John J. Cebra

Influences of microbiota on intestinal immune system development

The normal colonization of the mammalian intestine with commensal microbes is hypothesized to drive the development of the humoral and cellular mucosal immune systems during neonatal life and to maintain the physiologically normal steady state of inflammation in the gut throughout life. Neonatal conventionally reared mice and germ-free, deliberately colonized adult mice (gnotobiotic mice) were used to examine the efficacy of certain intestinal microbes.

Cutting Edge: Recent Immune Status Determines the Source of Antigens That Drive Homeostatic T Cell Expansion

Homeostatic proliferation of naive T cells transferred to T cell-deficient syngeneic mice is driven by low-affinity self-MHC/peptide ligands and the cytokine IL-7. In addition to homeostatic proliferation, a subset of naive T cells undergoes massive proliferation in chronically immunodeficient hosts, but not in irradiated normal hosts. Such rapid T cell proliferation occurs largely independent of homeostatic factors, because it was apparent in the absence of IL-7 and in T cell-sufficient hosts devoid of functional T cell immunity. Strikingly, immunodeficient mice raised under germfree conditions supported only slow homeostatic proliferation, but not the marked T cell proliferation observed in conventionally raised immunodeficient mice. Thus, polyclonal naive T cell expansion in T cell-deficient hosts can be driven predominantly by either self-Ags or foreign Ags depending on the host’s previous state of T cell immunocompetency.

Nasal-Associated Lymphoid Tissue Is a Mucosal Inductive Site for Virus-Specific Humoral and Cellular Immune Responses

Peyer’s patches are known as mucosal inductive sites for humoral and cellular immune responses in the gastrointestinal tract. In contrast, functionally equivalent structures in the respiratory tract remain elusive. It has been suggested that nasal-associated lymphoid tissue (NALT) might serve as a mucosal inductive site in the upper respiratory tract. However, typical signs of mucosal inductive sites like development of germinal center reactions after Ag stimulation and isotype switching of naive B cells to IgA production have not been directly demonstrated. Moreover, it is not known whether CTL can be generated in NALT. To address these issues, NALT was structurally and functionally analyzed using a model of intranasal infection of C3H mice with reovirus. FACS and histological analyses revealed development of germinal centers in NALT in parallel with generation and expansion of IgA+ and IgG2a+ B cells after intranasal reovirus infection. Reovirus-specific IgA was produced in both the upper respiratory and the gastrointestinal tract, whereas production of reovirus-specific IgG2a was restricted to NALT, submandibular, and mesenteric lymph nodes. Moreover, virus-specific CTL were detected in NALT. Limiting dilution analysis showed a 5- to 6-fold higher precursor CTL frequency in NALT compared with a cervical lymph node. Together these data provide direct evidence that NALT is a mucosal inductive site for humoral and cellular immune responses in the upper respiratory tract.

Development and maintenance of the gut-associated lymphoid tissue (GALT): the roles of enteric bacteria and viruses.

GALT can be subdivided into several compartments: (a) Peyers patches (PP); (b) lamina propria (LP); and (c) intraepithelial leukocyte (IEL) spaces. The B-cell follicles of PP are quiescent in neonatal and germ-free (GF) adult mice. Germinal centers (GC), including sIgA+ blasts, appear in the B follicles of formerly GF adult mice about 10-14 days after monoassociation with various gut commensal bacteria. The GC wax and wane over about a 3-week period, although the bacterial colonizers remain in the gut at high density. Neonatal mice, born of conventionally reared (CV), immunocompetent mothers, display GC reactions in PP postweaning, although pups of SCID mothers display precocious GC reactions at about 14 days of life. Normally, gut colonization of neonates with segmented filamentous bacteria (SFB) leads to explosive development of IgA plasmablasts in LP shortly after weaning. Commensal gut bacteria and the immunocompetency of mothers also appears to control the rate of accumulation of primary B cells from “virgin” B cells in neonates. Enteric reovirus infection by the oral route can cause the activation of CD8+ T cells in the interfollicular regions of PP and the appearance of virus-specific precursor cytotoxic T lymphocytes (pCTL) in the IEL spaces. Such oral stimulation can also lead to “activation” of both CTL and natural killer (NK) cells in the IEL spaces. More normally, colonization of the gut with SFB also leads to similar activations of NK cells and “constitutively” cytotoxic T cells.

