But with the use of a roof pitch in degrees converter, you will be able to get the correct measurement. With the use of this converter, it will be easier for you to locate a 30 degree roof pitch, 20 degree roof pitch, 25 degree roof pitch or a 15 degree roof pitch.
Published online 2012 Oct 4. doi: 10.3389/fimmu.2012.00313
PMID: 23060888
This article has been cited by other articles in PMC.
Abstract
As of now it looks promising, when the new devteam keeps up the pace with patches - essentially a big patch every month for now. Golemlabs was the original devteam for TG3, which were not responsible for TG2 (which was originally made by 4HEAD Studios - TG2R had been a fan mod, which then was adapted and incorporated and rereleased. The WSDOT specifications state PG64-22 asphalt at hot mix plants may be heated to a maximum temperature of 177°C (350°F). However, the preferred storage temperatures for PG64-22 is 140°C to 168°C (285°F to 335°F). Unnecessarily high temperatures result in increased hardening and heating costs. Choose to 'unzip/install' the patch file into that directory, and when you start your game again it should in the main menu say patch 4.17b. VERY IMPORTANT. Start a new savegame, do not use an old version since the changes the patch make only get activated in a new savegame. Tg2_patch_40_to_41[1].exe is a Microsoft or Windows process but some versions of this exe carry viruses. Check your PC for the bad versions of this file - with this Free Security Scan.
The function of intestinal immunity is to provide protection toward pathogens while preserving the composition of the microflora and tolerance to orally fed nutrients. This is achieved via a number of tightly regulated mechanisms including production of IgA antibodies by intestinal plasma cells. Celiac disease is a common gut disorder caused by a dysfunctional immune regulation as signified, among other features, by a massive intestinal IgA autoantibody response. Here we review the current knowledge of this B-cell response and how it is induced, and we discuss key questions to be addressed in future research.
Keywords: celiac disease, autoantibodies, mucosal immunity, intestinal mucosa, B cells
The immune system has evolved multiple strategies to maintain intestinal homeostasis. A unique feature of intestinal immunity is the ability to provide protection toward pathogens while preserving the number and composition of the commensal bacteria in a state of mutualism (). Secretory IgA is considered to be one of the crucial immune effector mechanisms in maintaining homeostasis at mucosal surfaces (; ). Therefore, it is not surprising that the mucosal immune compartment is tightly regulated. In this review, we aim at summarizing and discussing the basic concepts of intestinal immunobiology in the context of a prevalent disorder, celiac disease. The study of this condition provides unique understanding of how an intestinal IgA response is induced and reshaped from the healthy to the affected intestinal state, as well as it pinpoints areas of scarce knowledge and poses key questions for future research.
FEATURES OF INTESTINAL IMMUNITYTHE INTESTINAL IMMUNE SYSTEM![]()
The generation of secretory IgA is confined to intestinal lymphoid microenvironments that are composed of the inductive and the effector tissue compartments (). The inductive compartment of the intestinal immune system consists of gut-associated lymphoid tissue (GALT) and the regional lymph nodes, whereas the effector compartment consists of the lamina propria (LP) and surface epithelia. Together, these form the largest effector organ of humoral immunity, containing at least 80% of the body immunoglobulin (Ig)-producing cells ().
GUT-ASSOCIATED INDUCTIVE LYMPHOID TISSUE
The GALT is the main site for the induction of mucosal IgA B cells. The GALT is comprised of aggregated lymphoid follicles, termed Peyer’s patches and isolated lymphoid follicles. Gut-associated lymphoid follicles are organized structures covered with a specialized follicle-associated epithelium that contains microfold cells (M cells; ). In the canonical pathway, antigens from the gut lumen are internalized and delivered to subepithelial dendritic cells (DCs) via M cells or receptor-mediated endocytosis by epithelial cells (). Antigen-loaded DCs migrate from the subepithelial dome into the perifollicular T cell-rich area, where they can induce a response of helper T cells (). B cells become activated by the presentation of antigens from follicular DCs () and by CD40-mediated signals from antigen-primed helper T cells (). Gut-associated lymphoid follicles are, therefore, characterized by germinal centers (GC) that promote antigen-specific interaction between T and B cells, an essential mechanism for B cell differentiation and diversification. The GALT further drives intestinal IgA production by providing cytokines with IgA-inducing functions, including transforming growth factor-β (TGF-β; ; ), retinoic acid (RA; ), IL-6 (), and inducible nitric oxide synthase (iNOS; ). These events lead to up-regulation of the gene encoding for the enzyme activation-induced deaminase (AID), which is central to both class-switch recombination and somatic hypermutation of Ig genes (). This T cell-dependent pathway usually results in generation of plasma cells (PCs) producing intestinal IgA antibodies with high rates of somatic hypermutation (SHM), as well as memory B cells (). CD40-mediated signal from T cells is crucial to GALT GC initiation (), although it is possible that human intestinal GC-associated B cell responses are not exclusively dependent on cognate T cell/B cell interactions (). Studies have indicated that GCs can appear in Peyer’s patches and mesenteric lymph nodes without the need for the classic T cell/B cell interaction based on BCR specificity (). Accordingly, SHM can take place as an antigen-independent process without being necessarily linked to affinity maturation (; ). B cells that are activated in this way are thought to depend on “bystander” T cell help in the form of cytokines such as IL-5, IL-6, and IL-10 in order to induce an IgA response ().
