively contain an infection of the mucosal epithelium by a bacterial intracellular pathogen.