B1 cells contribute to serum IgM, but not to intestinal IgA, production in gnotobiotic Ig allotype chimeric mice

B1 cells are a significant source of natural serum IgM, thereby serving as a first line of defense against systemic bacterial and viral infections. They can migrate to the intestinal lamina propria and differentiate into IgA-producing plasma cells and thus might play a similar role in mucosal immunity. To investigate the contribution of B1 cells to the intestinal IgA response induced by the commensal flora in immunocompetent animals, we generated gnotobiotic and conventionally reared Ig allotype chimeric mice. In this system B1- and B2-derived Abs can be distinguished based on different allotypes. FACS analysis of peritoneal cavity cells and analysis of B1- and B2-derived serum IgM indicated stable B1/B2 chimerism and the establishment of a functional B1 population. Monoassociation with either Morganella morganii, Bacteroides distasonis, or segmented filamentous bacteria induced germinal center reactions in Peyer’s patches and led to the production of intestinal IgA, partially reactive with bacterial Ag. A considerable amount of serum IgM was B1 cell derived in both monoassociated and conventionally reared mice. However, most of the total as well as bacteria-specific intestinal IgA was produced by B2 cells. These data suggest that intestinal IgA production induced by commensal bacteria is mainly performed by B2, not B1, cells.

Restricted IgA repertoire in both B-1 and B-2 cell-derived gut plasmablasts

Mucosal IgA is the most abundantly produced Ig upon colonization of the intestinal tract with commensal organisms in the majority of mammals. The repertoire of these IgA molecules is still largely unknown; a large amount of the mucosal IgA cannot be shown to react with the inducing microorganisms. Analysis of the repertoire of used H chain Ig (VH) genes by H-CDR3 spectrotyping, cloning, and sequencing of VH genes from murine intestinal IgA-producing plasma cells reveals a very restricted usage of VH genes and multiple clonally related sequences. The restricted usage of VH genes is a very consistent observation, and is observed for IgA plasma cells derived from B-1 or conventional B-2 cells from different mouse strains. Clonal patterns from all analyzed VH gene sequences show mainly independently acquired somatic mutations in contrast to the clonal evolution patterns often observed as a consequence of affinity maturation in germinal center reactions in peripheral lymphoid organs and Peyer’s patches. Our data suggest a model of clonal expansion in which many mucosal IgA-producing B cells develop in the absence of affinity maturation. The affinity of most produced IgA might not be the most critical factor for its possible function to control the commensal organisms, but simply the abundance of large amounts of IgA that can bind with relatively unselected affinity to redundant epitopes on such organisms.

Activation of RegIIIβ/γ and interferon γ expression in the intestinal tract of SCID mice: an innate response to bacterial colonisation of the gut

Background and aims: The mechanisms by which commensal bacteria provoke intestinal inflammation in animal models of inflammatory bowel disease (IBD) remain incompletely defined, leading to increasing interest in the innate immune response of the colonic mucosa to bacterial colonisation. Methods: Using gene expression profiling of colonic RNA from C.B17.SCID germ free mice and those colonised with altered Schaedler’s flora, we investigated the innate immune response to bacterial colonisation in vivo. The two most consistently induced gene groups were RegIIIβ and γ as well as interferon γ (IFN-γ) response genes. Results: Using quantitative reverse transcription-polymerase chain reaction, we showed that RegIIIβ, RegIIIγ, and IFN-γ were constitutively expressed in the colon of conventionally housed SCID mice compared with either germ free SCID or conventionally housed BALB/c mice. Induction of these genes was reproduced by chronic monoassociation of germ free SCID mice with either of two separate gut commensal bacterial species—segmented filamentous bacteria and Schaedler’s Escherichia coli. The cellular source for IFN-γ on monoassociation of SCID mice with Schaedler’s E coli was localised to a subset of intraepithelial natural killer (IENK) cells that express asialo-GM1. In vivo IFN-γ immunoneutralisation studies failed to demonstrate any alteration in RegIIIβ or γ expression. Conclusions: Thus bacterial colonisation of the colon independently activates two distinct innate immune cell types at the mucosal interface with the colonic lumen, intestinal epithelial cells, and IENK cells, a response that may be regulated by the adaptive immune system. These innate immune responses may play a role in the pathogenesis of colitis in SCID adoptive transfer models in mice and possibly in patients with IBD.