T cell-independent intestinal IgA responses have been reported in both humans (; ) and mice (; ). In mice a T cell-independent, antigen-driven pathway in response to highly conserved microbial antigens recognized by Toll-like receptors (TLRs; ) generates a primitive intestinal IgA antibody repertoire to commensal bacteria (; , ). TLR-triggered class switching to IgA is mediated by BAFF and APRIL expression in DCs, monocytes, macrophages, granulocytes, and intestinal epithelial cells (; ; ). T cell-independent intestinal IgA responses in mice originate from peritoneal and intestinal LP B-1 cells as well as conventional follicular B-2 cells (). Thus, it has been suggested that the intestinal LP may act as a potential site for T cell-independent IgA induction. A lack of consensus, as well as a lack of homology between murine and human models () as the human equivalent of mouse B-1 cells is still not well described (), does however exist when it comes to the LP being a site for IgA class switching and diversification (; ; ; ; ; ; ; ). Although T cell-independent responses are biologically possible and certainly relevant in mice, in humans their contribution to the intestinal IgA repertoire remains uncertain.
Rv Trailer 20 To 22 FtGUT-ASSOCIATED EFFECTOR LYMPHOID TISSUE
The intestinal LP acts as the effector compartment of the humoral mucosal immune response, and contains predominantly terminally differentiated PCs (). These are characterized by the expression of the markers CD138 and CD27, whereas levels of CD19 and CD45 are more heterogeneous; the lack of expression of the Ki-67 marker indicates that these cells are not blasting and should therefore be referred to as PCs rather than as plasmablasts (). Human intestinal PCs preferentially produce IgA dimers (80%) and IgM pentamers (20%), whereas a small fraction produces IgG (; ). The amount of Ig produced by intestinal PCs is massive, being estimated at 3 g/day in adults (). Dimeric IgA is formed of two monomeric IgA units joined by a bridging J chain that is recognized by the polymeric Ig receptor (pIgR), an antibody transporter expressed on the basolateral surface of epithelial cells (; ). Upon interaction with pIgR, dimeric IgA is translocated to the surface of epithelial cells as secretory IgA where it exerts its function of immune exclusion, intracellular neutralization, and antigen excretion (; ). The intestinal LP in addition to PCs contains a variety of other cell types, including macrophages, DCs, and neutrophils which in association with epithelial cells may play a crucial role in creating and maintaining the niche for PC survival ().
Antigen-specific IgA antibodies generated upon immunization can be identified only locally () or both systemically and locally () depending on the immunization strategy. These observations, together with the fact that IgA reactive to commensal bacteria is exclusively present in gut secretions (), support the notion of mucosal immunity as an independent compartment. Rotavirus-specific memory B cells were shown to have different antibody repertoires () than effector mucosal PCs (), which raises some questions about the nature of the interplay between these two compartments.
THE INTESTINAL IgA REPERTOIRE
In principle, both unmutated IgA antibodies with broad reactivity to self and non-self antigens and somatically mutated antigen-specific IgA antibodies could contribute to the intestinal PC repertoire. There are several observations suggesting that the latter pathway may be dominating. reported results supporting GC origin of human intestinal IgA-producing PCs. Cloning and sequencing of Ig variable region genes of IgA PCs from LP of human small intestine revealed uniformly high degrees of SHM and high ratios of replacement to silent (R/S) mutations in complementarity determining regions (CDR), which argues in favor of antigen-mediated selection (; ). Our group isolated human intestinal PCs specific to rotavirus and cloned antibody genes, observing high numbers of mutations (17 mutations per sequence on average, with an R/S of 2.3, in the VH only; ). Similarly, cloned hundreds of human monoclonal antibodies (hmAbs) from IgA and IgG intestinal plasmablasts from the ileum of healthy donors. Regardless of their reactivity, the antibodies had many mutations, averaging 23 in the VH. The majority of the antibodies were specific for foreign or self antigens whereas 25% were polyreactive. Only 7% of IgA displayed cross-reactivity with diverse bacterial strains. To note, the polyreactive antibodies also had high degree of SHM suggesting that antibody polyreactivity of gut plasmablasts may be acquired by somatic mutations. Selection of somatically mutated variants of polyreactive antibodies may simultaneously act as a parallel mechanism in defining and contributing to antigen-specific immune response against foreign antigens. Revision of light chains expressed by IgA PCs is another distinct feature of human intestinal PCs and it is confined to gene rearrangements at the lambda loci (). This has been suggested as a beneficial mechanism in order to diversify the intestinal IgA repertoire and to remove non-functional or autoreactive antibodies.
