Article history:Received 21 April 2010Received in revised form 21 August 2010Accepted 1 September 2010Available online 15 September 2010
This article reviews the multiple uses of flow cytometry in the diagnosis, monitoringand research of celiac disease, themost prevalent chronic autoimmune gastrointestinal disease.The phenotyping of intraepithelial lymphocytes (IELs) is of clinical relevance in the diagnosis ofthe disease given the characteristic features of elevated CD3+ IELs (αβ and γδ TcR) and thedecrease in CD3− IELs. IEL biomarkers are also useful in the assessment of the response to thegluten-free diet and, importantly, in the diagnosis of the severe complications of celiac disease:refractory celiac disease and enteropathy-associated T-cell lymphoma. Novel applications offlow cytometry for the detection of anti-transglutaminase antibodies (a validated biomarker ofceliac disease) and of gluten (the triggering antigen of the autoimmune process) are alsodiscussed. The assessment of diagnostic and prognostic biomarkers by flow cytometry in celiacdisease is performed routinely in a growing number of centers and it is an example of theversatility of this technique and its applicability to the research and clinical study of solidtissues.
Flow cytometry is a powerful analytical tool that allowsfor high-volume analysis of themorphometry and phenotypiccharacteristics of heterogeneous individual cells. Since cellsneed to be in suspension in order to be analyzed by thistechnology, clinical practice of flow cytometry has beenmostly focused on the analysis of circulating cells inperipheral blood.
Solid tissues have been widely studied with flow cyto-metry in research applications, but the use of flow cytometryfor solid tissue study is relatively limited in the clinics, themain reason being the complexity and difficulty of thepreparation of cell dispersions from solid tissues within therestricted time frame for analysis of viable cells in a clinical-decision setting. One of the best examples of solid tissue flow
gluten-free diet; RCD,T-cell lymphoma; IEL,Tc, T cell.
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cytometry is the analysis of bonemarrow cells, relatively easyto disperse. Laser scanning cytometry is specifically designedto study solid tissue sections (Tarnok and Gerstner, 2002),however does not support the high throughput necessary toreach the high sample size, and thus accuracy, of flowcytometry.
The current review focuses on one relatively lesser knownsolid tissue application of flow cytometry: the flow-cyto-metric study of small intestinal immune cells, and inparticular of intraepithelial lymphocytes (IELs), of clinicalvalue in the management of celiac disease, the most frequentchronic gastrointestinal disorder. While a highly specializedtest, flow cytometry of intestinal intraepithelial lymphocytesis used routinely in a growing number of hospitals worldwidenot merely in the research study of the pathogenesis of celiacdisease, but also as a diagnostic and prognostic biomarker andin the follow-up of celiac disease and its complications.
2. Celiac disease
Celiac disease is an immunologically-mediated intoler-ance to dietary prolamins which evolves into a systemicautoimmune disease (Green and Cellier, 2007). Prolamins are
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the proteic and alcohol-soluble fraction of gluten, a majorcomponent of some of the commonest cereals in the westerndiet: wheat, rye and barley (Dicke et al., 1953). In celiacsubjects, ingestion of gluten induces a chronic inflammatoryresponse in the gastrointestinal tract that triggers theproduction of specific autoantibodies and extra-intestinalcomplications (Green and Cellier, 2007) and, in a majority ofpatients, a flattening of intestinal villi and malabsorption[Figs. 1 and 2]. Celiac disease is the most prevalent chronicgastrointestinal disease in Caucasians (1% prevalence) andone of the most common gastrointestinal diseases worldwidewith an estimated 20–30 million affected individuals globally.