T cell control of the gut IgA response against commensal bacteria

In humans and most experimental mammals the gut lamina propria (LP) is the site of prodigious synthesis of the IgA isotype of immunoglobulin (Ig) and its secretion into the lumen. The major part of total Ig synthesis occurs here, leading to questions concerning the possible specific and non-specific stimuli of its production and the usefulness of this product to the host. Clearly, this typically continuous output of IgA is not constitutive as axenic (germ free (GF)) and newborn humans and other mammals display few secretory IgA plasmablasts in gut LP and minimal levels of secreted IgA in their gut lumen.1 2 In some way, colonisation with members of the normal gut microbiota seem to initiate the development and chronic activity of certain elements of the humoral mucosal immune system.3 4 This IgA consists of specific antibodies identifiably reactive with colonising bacteria, as well as of large quantities of IgA that cannot be shown to have been stimulated by or be reactive with particular antigens (Ags) present in food or microbes—so called “natural” IgA. In the mouse, the apparent duality of the IgA response might be explained by a difference in origin: firstly, conventional B cells (also called B2 cells) are specifically stimulated by microbial Ags and benefit from cognate interaction with Ag specific CD4+ T cells. They are clonally expanded in germinal centre reactions (GCR) in Peyers patches (PP) and mesenteric lymph nodes, and benefit from the positive selection process occurring in GCR leading to affinity maturation that results in specific IgA antibodies.5 6 Secondly, a separate lineage of B cells, termed B1 cells, can be observed in the mouse. These cells, originally defined by expression of the surface marker CD5 and high expression of IgM, arise early in ontogeny, reside in the peritoneal and pleural …

Development of mucosal humoral immune responses in germ-free (GF) mice.

GF mice can be effectively mono-associated with gram-negative bacteria Morganella morganii. This colonization results in hypertrophy of Peyer’s patches (PP), including germinal center reactions (GCR), and the development of specific IgA responses detected in vitro in PP fragment cultures. IgA antibody against the phosphocholine (PC) determinant characterizes this response. Although the mucosal response to M. morganii develops in 14 days, colonization with another, related gram-negative bacteria-proteus spcs., fails to elicit GCR or IgA antibody responses over a period of weeks. However, oral administration of cholera toxin (CT) during this period results in a prompt mucosal immune response vs. both the CT as well as the commensal bacteria. We are employing a T-B clonal microculture to quantitate and determine the kinetics of development of specific IgA pre-plasmablasts and memory B cells in relation to GCRs.

Gut colonization of mice with actA-negative mutant of Listeria monocytogenes can stimulate a humoral mucosal immune response

ABSTRACT We used Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, to study the gut mucosal immune responses following oral infection. We employed a germfree (GF) mouse model to try to accentuate the development of a humoral mucosal immune response in the gut, and we used oral colonization with one of the mutants, actA-negative (ΔactA) L. monocytogenes, to restrict infection largely to the gut. The ΔactA mutant was able to colonize the intestinal mucosa of formerly GF mice for long periods of time without causing disease while eliciting secretory immunoglobulin A (IgA) responses, as evidenced by gut tissue fragment culture assays. Flow cytometric analyses and immunohistochemical methods showed the development of only minimal germinal center reactions (GCR) in Peyers patches and more robust GCR in mesenteric lymph nodes. Pronounced increases in total (natural) IgA production occurred in gut tissues by day 7 and were maintained for up to 90 days. Levels of specific IgA were modest in gut tissues on day 14, increased until day 76, and stabilized at day 90. We also observed a significant rise in serum IgA and IgG1 levels following oral infection by listeriae. Upon colonization, the organisms mainly infected the intestines and intestinal lumen, and we only sporadically observed few colony-forming bacteria in the liver and spleen. We observed a marked rise in IgA-secreting cells, including listeria-specific IgA antibody-secreting cells, in the lamina propria of the small intestine by enzyme-linked immunospot assays. To ascertain whether some of the IgA was specific for listeriae, we performed Western blot analysis to test the reactivity of IgA from fragment cultures to antigens in sonicates of L. monocytogenes. We detected IgA binding to antigenic proteins with molecular masses of 96, 60, 40, and 14 kDa in theListeria sonicates.