Contrary to initial studies suggesting that the intestinal IgA repertoire is oligoclonal and of low diversity (; ; ), using a high-throughput sequencing method, recently demonstrated that the intestinal IgA population in mice is highly polyclonal. The repertoire is comprised of both highly expanded and low frequency clones, and with age new clones are introduced. Notably, expanded clones with previously selected specificities repopulated the small intestine after PC depletion and showed similar SHM frequencies, hence indicating the presence of a functional intestinal IgA memory compartment. A polyclonal and highly diverse IgA repertoire would parallel the broad range of intestinal antigens to which the intestinal mucosa is constantly exposed, although it also appears that the repertoire continuously adapts to the current composition of the microflora (). The high-throughput analysis of the intestinal IgA repertoire performed by also suggested that clonal expansion is likely to occur predominantly in the periphery rather than locally in the LP, which has been a subject of debate in previous studies ().
Taken together, these studies strongly indicate that the great majority of gut IgA antibodies develop from antigen-specific B cell responses, which evolve by acquisition of somatic mutations to confer effectiveness and high specificity.
LONGEVITY OF INTESTINAL IgA PLASMA CELLS
Another matter of debate has been whether human intestinal IgA PCs can provide long-term humoral immunity. Evidence for a long-lived, commensal-specific IgA response was observed when germ-free mice were reversibly colonized by bacteria (). In absence of competition from newly generated cells, IgA PCs were shown to have a half-life of at least 16 weeks. The dynamic of such IgA response, however, reflected the contents of the intestinal lumen, suggesting that the number of long-lived PC niches is limited. As a consequence, in presence of competition such as that deriving from the continuously evolving microflora, “older” PCs – despite their long-lived potential – are constantly displaced by new ones, which are generated in response to the most recent stimuli. In agreement with these observations, we have demonstrated that the human small intestine harbors a population of non-proliferating PCs that are maintained by the local supportive microenvironment for long-term survival (). Moreover, an inflammatory microenvironment may enhance the niche capacity, resulting in more robust PC responses ().
MUCOSALLY INDUCED TOLERANCE
The homeostatic role of the intestinal immune system is to provide suppressed immune responses as to generate mucosally induced tolerance. Such tolerance can be directed toward orally administered antigens or toward gut bacteria. Thus, there are two layers of intestinal anti-inflammatory homeostatic mechanisms: immune exclusion of commensal bacteria by secretory antigen-specific IgA and immune suppression to avoid hypersensitivity to innocuous food antigens. These two mechanisms of mucosally induced tolerance appear to act independently in order to attenuate a broad range of immune responses (; ). The lack of such homeostatic tolerance results in intestinal immune pathology. Active proinflammatory immune responses directed toward the gut microbiota, inducing imbalance in IgA and IgG repertoires, are associate with the development of inflammatory bowel disease, such as Crohn’s disease and ulcerative colitis (; ). In celiac disease (CD) there is an active proinflammatory immune response to cereal gluten antigens.
CELIAC DISEASE
Celiac disease is a common intestinal disorder affecting 1% of the population in Europe and the US, although only a fraction of patients is readily diagnosed due to the highly variable clinical presentation of the disease (). CD can be considered a food intolerance to wheat gluten (consisting of the gliadin and glutenin subcomponents) and related proteins from rye and barley. In genetically predisposed individuals, gluten ingestion can cause an inflammatory reaction in the upper small intestine which gives tissue damage leading to villous atrophy (). The lesion and inflammatory changes disappear after weeks or months when patients for treatment purpose commence a gluten-free diet (GFD). The inflammatory reaction appears to be driven by activation of Th1-like CD4+ T cells (see Box 1) that recognize gluten peptides post-translationally modified by the enzyme transglutaminase 2 (TG2; ; ). What initiates this “aggressive” T cell response and lack of oral tolerance to gluten is not known. It has been suggested that some part of gluten may have innate properties or that infections may play a role (reviewed in ). In steady-state conditions, the maintenance of intestinal homeostasis is initiated by intestinal DCs that are affected by enterocyte-derived factors, such as retinoic acid and TGF-β, conferring tolerogenic properties on the DCs. Tolerogenic DCs educate the intestinal immune system to respond in a non-inflammatory manner to orally administered proteins by the induction of regulatory T (Treg) cells. An alteration of the intestinal environment, as observed in CD, characterized by a high level of inflammatory cytokines such as IL-15 and IFNα, may affect the acquisition of the tolerogenic phenotype of intestinal DCs. This will prevent the induction of Treg cells, further promoting the differentiation of proinflammatory T cells (). Interestingly, beside the strong gluten-specific T cell response, CD presents autoimmune features, most notably the production of autoantibodies. These antibodies are primarily directed against TG2 (), but antibodies specific for other autoantigens like actin, collagen and others have also been described (). Whereas the T cell response to gluten has been thoroughly characterized and is relatively well understood, significantly less is known about the B cell responses in CD.
