CeD is a chronic small intestinal immune-mediated enteropathy precipitated by exposure to dietary gluten in genetically predisposed individuals (Ludvigsson et al., 2013). CeD affects around 1-2% of Western population and occurs exclusively in genetically susceptible individuals. The great majority of CeD patients are HLA-DQ2.5-positive while HLA-DQ2.2 and HLA-DQ8 variants are less common among the patients. Gastrointestinal symptoms of CeD include harmful autoimmune reaction that causes structural alterations of the gut mucosa (Figure 5). Moreover, gastrointestinal manifestations of CeD are often accompanied by extraintestinal symptoms of which diarrhea, chronic abdominal pain, distended abdominal and weight loss are among the most common complications experienced by CeD patients. Life-long adherence to gluten-free diet is currently the only available treatment that can improve clinical symptoms of CeD and reverse gluten-driven damage of small intestinal mucosa (Figure 5). CeD is now considered a widespread public health problem due to substantial reduction in the quality of life of affected individuals (Lindfors et al., 2019).
CeD-associated HLA-DQ variants are the major risk factors predisposing to CeD. Yet, only a small fraction of individuals positive for HLA-DQ2.5, HLA-DQ2.2 or HLA-DQ8 develop the disease. Thus, it is likely that non-HLA genetic elements are required for the disease to develop (Withoff et al., 2016). Over 50 non-HLA candidate loci were identified to be associated with CeD, such as genes encoding interleukin receptors
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(IL18R1 and IL18RAP), interleukins (IL12A, IL2 and IL21), chemokine receptors (CCR1, CCR2, CCR3 and CCR4) and co-signalling receptors (CD28, CTLA4 and ICOS) (van Heel et al., 2007; Dubois et al., 2010; Trynka et al., 2011; Gutierrez-Achury et al., 2015). The effect size of the non-HLA genes is generally very small. In addition to genes, environmental factors, for instance yet unknown intestinal insults that are able to induce danger signals in the gut, could contribute to CeD development (Withoff et al., 2016). The clinical diagnosis of CeD is based on serological testing and morphology assessment of biopsies collected from the upper small intestine.
HLA typing is often used to exclude the disease. Duodenal biopsy is considered burdensome both by the healthcare system and patients, thus efforts are being made to find less invasive diagnostic procedures (Sallese et al., 2020).
Figure 5. Ingestion of gluten causes selective destruction of intestinal epithelial cells in CeD individuals. CeD lesion develops over time, beginning as crypt hyperplasia (middle-left section) and eventually taking form of villous atrophy with crypt hyperplasia (middle-right and far-right panels). Strict and lifelong adherence to a gluten-free diet helps to revert gluten-induced damage of small intestine mucosa that slowly recovers and returns to normal morphology (far-left panel). Adapted from Lindfors et al., 2019 with permission.
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• Dietary gluten and immunodominant gluten epitopes
The term gluten is used to denote grain storage proteins rich in proline and glutamine that are present in wheat. However, gluten-like proteins with similar amino acid composition can be found in other cereals, that is secalins in rye, hordeins in barley and avenins in oats. Gluten, hordeins and secalins evoke strong immune response in CeD individuals. On the other hand, harmful immune reaction to avenins is uncommon and thus oats are considered safe for CeD patients. Wheat gluten represent a complex protein mixture of alcohol-soluble gliadins (α-gliadins, γ-gliadins and ω-gliadins) and alcohol-insoluble glutenins (high-molecular-weight glutenins and low-molecular-weight glutenins). Gliadins and glutenins are fairly resistant to enzymatic digestion in the gut lumen because of their high proline content. As a result, various long peptides ranging from 15 to 50 residues are generated. Subsequently, gluten peptides pass through the epithelial cell layer and enter the lamina propria where selected glutamine residues in “native” gluten peptides are deamidated by transglutaminase 2 (TG2). In consequence, glutamine residues are replaced by negatively charged glutamic acid, leading to generation of
“deamidated” gluten peptides that are characterised by increased binding affinity to the HLA-DQ2.5, HLA-DQ2.2 and HLA-DQ8 molecules (Sollid & Jabri, 2013).