Celiac disease is a genetic disorder and virtually allpatients possess the HLA-DQ2 or HLA-DQ8 histocompatibilityantigen which mediates the autoimmune process. The mostaccepted pathogenic mechanism of celiac disease is thepresentation of modified gluten peptides by HLA-DQ2 and-DQ8-bearing antigen-presenting cells to T lymphocytes inthe intestinal mucosa. The modification which turns glutenpeptides into high-avidity binders for HLA-DQ2 and -DQ8 isdeamidation, carried out by the enzyme tissue transglutami-nase (tTG) in the gut wall (Shan et al., 2002). In short, gliadinpeptides would get access to the mucosal immune system ofthe small bowel by transcellular (Matysiak-Budnik et al.,2008) and paracellular transport and then undergo deamida-tion by tTG, extra-cellularly or in lamina propria antigen-presenting cells (reviewed in Green and Cellier, 2007). Thedeamidated gliadin peptides would then be presented withhigh affinity by antigen-presenting cells in HLA-DQ2 or DQ8to CD4+ lamina propria T-cell lymphocytes (LPLs). The CD4+LPLs would then produce Th1 and inflammatory cytokinessuch as INF-γ and TNF-α. These cytokines would inducedisassembly (opening) of the tight junctions of the intestinalepithelium and contribute to increased epithelial permeabil-ity and the entry of additional gluten peptides (Paterson et al.,2007).This intestinal permeability-inflammation positivefeedback loop results in a lamina propria CD4+ T-cell (Tc)response which stimulates B cells to produce autoantibodiessuch as anti-tTG antibodies, which contribute to multipleextra-intestinal manifestations (dermatitis, ataxia, infertility,etc). Additionally, the production of IL-15 in the gut mucosaand the higher sensitivity to its effects in celiac disease(Bernardo et al., 2008a) results in the activation of NK-
Fig. 1. Normal jejunal mucosa. Left image, haematoxylin-eosin (HE), original magmagnification 300×.Reproduced with permission from Leon et al. (2005b).
receptor-bearing IEL that exert cytolytic actions againstintestinal epithelial cells (IEC) via recognition of non classicMHC (Jabri et al., 2000) and contribute to the flattening of themucosa and ensuing malabsorption [Figs. 1 and 2].
There is considerable clinical heterogeneity in the pre-sentations of celiac disease. The classic presentation is withgastrointestinal symptoms and signs (distended abdomen,diarrhea, failure to thrive, muscle mass loss) and potentialmalabsorption. Particularly in adults, celiac disease canmanifest itself with a wide variety of presentations, includingmetabolic defects (vitamin deficiencies, iron-deficiency ane-mia), dermatological signs (dermatitis herpetiformis), repro-ductive or neuro-psychiatric abnormalities, etc (Green andCellier, 2007). For this reason, celiac disease if often termed a“clinical chameleon.”
Radical avoidance of gluten is the only available manage-ment of celiac disease, though it is extremely challenging inreal life. Avoidance of gluten from the diet facilitates therepair of the mucosal architecture, the normalization of totalIEL numbers and a clinical and functional recovery (“inactive”or “controlled” celiac disease or “celiac disease in remission”).Unfortunately, gluten-free diet (GFD) is expensive anddifficult to follow for most patients. The safety threshold isonly 50 mg of gluten per day (Catassi et al., 2007) and theaverage consumption in theWesternWorld is 13 g/day, manymultiples above the safe threshold. Gluten is present, as anadditive, in 80% of foods in the Western world and manyfoods in Asia (soy sauce, noodles, etc). For these reasons, amajority of patients with celiac disease fail to fully complywith the diet, and 30–80% of celiac subjects on a GFD have“active disease” at any given time (Lee et al., 2003; Rubio-Tapia et al., 2010). Occasionally, after a period of adequateresponse to therapy, the patient fails to respond any longer,situation that is termed “Refractory” celiac disease (RCD) andis often accompanied by signs of IL-15-driven lymphomagen-esis (Malamut et al., 2010) that can evolve into a full-blownlymphoma, the enteropathy-associated T-cell lymphoma(EATL) (Cellier et al., 2000; Ryan and Kelleher, 2000). Inaddition to active, inactive (on a successful GFD) and RCD,other recognized variants of the disease are “latent” and“potential” (defined by immune activation with autoantibo-dies and IEL changes but no histological abnormalities)(Arranz and Ferguson, 1993).
nification 200×. Right image, scanning electron microscopy (SEM),origina
l
Fig. 2. Schematic and histological representation of the stages of celiac disease enteropathy. From normality (1) to total atrophy (5). HE, original magnification200×.Reproduced with permission from Leon et al. (2005b).
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Complications arise in the absence of complete GFD andinclude osteoporosis, chronic anemia, increased susceptibilityfor autoimmune disease, reproductive abnormalities, obstet-ric complications and jejunal ulcers (reviewed in Green andCellier, 2007). Neoplasias appear in 10% of untreated patients,mainly digestive cancer (mouth, pharynx, and esophagus)and intestinal T-cell lymphomas as mentioned (Ludvigsson etal., 2009). In childhood, due to the loss of absorption throughthe villi complications are mainly nutritional, metabolic andgrowth abnormalities, but also a growing number ofmalignant complications.