CD is a multifactorial disease with a complex interplay between genetic and environmental factors eventually leading to chronic inflammation (see ; for review and references therein). Of these factors, the most significant genetic component is HLA; 90% of celiac patients carry a variant of DQ2 termed DQ2.5, whereas most of the remaining patients carry DQ8. The HLA association has been extensively investigated, and the study of lesion-derived T cell lines and T cell clones has allowed a detailed description of gluten T cell epitopes. Several epitopes exist and some epitopes are more frequently recognized than others (reviewed in ). An important feature of both the DQ2.5 and DQ8 molecules is their preference for binding of negatively charged amino acid residues (i.e., glutamate or aspartate) in certain binding pockets (P4, P6, and P7 for DQ2.5; P1 and P9 for DQ8). Gluten proteins have very few negatively charged residues, however they carry a high amount of glutamine and proline residues. Interestingly, glutamine can be deamidated to glutamate by the enzyme TG2, and TG2-modified gluten peptides show strong immunogenicity. This suggests that, under particular circumstances, deamidation happens in vivo, leading to the formation of post-translationally modified gluten peptides that are suitable for presentation by DQ2.5 or DQ8 molecules. The gluten T cell epitopes are furthermore hallmarked by the presence of multiple proline residues, and this is particularly so for epitopes presented by DQ2.5. Proline, in addition to influencing MHC binding, exerts a force in the selection of T cell epitopes at two additional levels. First, peptides rich in proline are resistant to proteolysis, and proline-rich gluten peptides survive gastrointestinal digestion allowing them to reach the LP where they can by loaded on HLA-DQ molecules expressed by antigen-presenting cells. Second, proline guides the specificity of TG2 so that glutamine residues in the motif glutamine-X-proline are targeted. Notably, the peptides harboring T cell epitopes in a complex gluten digest are the preferred TG2 substrates. How these forces work can be visualized by looking at the α2-gliadin, a representative α-gliadin. Upon treatment with gastric and pancreatic endopeptidases a relatively large fragment, the 33-mer LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (residues 57 to 89), survives digestion. Due to its length this peptide is resistant to digestion by small intestinal brush-border membrane ectopeptidases as well. Three glutamine residues within the 33-mer are deamidated by TG2. This deamidated 33-mer harbors six overlapping T cell epitopes and is an extremely potent antigen. Upon recognition of deamidated gluten peptides, the CD4+ T cells become activated and start producing cytokines including interferon-γ and interleukin-21. The priming of the gluten-specific CD4+ T cells likely takes place in GALT or mesenteric lymph nodes and the primed cells seed via the blood into the LP as effector cells. These T cells may be important for forming PC survival niches in the LP.
TG2 has several biological functions which include transamidation (cross-linking) and deamidation, and is involved in many physiological processes (). TG2 is present in large amounts in the gut LP, in particular in a sub-epithelial layer. It is hardly coincidental that TG2 is also the target of the autoantibodies in CD. Studies based on in situ detection with immunofluorescence () and phage display antibody libraries () suggested that anti-TG2 antibodies are produced locally in the small intestine, and recently we were able to visualize the intestinal PCs producing such antibodies (). In the following, we will describe the current knowledge and the future directions in the study of the intestinal B cell response in CD.
THE INTESTINAL B CELL RESPONSE IN CD
The celiac lesion is characterized by considerable expansion of the PC population (; ) and enhanced local immunoglobulin secretion (; ). In addition, there are IgA deposits at the epithelial basement membrane of the small intestine (; ) which can be observed without overt histological changes (). The plasmacytosis (increased median PCs per mucosal tissue unit of 2.1, 3.8, and 2.9-fold for IgA, IgM, and IgG respectively; ; ) may relate to bolstering of a PC survival niche. Local plasmacytosis in CD appears to be homeostatic with an unaltered immunoglobulin isotype distribution and marked preponderance of IgA PCs (). Notably, the duodenal IgA PC population in active CD maintains mucosal phenotype by J-chain expression and consists of a higher proportion of the IgA2 subclass than in the normal duodenal mucosa (). Upon dietary gluten restriction, intestinal PC numbers are reduced ().
ANTI-GLIADIN AND ANTI-TG2 ANTIBODIES
Early experiments performed by ELISA, ELISpot, and immunofluorescence indicated local intestinal secretion of anti-gliadin antibodies (; ; ; ). These studies suggested that gliadin-specific PCs account for 1–2, 10 and 5–10% of total IgA, IgM, and IgG PCs, respectively, in the small intestine of CD patients. Anti-gliadin IgA and IgG antibodies are detected in sera of untreated CD patients and can be harnessed as a diagnostic tool. These antibodies disappear after commencement of a GFD (; ), and they rise again when gluten is reintroduced into the diet (). Thus, their level seems to mirror the immune reaction triggered by gluten in the intestine and, further, their decrease is related to a clinical improvement of the intestinal mucosa (; ). IgA gliadin-specific B cells have been detected in peripheral blood of CD patients (; ); these possibly are circulating IgA plasmablasts homing to the LP.
Celiac disease patients also develop autoreactive antibodies originally identified as targeting connective tissue constituents, in particular the endomysium (). The enzyme TG2 was identified as the major endomysial autoantigen (). In the diagnostic workup of CD, assessment of serum of anti-TG2 autoantibodies has become an important tool for the diagnosis particularly in children where new recommendations allow the diagnosis to be made without histological examination of small intestinal biopsies (). Similarly to anti-gliadin antibodies, the production of anti-TG2 antibodies is dependent on dietary gluten exposure (; ). Anti-TG2 antibody titers have been shown to correlate with abnormal small intestine histopathology (). While serum anti-gliadin antibodies have a significant IgA2 component, only a minor portion of serum IgA antibodies reactive to the endomysium were found to belong to this subclass ().