Even though gluten contains many immunogenic peptides, of which the “33mer”
peptide from α-gliadin is considered the most immunogenic sequence, the T-cell responses to a handful of epitopes are observed across the great majority of CeD patients. These epitopes are widely known as immunodominant and six such epitopes have been defined so far. Five immunodominant epitopes are HLA-DQ2.5-restricted, namely DQ2.5-glia-α1, DQ2.5-glia-α2, DQ2.5-glia-ω1, DQ2.5-glia-ω2 and DQ2.5-hor-3. Among HLA-DQ8-restricted epitopes, DQ8-glia-α1 is regarded as immunodominant (Sollid et al., 2012; Sollid et al., 2020). The response to immunodominant epitopes was dominated by clonally expanded T cells over-using a biased TCR repertoire. V-gene bias towards TRBV7-2 (encoding TCR Vβ6.7 chain),
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TRAV26-1 and TRAV4 was observed among DQ2.5-glia-α2-reactive CD4+ T cells.
TRBV7-2 and TRAV26-1 were also preferentially used as a pair (Qiao et al., 2011;
Qiao et al., 2014; Dahal-Koirala et al., 2016). Biased usage of TRAV12, TRAV4 and TRBV4 was found among DQ2.5-glia-ω2-reactive CD4+ T cells, but no preferential pairing was detected (Dahal-Koirala et al., 2016). For DQ8-glia-α1-reactive CD4+ T cells, the TCR repertoire was biased towards TRBV9 and TRAV26-2 (Broughton et al., 2012). It was speculated that the development of CeD might depend on the random generation of high-affinity TCRs recognizing particular immunodominant epitopes (Jabri & Sollid 2017).
• Gluten-driven enteropathy of small intestine
The fundamental role of the gastrointestinal tract is to digest foodstuff, absorb nutrients and water, and eliminate undigested leftovers. The gastrointestinal tract is inhabited by a wide range of commensal microbes and is continually exposed to a multitude of antigens including dietary antigens. The gastrointestinal tract is usually divided into small intestine and large intestine. The small intestine can be further segregated into duodenum, jejunum and ileum whereas large intestine is made of caecum, colon and rectum. The small intestine and large intestine differ in size and mucosal architecture. The small intestine is a few meters long and is characterised by long and thin villi that provide extensive surface area of digestive/absorptive epithelium. On the other hand, the large intestine is wider in diameter but shorter than small intestine and is distinguished by flat surface without finger-like villi.
Therefore, the large intestine has little or no digestive abilities and is mainly involved in reabsorption of water. All parts of the intestine are constantly renewed by new epithelial cells arising from multipotent stem cells located near the bottom of the crypts (Mowat & Agace, 2014).
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The intestinal mucosa is composed of intestinal epithelium, lamina propria and thin muscle layer underneath lamina propria. The intestinal epithelium is formed mostly by absorptive enterocytes. However, other cell types can also be found such as Paneth cells producing antimicrobial peptides and mucus-producing goblet cells.
The lamina propria is a loosely packed connective tissue that harbours the blood supply, lymph drainage system and mucosal nervous system. Interestingly, the gastrointestinal tract is also a home to large number of immune cells in the human body with T cells residing in both intestinal epithelium and lamina propria. The intestinal epithelium is populated by intraepithelial lymphocytes (IELs) that are located at the basal membrane between enterocytes. The great majority of IELs in small intestine are antigen-experienced CD8+ T cells that are heterogenous in phenotype, antigen specificity and function. IELs establish a protective immune barrier that helps to preserve the integrity of intestinal epithelium by eliminating infected epithelial cells and promoting epithelial repair. T cells residing in lamina propria are often referred to as lamina propria lymphocytes (LPLs). LPLs consist mainly of CD4+ T cells. Both IELs and LPLs display significant variation in population size and phenotypic composition that are dependent on the region of gastrointestinal tract (van Wijk & Cheroutre, 2009).
In CeD, ingestion of gluten results in the development of harmful mucosal autoimmune response in the upper small intestine. Increased number of inflammatory immune cells in intestinal epithelium and lamina propria, dysregulated overproduction of IL-15 as well as selective destruction of intestinal epithelial cells manifested in the form of villous atrophy (blunting or flattening of the villi) and crypt hyperplasia (elongation of the crypts) are clinical hallmarks of CeD (Figure 5) (van Bergen et al., 2015). IL-15, a cytokine upregulated in distressed intestinal epithelium, promotes the expression of NKG2D on the surface of IELs that is critical in licensing IELs to kill intestinal epithelial cells. NKG2D is a co-stimulatory C-type lectin-like receptor that, besides augmenting TCR-dependent responses, can also directly
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induce cellular cytotoxicity independently of signalling via TCR. NKG2D interacts with MHC class I polypeptide-related sequence A (MICA) which in normal conditions is present only on a small percentage of enterocytes. However, MICA is a stress-induced molecule and significant increase of surface MICA is observed in inflamed small intestine of CeD individuals. The NKG2D-MICA interaction, supported by IL-15, facilitates indiscriminate, TCR-independent killing of MICA-expressing enterocytes by NKG2D-positive IELs through perforin/granzyme B pathway that eventually leads to epithelial pathology typical of CeD (Hüe et al., 2004; Meresse et al., 2004). IELs in CeD inflamed intestinal environment likely undergo a shift in their genetic program. Reprogrammed IELs acquire an activated NK phenotype that is manifested by aberrant upregulation of multiple NK receptors, including aforementioned NKG2D as well as CD94/NKG2C, that render them capable of not only killing intestinal epithelial cells but also proliferating and producing inflammatory cytokines in an antigen-nonspecific, TCR-independent manner (Meresse et al., 2006). However, overproduction of IL-15 by epithelial cells was alone insufficient to fully activate IELs. The interplay between IL-15 and anti-gluten immunity was necessary for IELs to acquire a fully activated cytotoxic phenotype (Setty et al., 2015). Indeed, distinctive histological alterations together with expansion of IELs are accompanied by infiltration of gluten-reactive CD4+ T cells that constitute around 2% of all CD4+ T cells in CeD lamina propria (Bodd et al., 2013; Qiao et al., 2021). It remains unclear what exactly causes intestinal epithelial distress in CeD. Some studies suggested that gluten peptides might possess toxic properties that would induce overproduction of IL-15 and upregulation of stress-induced surface molecules in the small intestine of HLA-predisposed individuals.
Furthermore, anti-TG2 antibodies deposited in the small intestine mucosa of CeD patients might increase permeability of the epithelial barrier, thereby allowing gluten peptides to reach the lamina propria and subsequently affect local gut biology (Abadie et al., 2012).
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• Gluten-reactive CD4+ T cells
Gluten-reactive CD4+ T cells in peripheral blood and gut of CeD patients are effector memory cells (CD45RA-CD62L-CCR7-) with a narrow phenotype resembling that of TFH cells except being negative for CXCR5. These cells are CXCR3+CD39+CD161+, express co-stimulatory receptors CD28, ICOS and OX40 as well as co-inhibitory receptors PD-1 and CTLA-4. CD69 is expressed only by gluten-reactive CD4+ T cells isolated from the gut (Christophersen et al., 2019a). On the other hand, gluten-reactive CD4+ T cells isolated from peripheral blood express the proliferation marker Ki-67 (Christophersen et al., 2019a) and are positive for the gut-homing marker integrin α4β7 (Christophersen et al., 2014). The cytokine production by gluten-reactive CD4+ T cells is dominated by IFN-γ (Nilsen et al., 1995). In addition, some gluten-reactive CD4+ T cells also produce IL-21 (Bodd et al., 2010), IL-10, IL-4 (Christophersen et al., 2016) and IL-2 (Goel et al., 2019), but not IL-17 nor IL-22 (Bodd et al., 2010).
Numerous studies showed that HLA-DQ:gluten tetramer staining of peripheral blood gluten-reactive CD4+ T cells (in this paragraph referred to as tetramer test) is highly specific and sensitive in detection of CeD patients. Tetramer test could distinguish treated CeD (TCeD) patients from HLA-matched controls and individuals without CeD-predisposing HLA. This discrimination was possible after short oral gluten challenge that increased the frequency of gluten-reactive CD4+ T cells in the bloodstream of TCeD, but not control individuals, on day 6 after first gluten ingestion (Ráki et al., 2007). Moreover, tetramer test coupled with short oral gluten challenge was superior to the evaluation of histological deterioration that could be observed in some TCeD on day 4 after consuming gluten (Brottveit et al., 2011).
Tetramer test was also able to detect gluten-reactive CD4+ T cells in peripheral blood of both untreated CeD (UCeD) patients and TCeD patients without oral gluten challenge when combined with bead enrichment of gluten-reactive CD4+ T cells (Christophersen et al., 2014). Although only a few gluten-reactive CD4+ T cells could
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be found in peripheral blood of healthy individuals carrying CeD-predisposing HLA-DQ2.5, those tetramer+ T cells differed from blood gluten-reactive CD4+ T cells from CeD patients by virtue of lower tetramer-binding intensity and no signs of biased TCR usage (Christophersen et al., 2016). All in all, tetramer test might supplement or perhaps replace the gold-standard histological examination of small bowel biopsies (Sarna et al., 2018a; Sarna et al., 2018b). Even though gluten-free diet normalises histology and disease-specific antibodies, gluten-reactive CD4+
memory T cells can still be detected in TCeD (Christophersen et al., 2014). Moreover, particular gluten-reactive CD4+ T-cell clonotypes, that is gluten-reactive CD4+ T cells expressing identical TCRα and/or TCRβ chains, persisted for decades in TCeD patients (Risnes et al., 2018). This indicates that even when on gluten-free diet, some TCeD patients experience a continuous low-level immune response that could arose from incidental/occasional exposure to gluten which in turn might have contributed to the maintenance of those specific clonotypes. As gluten-reactive CD4+ T cells are considered central in the pathogenesis of CeD (Figure 6), they represent an interesting target for potential therapeutic intervention (Christophersen et al., 2019b).
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Figure 6. The role of gluten-reactive CD4+ T cells in CeD. Undigested gluten peptides enter the lamina propria where they are deamidated by TG2. In consequence, glutamine residues are replaced by negatively charged glutamic acid which increase the affinity of deamidated gluten peptides (DGPs) to the HLA-DQ2 and HLA-DQ8 molecules. DGPs are taken up by APCs, for instance dendritic cells, that present them to reactive CD4+ T cells. Also, both specific and TG2-specific B cells may act as APCs for gluten-reactive CD4+ T cells. Upon interaction, gluten-gluten-reactive CD4+ T cells proliferate and secrete distinctive cytokines, mainly IFN-γ and IL-21. Moreover, activated B cells differentiate into plasma cells that secrete antibodies against DGPs and TG2. Adapted from Lindfors et al., 2019 with permission.
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• T cell-B cell collaboration in CeD
In CeD, gluten-reactive CD4+ T cells might provide help to gluten-specific B cells as well as TG2-specific B cells and therefore promote their differentiation into plasma cells that secrete antibodies against DGPs and TG2, respectively (Figure 7). Both anti-DGP antibodies and anti-TG2 antibodies are hallmarks of CeD and are often used to diagnose CeD in the clinic (Jabri & Sollid, 2009). The occurrence of antibodies against TG2 in CeD patients consuming gluten is puzzling. The model explaining this conundrum is based on the fact that TG2 can form complexes with gluten peptides, thereby forming hapten-carrier-like structures. In this model, TG2-specific B cells take up TG2-gluten complexes through B cell receptor-mediated internalization. Subsequently, gluten peptides are presented on HLA-DQ molecules to gluten-reactive CD4+ T cells. As a result, TG2-specific B cells receive T-cell help that facilitates the production of anti-TG2 antibodies while simultaneously serving as APCs amplifying the T-cell responses against gluten (Figure 7) (Sollid et al., 1997).
The role of B cells as APCs for gluten-reactive CD4+ T cells is supported by the phenotype of the latter that closely resembles that of TFH cells which are considered the dominant providers of activation signals to B cells (Christophersen et al., 2019a).
Moreover, plasma cells were identified as the most abundant APCs displaying gluten peptides in the small intestine lamina propria of CeD patients (Høydahl et al., 2019). Complementary to this, gene expression profile of TG2-specific plasma cells from CeD small intestine biopsies indicated a possible cross-talk with CD4+ T cells due to the expression of HLA class II as well as CD86 that serves as the ligand for CD28 (Snir et al., 2019; Lindeman et al., 2021). On the whole, efficient collaboration between gluten-reactive CD4+ T cells and B-lineage cells appears to be important for the pathogenesis of CeD. Therefore, it was recently proposed that B cells could serve as main APCs for gluten-reactive CD4+ T cells in lymphoid structures whereas plasma cells might occupy similar role in nonlymphoid tissues. However, the ability
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of plasma cells to stimulate CD4+ T cells is still to be demonstrated experimentally (Iversen & Sollid, 2020).
Figure 7. A hapten-carrier model explaining the occurrence of anti-transglutaminase 2 antibodies in CeD patients consuming gluten. TG2 can form covalently linked complexes with gluten peptides. Such TG2-gluten complexes can be bound by B-cell receptor (BCR) of TG2-specific B cells. The complexes are internalized and released gluten peptides are displayed on the HLA molecules to gluten-reactive CD4+ T cells. In this way, TG2-specific B cells act as antigen presenting cells for gluten-reactive CD4+ T cells that become activated, release cytokines and proliferate. In return, gluten-reactive CD4+ T cells provide help signals to TG2-specific B cells that differentiate into plasma cells secreting anti-TG2 antibodies. Adapted from Sollid et al., 1997 with permission.