The gut epithelium is a unique immunological compart-ment that supports extrathymic T-cell selection and matura-tion processes. The gut lymphoid system is exposed tocontinuous antigenic challenge; the local immune response,via the secretion of different cytokines by effector immuno-competent cells, leads to inflammation or tolerance. IELsrepresent one of the most abundant lymphocyte populationin the body and comprise phenotypically heterogeneouslymphoid subsets.
Asmentioned in the previous section, there is evidence thatboth LPLs and IELs contribute to the pathogenesis of celiacdisease. IELs promote innate immunity while LPLs are adaptive
immune effectors, and the relationship between these tworesponses in celiac disease is not well understood (Jabri et al.,2005). Despite the role of LPLs in the pathogenesis, their studyby flow cytometry is less useful in clinical practice of celiacdisease, for two reasons: 1. The changes in LPL phenotype(mostly activation) are less characteristic than the IEL changeswhich will be described below; and 2. The intraepithelialcompartment is more readily available to cell dispersion anddoes not require the aggressive collagens' treatment of thebiopsies needed to release LPLs.
3.2. Isolation and staining of IELs for flow cytometry
Epithelial (IEC) and intraepithelial lymphocyte (IEL) cellsare usually isolated from intestinal biopsies by vigorousshaking in Ca 2+-free RPMI medium supplemented with 10%Fetal Calf Serum and calcium chelants such as DDT, EDTA orEGTA at ~1 mM (Cerf-Bensussan et al., 1985; Madrigal et al.,1993; Lefrançois, 2001). The calcium chelants induce thedisassembly of inter-epithelial junctions and the release ofthe epithelial cells and the IELs, which will constitute ~5% (1–10% range) of the released cells. These cells in suspension arethen washed and labeled for flow-cytometric analysis, asdescribed below. There is no unified protocol for thepreparation of IEL suspensions but in general most groupsuse a rocker or high-speed shaker to vigorously disrupt theepithelium in the presence of the calcium chelants for~60 min at room temperature or 37 °C (note the separationshould not be done at 4 °C).
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In practical terms, one can expect ~100,000 IELs per cubicmillimeter small bowel biopsy (1×1×1 mm), and this isenough material for the basic staining of IELs required fordiagnosis and monitoring of celiac disease. If additionalresearch markers are desired, more than one biopsy fragmentshould be obtained and included in the procedures.
In the clinics, the most widespread cytometers are three-color instruments which allow for an accurate measurementof the simultaneous expression of 2 fluorescent markers on apreviously selected cell population, what is needed tocharacterize IEL subsets and cannot be easily accomplishedwith immuno-histochemistry. IELs are identified on the basisof low side scatter and high CD45 expression, and theaccuracy of the analysis gate is assessed by staining withCD103 and CD7 to confirm the intraepithelial and bonemarrow-derived nature, respectively, of the cells gated (Eiraset al., 1998).
It should be noted that, while multi-marker analysis is thecompetitive advantage of flow cytometry as compared toothermethods, the results are expressed in relative terms andno absolute quantification is provided. Quantification of cellnumbers can be performed by light microscopy in anhemocytometer chamber and should particularly be per-formed if the IELs are to be frozen for future use. Thislimitation of flow cytometry is not an obstacle for theestablishment of a robust clinical method if the assay isperformed consistently, but it has to be taken into accountwhen evaluating the results obtained since theremay not be acorrelation between absolute and relative numbers ofimmune cells as the IECs are also changing in proportionand absolute number during the course of the disease (Cartonet al., 2004).
3.3. Normal phenotype of IELs
IEL represent 5–15% of the cells isolated from theepithelium (Camarero et al., 2007) and most IELs expressthe αE/β7 (CD103) epithelial-homing integrin. The majorityof IELs (N70%) are CD3+ T cells and a minority (10–20%) areTcR-CD3− lymphocytes. Of the CD3+ IEL, the majorityexpresses TcR-αβ (80%) and these T cells express mostlythe CD8 antigen (N85%), while only ~10% express CD4. Theremainder of the CD3+ IELs (5–15%) expresses the TCR-γδ1T-cell receptor with variable expression of CD8 (40–80%). Thefunction of the γδ IEL subset in celiac disease is not entirelyclear, and recent investigations by means of flow cytometrypoint towards a regulatory function (Bhagat et al., 2008).