Recently, we demonstrated that TG2-specific PCs can be visualized by immunofluorescence of tissue sections (Figure Figure11) and by flow cytometry of single-cell suspensions from duodenal biopsy specimens (). To note, TG2-specific PCs comprise 4–24% of the total IgA PC population in the celiac lesion. This massive accumulation of TG2-specific PCs is further supported by the notion that IgA intestinal antibody deposits target the same antigen in the extracellular matrix and the endothelium of the small blood vessels (), thus reflecting an abundant local antibody production. Notably, TG2-targeted IgA intestinal deposits are present at all stages of CD, including early developing CD (prior to villous atrophy; ; ; ) as well as the advanced lesion stage in rare seronegative patients ().
A patch with high frequency of TG2-specific PCs as revealed by immunofluorescence analysis on a cryosection of the duodenal mucosa of a patient with active CD. Staining performed with biotinylated TG2 (biot-TG2, followed by fluorescent streptavidin, green), a plasma cell marker (anti-CD138, red), and a nuclei stain (DAPI, blue).
CHARACTERISTICS OF THE ANTI-TG2 ANTIBODY REPERTOIRE
In our recent, thorough dissection of the antibody (Ab) repertoire of the intestinal autoimmune response to TG2, we found that despite extensive class switch to IgA, antibody genes had a very limited amount of somatic mutations (). From the analysis of 60 sequences of heavy chain variable region genes cloned from single intestinal PCs of CD patients, we observed on average less than half the number of mutations found in the rest of the intestinal PC compartment. Interestingly, anti-TG2 antibodies with heavy chain encoded by the VH5 gene, which accounted for 44% of the response, were significantly less mutated than those encoded by other genes, including several that were entirely germ-line encoded. A trend of low mutation in VH5 genes in the gut was observed before (), but the reason for this and for the preferential recruitment of cells expressing antibodies encoded by VH5 in the autoimmune repertoire is not known. It may be related to structural properties of the VH5 region and/or to the fewer mutational hotspots in the VH5–51 gene. Worth noting is that a number of VH5 antibodies with different specificity did not bind TG2 to any extent, thus showing that the anti-TG2 reactivity is CDR-encoded and not depending on unspecific binding of the VH5 framework regions ().
This significantly differs from all other intestinal human antibody repertoires described to date, as presented and discussed above, in which antibody genes with such a limited number of mutations were seldom observed. This represents a unique feature of the anti-TG2 response and may therefore provide clues into its generation.
GENERATION OF TG2-SPECIFIC B CELLSTg2 Patch 20 To 22 Degrees Celsius To Fahrenheit
A schematization of the current knowledge and hypotheses for the generation of the intestinal TG2-specific B cell response is shown in Figure Figure22. The scheme assumes a T cell-dependent mechanism for generation of the response. Does instead the observed phenotype, with scarce SHM, indicate a T cell-independent one? Indeed, despite numerous efforts, to date there is no convincing evidence for the existence of TG2-specific T cells. However, the clinical observation of the strict HLA-dependent appearance of TG2-specific antibodies speaks against this model. In a population based cohort study, compared the presence of TG2-specific antibodies in children with or without the DQ2 or DQ8 HLA-risk alleles, and found that 73 of 1620 (4.5%) individuals with HLA-risk alleles were positive for anti-TG2 antibodies, versus none of 1815 subjects without HLA-risk alleles. This strongly suggests T cell involvement. How to reconcile these data with the inability to identify TG2-specific T cells? Once again, clinical observations provide clues: anti-TG2 antibodies rapidly disappear when gluten is removed from the diet (; ). The regulation of the B cell response by gluten intake is the foundation of the “hapten-carrier-like model” proposed several years ago (). In this model, intestinal gluten-specific T cells provide help to TG2-specific B cells. This is possible because of the enzymatic activity of TG2. The enzyme can form covalently linked complexes between itself and gluten peptides (), which then can act as “hapten-carrier-like” complexes. Upon withdrawal of gluten from the diet, the T cell help will cease and anti-TG2 antibodies will disappear. Although this model has not been formally demonstrated in vivo, we recently provided in vitro evidence that TG2-specific B cells indeed can present gluten peptides to gluten-reactive T cells when offered TG2–gluten peptide complexes ().
Schematic representation of the inductive and effector sites for the mucosal B-cell response in CD. Through yet unknown mechanisms, gluten peptides (GP) cross the epithelium and become deamidated gluten peptides (DGP) by the action of transglutaminase 2 (TG2). These peptides are then transported, possibly by dendritic cells, to local lymphoid tissue structures where a strong, proinflammatory T cell response takes place. Some native peptides may reach the lymphoid tissue and may become targeted by TG2 there. In lymphoid tissues TG2-specific B cells likely interact with gluten-specific T cells, according to the hapten-carrier-like model described in the text which builds on the ability of TG2 to form TG2-GP/DGP complexes. The B cell response generated follows either a canonical germinal center (GC) pathway or an extrafollicular pathway, resulting in massive plasmablast generation. The plasmablasts migrate via the blood to the lamina propria, where they mature as plasma cells (PC), secrete antibodies (Abs) and survive for extended time in what is called a PC survival niche. In active CD, this results in plasmacytosis and sustained secretion of IgA dimers (dIgA) – as well as IgM pentamers – which are transported across the epithelium to the lumen as secretory IgA (sIgA). TG2-specific antibodies produced at this site are also deposited on the subepithelial layer of TG2. BCR, B cell receptor; FDC, follicular dendritic cell; TCR, T cell receptor.
Based on strong clinical evidence, T cells thus appear to be involved in the generation of the anti-TG2 B cell response. In what is regarded as the “canonical” response, T and B cells cooperate in the GC, where B cells not only class-switch, but also accumulate mutations so that clones with increased affinity are selected (affinity maturation) in a process that ultimately generates memory B cells and long-lived PCs. In particular, B cell memory is “designed” to respond more rapidly to a secondary antigen challenge, and repeated stimulation leads to the generation of a switched response composed of high affinity clones which have acquired many mutations. In humans, this is clear in the cases of HIV, influenza, and rotavirus infections (; ; ).
A possible scenario to explain the phenotype of the anti-TG2 response is that it is generated at extrafollicular sites, thus bypassing GC formation () but nonetheless maintaining the requirement for T cell involvement. In mice, this has been observed in more than one context, such as Salmonella infection () and, interestingly, the autoimmune RF response (). Contrary to what commonly thought, AID can be expressed at EF sites (), and in the autoimmune context SHM has clearly been shown to vigorously take place at this site (). The intestinal environment may play a role in this process. AID-dependent class switching to IgA at the LP level has been shown in mice () and studies in humans suggested that proliferation, class-switching, and SHM could take place in the intestinal LP (; ). As discussed above, a study of mice based on analysis of IgA sequences obtained by high-throughput sequencing suggested instead that extra-mucosal expansion is followed by massive seeding in the LP, of which clonal relatedness is a consequence (). Although still being a matter of major debate, it cannot be excluded that the TG2-specific response expands and evolves in the LP, and that B cells do not enter GCs at all. If they do, the scarce number of mutations suggests that they undergo limited rounds of selection, possibly a single one, in the GC. The scope of the GC is to ultimately produce memory cells and long-lived PCs. Does this happen in the case of the anti-TG2 response? For both cell populations evidence is limited and further studies are required. Upon gluten removal the TG2-specific IgA serum titer decreases within months (), suggesting that the long-lived PC compartment is limited. Although finally differentiated to a PC phenotype, IgA PCs in the small intestine are only relatively long-lived, with a lifespan of some months (); the microenvironment is likely to play a major role. Similarly, we have only partial evidence for the existence of a TG2-specific, IgA memory compartment in CD, which appears to be more substantial in GFD-treated rather than active CD patients. It has to be noted that small amounts of memory B cells seem to be produced also in the absence of GCs (; ).
POSSIBLE REGULATION MECHANISMS OF THE ANTI-TG2 IMMUNE RESPONSE![]()
Whether cells are or not generated in GCs, undoubtedly specific factors limit GC activity. To note, the diagnosis of CD (and thus specimen sampling) often occurs many months or years after the appearance of the symptoms. Moreover, the anti-TG2 response precedes symptoms and intestinal damage. All the patients from whom we obtained intestinal specimens were adults (); the anti-TG2 response had likely been present for several months or years. In this time frame, according to the canonical model of immune response, GCs should be formed, memory generated, upon secondary stimulation memory cells preferentially and rapidly reactivated, resulting in extensive affinity maturation and accumulation of mutations. What inhibits this? Our current knowledge only allows speculation. Several different, and not mutually exclusive, scenarios can be envisaged.
(i) Feedback mechanisms. These could happen both at the antibody and the antigen level, as well as at the cellular level. The extent of the anti-TG2 response (10% of intestinal PCs, on average) and the consequent massive antibody production may provide a negative feedback that inhibits GC activity. This could be seen as a self-regulating mechanism of the immune system. With regard to antigen availability, chronic stimulation (i.e., continuous gluten ingestion) may be different than repeated antigen challenge (i.e., seasonal flu infections). Another feedback mechanism may result from antigen-specific, isotype-switched PCs that before homing to the LP act as potent antigen presenting cells. PC-controlled antigen presentation was shown to suppress the development and function of antigen-specific follicular helper T cells (). Such function of PCs would create bidirectional regulation of adaptive immunity by providing a negative cognate regulatory mechanism which may serve as a functional sensor of PC production that can control ongoing GC B cell responses.
(ii) Affinity. The binding strength of the anti-TG2 antibodies may be a factor. Even when a panel of mutated anti-TG2 hmAbs was reverted to their germline counterparts, strong binding to TG2 was observed (). This is atypical as in the case of anti-flu antibodies removal of mutations gave a dramatic loss in affinity (). It has been previously shown in mice that high affinity may favor EF T cell-dependent PC responses (). Alternatively, it may be that inside GCs it is affinity that regulates the fate of B cells – i.e., whether to become a memory B cell or PC – and that high affinity favors the latter. In both scenarios, this would result in generation of a scarce memory compartment and, irrespective of the specific mechanism, to continuous activation of naïve B cells thus explaining scarce SHM. As an alternative explanation, it has been shown that the initial affinity of the B cell receptor (BCR) is inversely correlated with accumulation of mutations in GC T cell-dependent antibody responses in vivo (). This effect was due to GC selection, as both high and low affinity B cells had the same frequency of mutations in non-coding sequences.