The second subset in size after the T-cell IELs is the CD3−lymphoid IEL population (10–20% in adults, 20–40% inchildren (Spencer et al., 1989) reviewed in (Eiras et al.,1998; Camarero et al., 2007). The CD3− IEL subset is beststudied by flow cytometry, given the need for multi-colorstaining since these cells possess no specific markers. TheseCD3− cells express CD7, CD45R0, CD122, CD69 and CD38.About half of these lymphocytes express CD56. CD3− IELsappear to comprise different functional subsets: some CD3−IEL appear to be precursors of CD3+ Tc in the human fetal(Howie et al., 1998) and adult (Jarry et al., 1990; Lundqvist etal., 1995) guts. Some have a distinct NK profile (CD161, CD94,CD16, CD56) and express also the IL-2R-alpha chain (CD122)and intracytoplasmic CD3 and perforin (Eiras et al., 2000;
Leon et al., 2003). Finally, the expansion of clonal CD3− IEL inRCD and EATL has been described (Cellier et al., 1998) andthese expanded lymphocytes have been found to be T cellsdue to the existence of TcR rearrangements.
3.4. IEL changes in celiac disease
The first detectable immune abnormality in celiac diseaseis an increase in the absolute and relative numbers of αβ andγδ T-cell IELs (Halstensen et al., 1993; Schuppan, 2000). Theincreased % of IEL in the epithelium is due both to theirincreased proliferation and to a reduction in epithelial cellsduring the flat mucosa stage of celiac disease (Eiras et al.,1998; Camarero et al., 2000; Carton et al., 2004). The increasein CD3+ IEL is also the first detectable event during thegluten challenge test.
A second abnormality observed in celiac disease is theincrease in TcR-γδ+ IEL (Spencer et al., 1989; Holm et al.,1992). Both αβ and γδ IEL proliferate in situ in celiac disease.However, while the increase in αβ IEL correlates with diseaseactivity (Camarero et al., 2000) and is corrected by the GFD,the increase in γδ IEL remains stable in relative terms: whilethese cells average 4% of all IEL in healthy controls, theyrepresent an average of 25% in celiacs (Camarero et al., 2000),in all phases of disease [Fig. 3]. In Finland, where the GFD isfollowed exceptionally well for social and healthcare reasons,γδ IEL still remain elevated in 60% of celiacs on a GFD(Koskinen et al., 2009). The γδ IEL increase is not totallyspecific of celiac disease, since it has been occasionally foundin other situations, such as cow's milk intolerance, foodallergy, cryptosporidiasis, giardiasis, Sjögren Syndrome andIgA deficiency (reviewed in Leon et al., 2005b). However, theincrease in γδ IEL in a minority of patients with theseconditions tends to be mild and transient (Kokkonen et al.,2000). Or in some cases may correspond to latent celiacdisease (Arranz and Ferguson, 1993). The γδ IEL increase isnot observed in other common intestinal disorders (Trejdo-siewicz et al., 1991), and it can be stated that celiac disease isthe only disease in which γδ IEL are increased systematically,permanently and intensely (Leon et al., 2005b). The increasein γδ IEL is a helpful diagnostic biomarker in latent andpotential celiac disease (Camarero et al., 2000) [Fig. 3] andalso in those circumstances when patients start the GFDbefore reaching a formal diagnosis and there are doubts aboutdifferential diagnosis.
The third and last main abnormality described in IELsubsets in celiac disease is the decrease in CD3− CD103+ IEL(Spencer et al., 1989). As mentioned, CD3− IEL areproportionally the second subset in the healthy smallbowel, and in active celiac disease they become almostundetectable [Fig. 3]. The combined determination of γδ andCD3− IEL provides specificity to the flow-cytometric analysisof tissue biopsies, specificity which pathology alone cannotprovide to the same extent (Eiras et al., 2000).
The analysis by flow cytometry of these 3 lymphoidsubsets (αβ, γδ and CD3− IEL, what has been called the “IELlymphogram”) has a high specificity and sensitivity in theclinics in the diagnosis of celiac disease, and is particularlyhelpful in atypical presentations or when there are diagnosticdoubts (Eiras et al., 1998; Leon et al., 2002).
Fig. 3. IEL changes in the stages of celiac disease. The first abnormality detected in the mucosa of celiac disease patients, even prior to histologic or serologicfindings, is an increase in TcR-γδ IEL. When exposure to gluten stimulates the immune system of the gut, TcR αβ IEL increase in numbers and activation markers,and CD3− IEL vanish. These two parameters, unlike the increase in γδ IEL, tend to normalize during gluten-free diet (GFD). The changes in IEL subsets are verycharacteristic of celiac disease and the IEL “lymphogram” has a high sensitivity and specificity in the diagnosis of celiac disease including its potential and latentforms.Reproduced with permission from Leon et al. (2005b).
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4. Flow cytometry applications for the study ofceliac disease
4.1. Diagnosis: diagnostic biomarkers assessed by flowcytometry in celiac disease
4.1.1. IELs in the diagnosis of celiac diseaseThe initial screening of celiac disease is generally
performed in the clinics by means of the detection of theassociated serum anti-tTG or other antibodies (Green andCellier, 2007). The confirmation of the diagnosis is basedupon the demonstration of a causal relationship betweengluten intake and the characteristic enteropathy of celiacdisease. While the necessity to observe the enteropathy toreach the diagnosis is currently under discussion, a smallbowel biopsy is still considered mandatory for the diagnosisof celiac disease by most medical guidelines. The biopsy isobtained by oral endoscopy. The histological alterations arenot pathognomonic of celiac disease, and can also beobserved in other childhood diseases such as in gastroenter-itis and post-enteritis syndrome, food allergy — particularlyto soy and cow milk-, and parasite infestations (Giardia,Cryptosporidium and nematodes). In adults, similar histolog-ical changes can be observed in tropical sprue, immuno-deficiencies, lymphomas, Crohn's disease, and other diseases(reviewed in Leon et al., 2005b). This lack of specificity of theintestinal biopsy makes the study of intestinal intraepitheliallymphocytes (IELs) by flow cytometry the element whichprovides specificity to the analysis of the tissue specimen.
As mentioned, celiac disease is characterized by animportant increase in the TcR-γδ+ IEL subset, a decrease inthe CD3− subset and, depending on gluten intake, aconsiderable increase in the TcR-αβ+ IEL subset whichconstitute the majority of IEL [Fig. 3]. The increase in γδ IEL(average 4% in controls v 25% in celiacs, with respect to totalIEL) is not strictly diagnostic of celiac disease as it has beenobserved, although to a lower extent, in food allergy andoccasionally in other conditions. The γδ IEL decrease withGFD in absolute terms as measured by immuno-histochem-istry (Jarvinen et al., 2003) though they do not fully normalizein a majority of patients (Koskinen et al., 2009).
The increase in γδ IEL can also be a helpful diagnosticbiomarker in latent and potential celiac disease (Camarero etal., 2000) and in the cutaneous manifestation of celiac disease(dermatitis herpetiformis) which often times has no intesti-nal mucosal atrophy despite the elevation of IELs and γδ IELsin particular (Savilahti et al., 1997). Finally, IEL flowcytometry is useful in the differential diagnosis of celiacdisease versus other immune-mediated enteropathies such asgastrointestinal Tc lymphoma (Vega et al., 2006).
4.1.2. Assessment of other celiac diagnostic biomarkers by flowcytometry
In addition to the study of IELs, flow cytometry can be usedto measure other biomarkers, such as the highly specific anti-tTG antibodies. Multiplexed particle-based flow cytometrytechnology represents a new approach in the diagnosis ofceliac disease by its ability to quantitatively determine
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multiple autoantibodies in the same sample, simultaneouslyand rapidly. This technology offers similar performance toELISA assays in the detection of anti-tTG antibodies and evenbetter performance thanELISA in celiac patientswith the quitecommonhepatic dysfunction, possibly due to the avoidance ofdebris and unbound cross-contaminants creating “noise” inthe samples under analysis (Yiannaki et al., 2004).
4.2. Monitoring: disease activity biomarkers assessed by flowcytometry in celiac disease
Celiac disease is a lifelong disease that requires constantavoidance of gluten from the diet. At diagnosis, or when thepatient fails to comply with the diet, the intestinal mucosapresents with intraepithelial lymphocytosis, crypt hyperpla-sia and villous atrophy, and this is considered characteristic ofthe active phase of the disease. If the patient fully complieswith the diet the villous atrophy may disappear but theintraepithelial lymphocytosis usually persists, possibly due tolow levels of contaminating gluten in the GFD as assessed alsoby the presence of intestinal deposits of anti-tTG IgAantibodies (Koskinen et al., 2010). The titer of serum celiacautoantibodies decreases after the start of the GFD (Greenand Cellier, 2007) and constitutes a useful tool to monitordietary compliance and complications, however they are lesssensitive than IELs and deposited anti-tTG to smaller amountsof gluten such as contaminant gluten in a GFD (Koskinenet al., 2010).
In RCD, when the patient fails to improve despite amaintained gluten-free diet, the same histological character-istics seen on the active phase are seen. RCD is a chronic stagewhere disappearance of the villous atrophy needs ofimmunosuppressive drugs. RCD can have a poor prognosiswith about 5% of the patients evolving to a lymphoma (EATL).
Differentiating between these different stages — Active,inactive on a GFD, refractory and EATL — is therefore, veryimportant clinically and is in this area where flow cytometryhas played the most important role in celiac disease. Sinceceliac disease is an immunologically-mediated disease, flowcytometry studies have involved the intestinal lymphocytesof the intestine: IEL and LPL, the circulating lymphocytes inthe peripheral blood compartment and the cytokines secretedby these lymphocyte subsets.
4.3. Diagnostic and prognostic biomarkers in refractory celiacdisease types I and II
RCD is one of the least frequent (~1%) but most severecomplications of celiac disease. In RCD, symptoms andhistological damage persist or recur, after a former goodresponse to a strict GFD, despite adherence to the diet formore than 12 months (anti-tTG antibodies are often negativein RCD) (Trier et al., 1978; Daum et al., 2005). If celiac diseaseis not managedwith a strict GFD, the intestinal T lymphocytesare stimulated over time, until they become abnormal andlose the mechanisms that control their growth, a process inwhich IL-15 appears to play a major role (Mention et al.,2003) (Malamut et al., 2010). If left untreated, RCD has badprognosis (Rubio-Tapia et al., 2009) and can give rise toneoplasias, mainly of the gastrointestinal tract (mouth,pharynx, esophagus) or to intestinal T-cell lymphomas such
as the enteropathy associated T-cell lymphoma (EATL)(Catassi et al., 2005).
The pathological mechanism of RCD is not known but it issuspected that IELs play an important role. Indeed, RCD hasbeen classified in type I or II according to the IEL phenotype(Daum et al., 2005). While RCD I patients conserve a normalphenotype of IELs, RCD II patients have an aberrant IELphenotype population characterized by the loss of the normalsurface markers CD3, CD4, CD8 and TCR with preservedexpression of intracytoplasmic CD3 (CD3ε), CD7 and restrict-ed TCR (α or β) gene rearrangements (Cellier et al., 1998).These monoclonal and phenotypically abnormal IELs, can befound not only at the epithelial level, but also in thesubepithelial layer or lamina propria of the small intestineand colon and in extra-intestinal tissues such as the lung, skin,blood and bone marrow of patients with RCD II before eventhe onset of an overt lymphoma (Fine et al., 1998; Verbeeket al., 2009). A detection of N40% CD3ε+ CD8− by immuno-histochemistry (Liu et al., 2010) or of N20% CD103+, CD45+,CD7+, CD3−, CD4/CD8− by the more sensitive flowcytometry (Verbeek et al., 2008a) is considered diagnostic ofRCD II. Furthermore, the presence of persistent monoclonalitytogether with N80% CD3ε+CD8− IELs is a strong predictor ofEATL development in RCD patients (Liu et al., 2010).
While immunohistochemical methods have proved to beeffective in detecting the abnormal IEL phenotype in frozenand fixed tissues by double CD3/CD8 immunostaining (Cellieret al., 2000; Patey-Mariaud De Serre et al., 2000), flowcytometry has proved to be more accurate and a betterpredictor of prognosis than the detection of T-cell clonality(Goerres et al., 2003; Sanchez-Munoz et al., 2008; Verbeek etal., 2008a). Contrary to active celiac disease, in RCD II, the γδ-TCR cells are decreased in relative terms and they tend tonormalizewith effective treatment (Verbeek et al., 2008b). Anadditional aspect of the prognosis of RCD which can beevaluated with flow cytometry is the expression of CD30,since it has been proposed that IELs expressing CD30 areassociated with worse prognosis and development of lym-phoma (Farstad et al., 2002). In addition to changes in the IEL,RCD also shows changes in peripheral blood that can bedetected by flow cytometry. For example, RCD patients havedecreased numbers of iNKT cells in peripheral blood whencompared to patients responding to a GFD (Bernardo et al.,2008b). In conclusion, continual monitoring of both immu-nophenotype and clonality of IELs is very important thediagnosis and follow-up of RCD, and could provide a usefultool for surveillance of patients at risk of EATL (Liu et al., 2010).
4.4. Diagnostic and prognostic biomarkers in enteropathy-associated T-cell lymphoma
If left untreated, celiac disease can evolve into an overtlymphoma (Bagdi et al., 1999). Enteropathy associated T-celllymphoma (EATL) is the most common primary T-celllymphoma found in the gut and has a poor prognosis (Al-Toma et al., 2007). It arises from the IELs and is frequentlyfound in celiac disease due to the increased numbers ofactivated IELs present (Isaacson, 1994). Two different typesare now recognized based essentially on immunophenotype:EATL type 1, characterized by CD56 negativity, and EATL type2, characterized by CD56 expression.
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Early detection of celiac patients at risk for developingEATL and of EATL patients is essential, because EATL can betreated if diagnosed early, with chemotherapy and, in thefuture, with potentially more specific drugs currently underdevelopment, such as IL-15 inhibitors. The two mostfrequently methods used to diagnose EATL are flow cytome-try and the assessment of the oligoclonality or monoclonalityof intestinal T lymphocytes by PCR.
EATL type 1 is composed of CD3+,CD5−, CD7+, CD8−(20%+), CD4−, CD103+, TCRβ+/− cells that almost in allcases express CD30 (Deleeuw et al., 2007). EATL type 1 ismore frequent and is the only one that has been associated toCeliac disease and shows chromosomal gains in 1q and 5q(Deleeuw et al., 2007). EATL type 2 is composed of CD3+,CD4−, CD8+, CD56+, and TCRβ+ cells. The neoplasticcells of the EATL are most frequently CD3+, CD4−, CD8−,CD103+, TIA-1+ (T-cell intracellular antigen) (Bagdi et al.,1999), a immunophenotype similar to that of the aberrantIELs found in RCD II. EATL can develop directly from an activeceliac disease stage (primary EATL), but it is normallypreceded by a refractory celiac period, in particular, RCD II(Al-Toma et al., 2007). Indeed, the same monoclonal andaberrant IEL populations seen in RCD II have been found in theEATL and thus RCD II is considered a cryptic or low grade T-cell lymphoma of the intestine (Carbonnel et al., 1998; Bagdiet al., 1999; Cellier et al., 2000; Daum et al., 2000). Thedetermination of the phenotype of IELs is thus of clinicalimportance in the diagnosis an prognosis of EATL.
4.5. Use of flow cytometry in the investigation of thepathogenesis of celiac disease
4.5.1. Cytokine profile of IELsAll IEL subsets display a type I pro-inflammatory cytokine
profile in both control and celiac disease subjects, with TcR-αβ+ IELs being the main IFN-γ producers (Olaussen et al.,2002; Leon et al., 2005a). IL-4 is almost undetectable and IL-10 increases in patients on a GFD and in asymptomaticpatients, possibly indicating a functional regulatory role(Leon et al., 2005a).
IL-15 has been found to be massively increased in thelamina propria and epithelial layer of patients with active andrefractory celiac disease, probably perpetuating the damageto the enterocyte and promoting clonal expansion of IELs(Mention et al., 2003; Di Sabatino et al., 2006). Recent studieshave shown that a novel subset of T cells characterized byexpression of high levels of IL-17A, the Th17 cells, may beresponsible for pathogenic effects previously attributed toTh1 cells. Flow cytometry confirmed that IL-17A is over-produced in celiac disease mucosa and that CD4+ and CD4+CD8+ cells were major sources. The majority of IL-17A-producing CD4+ and CD4+ CD8+ cells co-expressed IFN-gamma but not CD161 (Monteleone et al.).
4.5.2. Study of lamina propria lymphocytes (LPLs)A detailed description of LPLs in healthy individuals and
celiac disease is beyond the scope of this review. Theavailability of phenotypic information on LPL and its clinicalusefulness are considerably lower than for IELs probablybecause of the extra processing required in order to analyzethese cells, a collagenase digestion of the biopsy specimens
after the epithelium has been stripped. In contrast to IELs, thatare normally CD8+, LPLs are mostly CD4+ CD45ROαβ T cells(Blumberg et al., 1993). It is suspected that IL-15 and CD30are associated with the intestinal immunologic activation ofceliac disease both at the intraepithelial and lamina proprialevel (Periolo et al.). It is also believed that LPL induceapoptosis of epithelial cells in celiac disease, like IELs (DiSabatino et al., 2001). The study of LPLs is the next frontier inthe flow cytometry of celiac disease and it may yield as muchclinically valuable information as the study of IELs.
4.6. Other uses in celiac disease
4.6.1. Peripheral blood flow cytometry in celiac diseasePhenotyping of peripheral blood lymphocytes in adult
celiac disease has been conducted by several groups. Adecrease of CD3+, CD4+, CD8+ and CD19+ lymphocyteswas found in untreated celiacs compared with GFD-treatedceliacs and healthy volunteers (Di Sabatino et al., 1998). Theabsolute reduction of peripheral lymphocytes in celiacdisease probably reflects their homing to the intestinalmucosa. Untreated celiac patients have been shown to havehigher percentages of circulating CD45RO+ TCR gammadeltacells in adults (Kerttula et al., 1998) and children (Klemolaet al., 1994). In addition, reduced numbers of circulatingregulatory T cells (Granzotto et al., 2009) and peripheraldendritic cells (Ciccocioppo et al., 2007) have been reportedin celiac disease.
More recently, up-regulation of T-bet and phosphorylatedsignal transducers and activators of transcription (pSTAT)1,key transcription factors for the development of T helper type1 (Th1) cells, has been described in the mucosa of patientswith untreated celiac disease. Flow cytometry analysis of T-bet, pSTAT1 and pSTAT3 expression showed that T-betexpression in CD4+, CD8+ T cells, CD19+ B cells andmonocytes and IFN-gamma production by PBMC was higherin untreated than in GFD-treated celiac patients and controls.Thus, flow-cytometric analysis of pSTAT1 and T-bet proteinexpression in peripheral blood mononuclear cells could beuseful and sensitive markers in the follow-up of celiacpatients to evaluate disease activity and response to dietarymanagement (Frisullo et al., 2009).
Finally, peripheral bloodanalysis of gluten-specific circulatingT cells has also beenutilized experimentally for the assessment ofthe immunologic effects of gluten challenge and the elucidationof the pathogenesis of celiac disease (Raki et al., 2007).
4.6.2. Flow cytometry for the detection of gliadin peptides andthe prevention of dietary contamination
There is concern that gliadin, even when present ingluten-free-foods within the limit fixed by the CodexAlimentarius, over the long term may become toxic to celiacpatients. Flow cytometry has been used to analyze thecontent of gliadin in gluten-free foods, and a study estab-lished the detection limit of gliadin by flow cytometry in10 pg/ml (Capparelli et al., 2005).
4.6.3. Ex vivo flow-cytometric analysis of basophil activation inthe differential diagnosis of celiac disease from wheat allergy
Allergy to wheat is a Type1 hypersensitivity processmediated by IgE and should not be confounded with Celiac
184 F. Leon / Journal of Immunological Methods 363 (2011) 177–186
Disease, a type 4 hypersensitivity to gluten mediated by Tcells. Despite the clear differences in pathogenesis and indisease course, these two entities are sometimes hard todifferentiate if the afflicted patient has started a gluten-freediet and lacks serum anti-tTG autoantibodies. The negativityof the serum celiac antibodies could be due to an effectiveGFD in a celiac or to the lack of celiac disease. Both entitiesdisplay mucosal atrophy in the small intestine, renderinghistology unable to differentiate between them. Finally,serum specific IgE to wheat is known to be a poor predictivemarker of disease, with high rates of false negatives.
As mentioned, flow cytometry can be useful in thissituation because wheat allergy does not present with thetypical IEL changes of celiac disease. In addition, a newapplication of flow cytometry is the measure of the antigen-specific activation of basophils, obtained from the patient andstimulated ex vivowith wheat in order to determine whetheran IgE-mediated process exists (Tokuda et al., 2009).This isyet another example of how flow cytometry can contribute tothe clinical evaluation and monitoring of patients with celiacdisease and other gastrointestinal diseases.
5. Conclusion
Flow cytometry has proven to be a powerful tool in theunderstanding of the immunological mechanisms leading toceliac disease. In addition to its research use, flow cytometryalso has important clinical applications and is currently usedroutinely in specialized laboratories for the assessment ofdiagnostic, prognostic and disease activity biomarkers of thedisease. IEL biomarkers are useful in the diagnosis, assess-ment of the response to the gluten-free diet and, importantly,in the diagnosis of the severe complications of celiac disease,namely Refractory Celiac Disease and Enteropathy-AssociatedT-cell lymphoma. While the number of specialized celiaccenters is growing, additional standardization is needed inorder for flow cytometry to become a mainstream techniquefor celiac disease. If performed in the same tissue obtained byendoscopy for standard histological and immunohistochem-ical analyses, flow cytometry of IELs does not add any burdenon the patient and should be incorporated to the armamen-tarium of celiac reference centers. The assessment ofdiagnostic and prognostic biomarkers by flow cytometry inceliac disease is an example of the versatility of this techniqueand its applicability to the research and clinical study of solidtissues.
Acknowledgement
The author thanks Dr. Carolina Argüelles-Grande (CeliacDisease Center, Columbia University, NY) for her expertreview of the manuscript and her insightful advice.
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