Tg2 Patch 20 To 22 Degrees
(iii) T cell control. T cells may represent an important regulator. It has been shown that a robust and efficient T cell response increases the magnitude of the PC response while preventing GC recycling and memory cell differentiation (; ; ). Furthermore, it has been suggested that the decision to become a PC upon receiving T cell help is antigen dose-dependent (). If the help to TG2-specific B cells indeed is provided by gliadin-specific T cells, the amount of antigen and strength of the T cell response would be compatible with a limited GC reaction.
(iv) Nature and location of the response. The nature (autoimmune) and the location (intestine) differ from any other B cell responses that have been characterized in humans, and hence models for comparison are not easily available. In mice, peritoneal reservoirs of B1 cells significantly contribute to intestinal responses () – often via T cell-independent mechanisms, that do not efficiently form GCs (). Whether similar mechanisms take place in humans is not fully understood (). Could instead the unique features of the anti-TG2 response relate to the self nature of the antigenic target? Does the immune system sense the antigen as self and redirects the response toward an EF one? Germ-line encoded autoreactivity has been described, for instance in RA (), but does not seem to be the general rule. In mice, there are notable examples of autoimmune responses developing at EF sites – the response to RF being such one situation (). Important insights will come from the analysis of the repertoire of the gluten-specific B cell response. Would gluten-specific PCs show SHM at the same level as TG2-specific PCs, or would they have high degrees of SHM as generally seen in intestinal IgA PCs? Together with the investigation of the CD-specific B cell memory, this is one of the most interesting immunological aspects toward which research should focus.
(v) Structural features. As mentioned above, anti-TG2 antibodies have high affinity even when germ- line encoded. What confers such high affinity and, in particular, what favors VH5 selection over other V regions is not known. On the surface of a B cell, BCR cross-linking is one potent mechanism for activation. We recently showed that, in vitro, TG2 can mediate covalent cross-linking of IgD (and, to a minor extent, IgM, but not of IgA or IgG) antibodies, providing a hypothetical model where continuous activation of naïve B cells is favored over IgA-switched memory cells, thus explaining lack of accumulated mutations (). Similarly, it is conceivable that the VH5 dominance is based on a similar mechanism.
20 To 22 Inch Bathroom Vanities
To address these questions, we need to know more about how antibodies bind to TG2. The hmAbs that we have cloned from intestinal IgA PCs represent a unique tool to better understand their interaction with TG2. We are currently investigating the epitopes recognized by the autoantibodies, as well as their ability to bind TG2 in its different forms (i.e., free vs bound to fibronectin, open vs close conformation, GTP vs Ca2+-bound, etc). Ultimately, fundamental insights will derive from efforts directed toward the generation of crystal structures of TG2-hmAb complexes.
TG2-SPECIFIC B CELLS AS ANTIGEN PRESENTING CELLS AMPLIFYING THE ANTI-GLUTEN T CELL RESPONSE
B cells can program CD4+ T cell responses (reviewed in ). This is so, much because B cells and T cells interact in an antigen-specific manner. By BCR-mediated uptake and concentration of antigen, B cells serve as potent antigen-presenting cells for T cells (). This mechanism is essential for B cells to receive cognate T cell help, but notably it has also direct consequences for the T cells. B cell-mediated antigen presentation leads to proliferation and clonal expansion of antigen-specific T cells (). If TG2-specific B cells are able to load deamidated gluten peptides for presentation to gluten-specific T cells in vivo (), this would likely result in expansion of gluten-specific T cell clones. These gluten-specific T cells would then be able to interact both with TG2-specific B cells as well as with B cells specific for deamidated gluten peptides. Collectively, these events will support the antibody responses to TG2 and deamidated gluten peptides and importantly lead to an amplification of the anti-gluten T cell response. In this scenario, B cells would be at the center stage of the immunopathogenesis of CD, and could therefore be a potential target for therapy even if CD is considered primarily a T cell-mediated disease.
PATHOGENIC ROLE AND INTERACTION OF AUTOANTIBODIES WITH INTESTINAL STRUCTURES
Since the discovery of autoantibodies in CD, and subsequently the identification of TG2 as the main target, there has been speculation about whether the antibodies themselves are pathogenic. Most research has focused on the effects of antibodies on enzymatic activity, with discordant results (; ; ). A reason for this could be the variety of assays and experimental conditions used to assess TG2 activity. Although in some cases weak inhibition has been reported, as of now there is no compelling evidence that the effects of autoantibodies on TG2 play a major role, either in the pathogenesis or in relation to the clinical features of CD. The great majority of the hmAbs from our newly generated panel neither inhibited nor enhanced TG2 activity, consistent with a central role for TG2 in the enzymatic deamidation of gluten peptides.
Among other proposed effects, anti-TG2 antibodies could contribute to the formation of the lesion by inhibiting angiogenesis () as well as by interfering with the differentiation of epithelial cells (). Effector function of antibodies could contribute to the tissue damage seen in CD. Complement-dependent inflammation has been observed in the CD lesion (). Unlike IgA, IgM does fix the complement, and TG2-specific IgM are indeed produced in patients, especially in those with IgA deficiency (; ).
Celiac disease patients with active disease have increased transport of gliadin peptides across the epithelium (), and IgA antibodies have also been suggested to have a role in such transport (). In the intestinal mucosa TG2 binds fibronectin, forming a sub-epithelial layer, and in patients IgA/IgM deposits are observed at this location. Moreover, IgA is found on the brush border. have shown that gliadin peptides can be retro-transcytosed as IgA–gliadin complexes via the transferrin receptor, which is abnormally expressed at the apical surface of enterocytes in active CD. TG2 localized at the apical side of the epithelium may as well play a role in this mechanism (). In the LP, intact immunostimulatory gliadin peptides might act by triggering a local immune response and promoting inflammation. We hypothesize that dimeric anti-TG2 IgA play a role in this mechanism. Anti-TG2 antibodies could detach TG2 from fibronectin and the complexes could be transported across the epithelium, where sampling of gliadin peptides by TG2 itself or by anti-gliadin antibodies could take place. We have preliminary evidence that a fraction of anti-TG2 antibodies can in fact compete for the fibronectin binding site.
In conclusion, as of now the evidence for a role of anti-TG2 antibodies is scarce, and it derives from in vitro or cell culture systems; when anti-TG2 antibodies were expressed in vivo in mice (), no obvious effect was seen. This highlights the need for the generation of an animal model of CD.
ANIMAL MODELS OF CD
Several animal models have been developed that try to recapitulate CD, however none of them entirely succeeded reproducing the complex mechanisms causing this disease (reviewed in ). Expression of anti-TG2 autoantibodies in vivo by means of adeno-associated virus-based gene transfer led to lifetime production of such antibodies, analogous to what observed in patients, but no clinical features were associated (). Some signs of disease were obtained when pre-sensitized CD4+ T cells were transferred in Rag-deficient mice, inducing weight loss and duodenitis (); however also this system was not able to recapitulate the majority of the immune features observed in CD. Interestingly, Jabri’s group has described a humanized HLA-DQ8-transgenic mouse model (), characterized by over-expression of IL-15 in the LP. When gliadin-fed, these animals develop IFN-γ-producing anti-gliadin T cells, anti-gliadin and anti-TG2 antibodies, and intraepithelial lymphocytosis. Despite lacking the hallmark of villous atrophy, this model does resemble early stages of CD. Mice are not the only species where CD models are investigated, as a screening of macaques also led to identification of animals with signs and symptoms of CD (); the usefulness of such model remains to be evaluated. In general, efforts are being made toward the generation of an animal model of CD, which will greatly facilitate research.
THE MICROFLORA AND ITS IMPACT ON INTESTINAL IMMUNITY
Genetic factors may account only for about half of the risk to develop CD thus leaving an important role for the environment in the pathogenesis. Gluten exposure obviously is critical, but environmental factors outside of gluten may be implicated as well. Such factors could be pathogenic infectious agents or commensal bacteria. Recently, it has become clear that the gut microflora profoundly influence intestinal immunity (). There is a complex interplay between intestinal immunity and the populations of commensal bacteria, and these two components regulate each other. Not only does the microflora regulate several aspects of the innate and adaptive immunity, as well as of several metabolic pathways, but it has also been shown that dysregulated immunity (for instance as a consequence of experimental manipulation of molecules such as PD-1 and AID; ; ) results in skewed gut microbial communities, and this in turn may have detrimental effects. In future, it will be of major importance to understand how the microbial communities contribute to the intestinal immune response in a context such as CD where both the T and the B cell intestinal populations seem altered as compared to healthy individuals.
CONCLUSION
Download zebra designer software windows 10. In the last two decades we have learnt a lot about CD and its interplay with intestinal immunity. Among the most remarkable discoveries, TG2 has been identified as the main autoantigen of CD, and its role in creating potent T cell epitopes has been unraveled. We have made huge steps forward in understanding the role of HLA genes, and many non-HLA susceptibility genes have been identified. Some limited progress has also been made in understanding the role of innate immunity factors in CD. Recently, the knowledge of the intestinal B cell response in CD has significantly improved. We have learnt that anti-TG2 antibodies form deposits in the small intestine, a number of highly sensitive serological tests based on serum antibodies have been developed, monoclonal antibodies have been isolated by phage display and single cell cloning, and the cells producing these antibodies have been visualized, characterized and enumerated. However, many important features of CD, in particular related to the intestinal environment in which the disease takes place, remain to be tackled. These include characterization of the anti-gliadin B-cell response, the IgG anti-TG2 repertoire, identification of the memory compartment, and others. A better understanding of human intestinal immunobiology is needed to address these questions.
Conflict of Interest Statement
Mortal kombat chess game. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgments
The authors have been supported by grants from a Marie Curie Research and Training Network (European Commission), the European Research Council, the Research Council of Norway, and the South-Eastern Norway Regional Health Authority.
REFERENCES
Articles from Frontiers in Immunology are provided here courtesy of Frontiers Media SA
Comments are closed.
|
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |