Tetramer visualization of gut-homing gluten-specific T cells in the peripheral blood of celiac disease patients
Edited by Michael Sela, The Weizmann Institute of Science, Rehovot, Israel, and approved December 18, 2006
Abstract
Tetramers of MHC–peptide complexes are used for detection and characterization of antigen-specific T cell responses, but they require knowledge about both antigenic peptide and the MHC restriction element. The successful application of these reagents in human diseases involving CD4+ T cells is limited. Celiac disease, an intestinal inflammation driven by mucosal CD4+ T cells recognizing wheat gluten peptides in the context of disease-associated HLA-DQ molecules, is an ideal model to test the potential clinical use of these reagents. We investigated whether gluten-specific T cells can be detected in the peripheral blood of celiac disease patients using DQ2 tetramers. Nine DQ2+ patients and six control individuals on a gluten-free diet were recruited to the study. Participants consumed 160 g of gluten-containing bread daily for 3 days. After bread-challenge, gluten-specific T cells were detectable in the peripheral blood of celiac patients but not controls both directly by tetramer staining and indirectly by enzyme-linked immunospot. These T cells expressed the β7 integrin indicative of gut-homing properties. Most of the cells had a memory phenotype, but many other phenotypic markers showed a heterogeneous pattern. Tetramer staining of gluten-specific T cells has the potential to be used for diagnosis of celiac disease.
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The development of multimeric MHC–peptide complexes has revolutionized the analysis of antigen-specific T cell responses. Tetramers are such reagents consisting of four soluble recombinant MHC molecules, each loaded with a single peptide and bound to a streptavidin molecule that is coupled with a fluorogenic marker (1). Multivalent engagement of the MHC-peptide complexes leads to a stable binding of the tetramer to T cell receptors on the T cell surface, allowing direct visualization of T cells with a defined specificity.
MHC class I tetramer technology has greatly facilitated our understanding of CD8+ T cell responses in viral infections and cancer (2). The benefit of tetramers for characterization and diagnosis of human autoimmune and infectious diseases has, however, been modest (3–6). This particularly relates to MHC class II tetramers used for the characterization of antigen-specific CD4+ T cells. Only a few studies of relevance to autoimmunity exist (7–9). MHC class II tetramers are more difficult to produce than MCH class I tetramers (10, 11), and CD4+ T cells of a given specificity appear to be present at much lower frequencies than their CD8+ counterparts (12, 13). Several criteria have to be met to be able to detect antigen-reactive T cells with MHC II tetramers: both the peptide epitope and HLA-restriction element have to be identified, and sufficient frequency and relative high avidity of reactive T cells are needed (14).
Celiac disease, a chronic inflammatory disorder of the small intestine precipitated by ingestion of cereal gluten proteins, presents as an ideal model to test the potential clinical use of MHC class II tetramers. The disorder is driven by intestinal gluten-reactive CD4+ T cells that uniquely recognize gluten peptides in the context of the disease-associated HLA-DQ molecules (15). The HLA association of the disease is very strong, and the majority (80–95%) of the patients express DQ2 (DQA1*05/DQB1*02) and a minority express DQ8 (DQA1*03/DQB1*0302). Over the last few years, T cell epitopes of gluten (consisting of α-, γ-, and ω-gliadin and glutenin components) have been extensively characterized (16, 17). Some of them appear to be more important, such as the immunodominant DQ2-αI and DQ2-αII epitopes of α-gliadin, which are recognized by T cells from almost all patients, whereas the epitopes of γ-gliadin are less often recognized (16). The identification of gluten T cell epitopes facilitated the generation of DQ2 tetramers specific for the celiac disease-relevant T cell epitopes (18). In this study, we have used these DQ2 tetramers harboring either the DQ2-αI or the DQ2-αII epitopes of α-gliadin to directly enumerate and characterize gluten-specific T cells in the peripheral blood of celiac patients after a short-term challenge with bread. This rapid and sensitive method may prove to be useful to establish a reliable diagnosis of celiac disease in patients in uncertain cases and may also allow testing of emerging new therapies of celiac disease.
Results
Gluten-Specific T Cells Can Be Directly Detected in the Peripheral Blood of Celiac Patients.
Pilot experiments suggested that we were unable to detect gluten tetramer-positive T cells in the peripheral blood of celiac disease patients. Gluten-specific T cells were detectable neither in untreated patients with active celiac disease nor in treated patients on a gluten-free diet (data not shown). We therefore took advantage of a study by Anderson et al. (19) that demonstrated a transient, DQ2-restricted CD4 T cell response against deamidated gluten peptides after a short gluten challenge in celiac patients by measuring IFN-γ production with enzyme-linked immunospot (ELISPOT) assay. We hypothesized that gluten-specific T cells were responsible for at least part of this IFN-γ production and adapted the bread challenge protocol for detection of DQ2-gliadin tetramer-positive cells.
Study participants ate four slices of bread daily for 3 days and then continued their usual gluten-free diet. On day 0 before the bread challenge, and day 6 after it, peripheral blood mononuclear cells (PBMC) were separated followed by positive selection of CD4+ T cells. The isolated CD4+ T cells (>98% purity) were then stained with DQ2-αI and DQ2-αII tetramers, as well as with the DQ2-control tetramer. We also stained for CD11c or CD14 to exclude false-positive tetramer staining by contaminating monocytes. Specificity of tetramers was assessed after each conjugation by staining T cell clones specific for the epitopes incorporated into the tetramers [supporting information (SI) Fig. 6].
On day 0 before the bread challenge, equally low-intensity background staining was detected in celiac patients with all three tetramers. On day 6, T cells reactive with the DQ2-αI epitope were found in all patients. T cells staining with the DQ2-αII tetramer were detected in 8 of 10 challenges. Samples stained with the control tetramer still showed background staining (Fig. 1 A and B).
Fig. 1.
We wished to confirm that the T cells stained by DQ2-αI and DQ2-αII tetramers represented celiac disease-relevant populations of CD4+ T cells, and that tetramer staining was also specific in these complex blood samples containing polyclonal T cells. A similar analysis was therefore performed with four DQ2+ and two DQ2− control persons who were on a gluten-free diet and underwent the same short-term bread challenge (Fig. 1B). Only background staining was observed in these samples both before and after the challenge, indicating that neither alloreactive nor other non-gluten-specific T cells were stained by the tetramers. To further ascertain the specificity of the staining, we examined CD4+ cells separated from the blood of four DQ2+ and two DQ2− individuals on a gluten-containing diet. Also here only background staining was observed (Fig. 1C).
We further attempted to culture tetramer-positive cells prepared from cryopreserved samples after flow-cytometric sorting with DQ2-αI and DQ2-αII tetramers in four patients. In the attempts of making T cell clones from sorted cells, cell growth was observed in very few wells (1 in 100–300 wells), and no gluten-reactive T cells could be detected in the screening of growing clones. We also tried to generate T cell lines from 50–500 tetramer-positive precursor cells of three patients. Some of the lines were not reactive to gluten antigen in the screening. Of the gluten-reactive lines, we obtained five lines, which were only reactive to the epitope corresponding to the tetramer used for the sorting. In two lines we found evidence for weak reactivity to gluten antigens different from the epitopes of the tetramers used for the sorting (data not shown).
DQ2-Gluten Tetramer-Positive T Cells in the Peripheral Blood Have Gut-Homing Characteristics but Otherwise Variable Phenotypes.
We examined the phenotype of T cells staining with the DQ2-αI tetramer in eight patients by using four-color flow cytometry. In addition to the tetramer staining and the CD11c or CD14 staining, antibodies against various activation and differentiation markers were used. The results are summarized in SI Table 2.
Of the activation markers, CD69 was expressed in <10% of the gluten-specific T cells. CD25 was expressed by 10–40% of DQ2-αI tetramer-positive cells, except for one patient where 70% of T cells reacting with the DQ2-αI tetramer expressed this marker (Fig. 2A and SI Table 2). Most DQ2-αI tetramer-positive T cells expressed the β7 integrin. The β7 integrin is expressed together with the α4 integrin on >99% of peripheral blood T cells (20) indicating that these cells home to the gut (Fig. 2B). We examined expression of CD95 (Fas) in three patients and found high expression in all DQ2-αI tetramer-positive cells (Fig. 2B).
Fig. 2.
As opposed to the relatively homogeneous level of the above-mentioned markers, expression of differentiation markers varied. In five of eight patients, T cells staining with the DQ2-αI tetramer were CD45RO+CD45RA− memory cells, whereas in three patients, most DQ2-αI tetramer-positive T cells expressed both CD45RO and CD45RA with low intensities (Fig. 3A). Expression of CD62L, CCR7, and CD27 varied (Fig. 3B), but most CD62L+ memory cells also expressed CCR7 and CD27 (Fig. 3C). More than 90% of DQ2-αI tetramer-positive T cells expressed the costimulatory molecule CD28, indicating that activation of these cells requires costimulation (data not shown).
Fig. 3.
Taken together, most T cells specific for the DQ2-αI epitope expressed the gut-homing β7 integrin, the costimulatory molecule CD28, and CD95, but their stages of differentiation varied.
One patient (p7) participated twice in the study. She was diagnosed at the age of 1 year and had been on gluten-free diet for 32 years at the time of the first challenge (p7a). At this point, she had relatively few DQ2-αI tetramer-positive T cells, and a high proportion of these were CD45RA+ and CD62L+ (SI Table 2). In agreement with these phenotypic features, few spot-forming units were counted on ELISPOT, suggesting that many gluten-specific T cells did not secrete IFN-γ. At the second challenge 3 months later (p7b), the frequency of DQ2-αI tetramer-positive cells was lower. Still, >90% of DQ2-αI tetramer-positive T cells showed the CD45RA−CD45RO+ phenotype, and ≈90% of the cells were positive for the β7 integrin. Moreover, a higher number of spot-forming units were observed on ELISPOT (SI Table 2).
Comparison of IFN-γ ELISPOT Results and Tetramer-Based Staining Characteristics.
We also looked at the relationship between signals of IFN-γ ELISPOT, as used by Anderson and coworkers (19, 21), and tetramer-staining. Specifically, we examined IFN-γ ELISPOT responses to the immunodominant 33-mer peptide (22), which contains altogether six copies of the DQ2-αI, DQ2-αII, and DQ2-αIII epitopes. We found an increased IFN-γ secretion after the bread challenge in 9 of 10 challenges (Fig. 4). The ELISPOT responses correlated positively with the frequencies of CD4† T cells positive for DQ2-αI tetramer (Fig. 5A). We observed a negative correlation between the proportion of CD45RA+ DQ2-αI tetramer-positive cells and IFN-γ production in the ELISPOT assay (Fig. 5B), indicating that not all DQ2-αI tetramer-positive T cells produce IFN-γ. Positivity for CD62L correlated negatively with ELISPOT signals and, as expected, positively with CD45RA (data not shown).
Fig. 4.
Fig. 5.
Discussion
Here, we show that gluten-specific CD4+ T cells can be visualized in peripheral blood of celiac disease patients after a short-term bread challenge by use of DQ2-gliadin peptide tetramers. The frequencies of T cells reactive with the DQ2-αI or DQ2-αII epitope were similar in most of the patients, ranging from 1:1,000 to 1:5,000 among CD4+ T cells (equivalent of 1:5,000 to 1:25,000 in PBMC). The cells staining with the DQ2-gliadin peptide tetramers were all expressing the β7 integrin indicative of gut-homing properties. This finding suggests that after a short-term bread challenge, a wave of gut-homing T cells reactive with gluten peptides bound to the disease-associated DQ2 molecule can be found in the peripheral blood. Our results demonstrate the potential use of MHC class II tetramers in a human disease where the antigen and MHC restriction element is known and suggest that detection of these cells can be used for diagnostic purposes in celiac disease.
Our tetramer staining allowed a direct detection of gluten-specific T cells based on antigen-specificity, and, importantly, this was done without a culture-amplification step. The culture-amplification step has often been used to detect CD4+ T cells by MHC class II tetramers, because antigen-specific CD4+ T cells, in contrast to CD8+ T cells, appear to be present at considerably lower frequencies (4, 12). In vitro culture may alter the composition and function of the T cell population of interest and may favor the growth of certain subpopulations. Functional tests such as ELISPOT, intracellular cytokine staining, or CFSE assay are also used to study antigen-specific T cell responses; however, these methods may not detect all antigen-specific cells, for instance naïve cells. Thus, the method we have used has several advantages: it is rapid, it does not rely on the function of the cells, and it allows direct enumeration and characterization of epitope-reactive T cells.
The specificity of the tetramers was checked after each conjugation by staining of gluten epitope-specific T cell clones derived from celiac lesions. We extended the specificity control by obtaining negative staining results of CD4+ T cells isolated from PBMC of various control subjects. We also tried to ascertain the specificity by culturing DQ2 tetramer-positive T cells from cryopreserved PBMC isolated by flow cytometry-assisted cell sorting. Although we did manage to grow T cell lines uniquely reactive with the epitope for which they were tetramer-sorted from one patient, the overall success of growing T cell clones and T cell lines was poor. This inefficiency could reflect poor specificity of the stainings, but in light of the other specificity controls, we rather believe that these results reflect poor growth of the tetramer-sorted cells. Poor growth may relate to the fact that we used cryopreserved cells for this procedure or that the conditions for T cell culture may not have been optimal for the growth of these recently activated T cells.
MHC tetramers allow for phenotypical characterization of antigen-specific T cells, and we performed such a characterization in seven of the patients recruited to the study. During the initial exposure to gluten of celiac patients, digested gluten peptides enter the lamina propria of the small intestine and are taken up by dendritic cells. The dendritic cells migrate to Peyer's patches and mesenteric lymph nodes and present gluten peptides to specific T cells. These naïve or central memory T cells (23) become activated and transit the efferent lymph and the thoracic duct to reach the systemic circulation, and so can return to the gut. The DQ2-gliadin tetramer-positive cells, which we detected, are likely represented by the T cells that transit systemic circulation. After returning to the gut, these T cells can be activated and exert effector functions after interaction with local antigen-presenting cells.
Many of the DQ2-gliadin tetramer-positive cells did not display an activated phenotype after the bread challenge. The relatively low proportion of activated gluten-specific T cells may be explained by the fact that the patients stopped eating bread 3 days before the blood was drawn and analyzed. Indeed, when effector T cells are deprived of stimulation, they quickly loose their CD25 expression (24).
In five of eight challenges, DQ2-αI tetramer-positive cells were CD45RA−CD45RO+ memory cells. In the other three, besides these memory cells, we found moderate or high (30–80%) percentages of CD45RAloCD45ROlo cells. Traditionally, naïve and memory cells are distinguished by their expression of the splicing variants of CD45, naïve cells expressing CD45RA and memory cells CD45RO. Nevertheless, previous studies have demonstrated that upon activation with PHA or alloantigen, naïve T lymphocytes first acquire CD45RO and then loose CD45RA (25, 26). Because of the slow turnover of surface CD45, it takes several (2–3) days for the CD45RA isoform to be replaced by CD45RO (27, 28).
In our study, both CD45RA−CD45RO+ and CD45RAloCD45ROlo cells had a varying expression of other surface markers, such as CD62L, CCR7, and CD27. These results are in agreement with recent studies (29, 30) that also found heterogeneity of such phenotypic markers, even in a system with synchronous activation of naïve T cell receptor transgenic T cells (30). During their differentiation, T cells down-regulate or lose CCR7, CD62L, and CD27. One- to two-thirds of DQ2-αI tetramer-positive cells in the patients we examined were CD27−, and these were presumably effector cells (31, 32). We found DQ2-αI tetramer-positive cells that were both positive for CCR7, a chemokine receptor needed for entering lymph nodes, and the gut-homing marker β7 integrin; these cells may be destined for homing to Peyer's patches. In contrast, DQ2-αI tetramer-positive cells, which were expressing CD45RO and β7 integrin but were negative for CCR7 and CD62L may be destined to migrate to the lamina propria of the small intestine. These cells also expressed CD95, a marker typical for lamina propria lymphocytes (33).
IFN-γ production in ELISPOT correlated well with tetramer staining, indicating that gluten-specific cells secrete IFN-γ. Moreover, DQ2-αI tetramer-positive T cells expressed the β7 integrin, in accord with the results of the study by Anderson and coworkers (21) showing that depletion of β7 integrin-expressing cells abolish IFN-γ production in ELISPOT assay.
Shortly after bread challenge, circulating gluten-specific T cells exhibited variable phenotypes corresponding to various stages of T cell differentiation. Indeed, acquisition of IFN-γ production by these cells seemed to be related to down-regulation of CD45RA and CD62L, indicating the presence of central and effector memory cells and cells with intermediate phenotype.
The limited observation on serial challenge in one patient may suggest that gluten-specific T cells stimulated at the first challenge became fully differentiated effector cells during the second challenge; in this case, these cells were sequestered in the gut, which could explain the low number of tetramer-positive T cells detected in the blood. More observations are needed, however, to settle this issue.
DQ2-gliadin tetramer staining has the potential to become a diagnostic adjunct for celiac disease. Staining of T cells with tetramers would be particularly valuable for patients who started a gluten-free diet without a clear diagnosis of celiac disease. These patients are frequent in clinical practice and can only be diagnosed by histological examination of intestinal biopsies after several months of gluten challenge. However, many of such patients are reluctant to begin long-term gluten provocation because they fear the return of symptoms and discomfort. Indeed, when we recruited participants to the study, most of the possible controls lacked intestinal biopsies while they were on a gluten-free diet and thus had an uncertain diagnosis. We managed to identify six subjects who undoubtedly did not have celiac disease but who were on a gluten-free diet. Importantly, only background staining was observed for all tetramers in these controls. Of the nine celiac disease patients, DQ2-αI tetramer-positive cells were found in all, and DQ2-αII tetramer-positive cells were found in seven. These results are encouraging, but to fully evaluate the performance of the DQ2 tetramer test for disease specificity and sensitivity, a larger study on patients with uncertain diagnosis is needed.
In conclusion, we have shown that after a short bread-challenge, gluten-specific T cells are detectable in the peripheral blood of celiac patients but not controls. These T cells uniformly express the β7 integrin, indicating that they home to the gut. This rapid and sensitive method may prove to be useful in establishing a reliable diagnosis of celiac disease in patients on gluten-free diet and may also allow testing of emerging new therapies of celiac disease.
Materials and Methods
Subjects and Study Protocol.
Ten DQ2+ celiac patients and six control persons (four DQ2+ and two DQ2−) on a gluten-free diet were recruited to the study. In addition, blood samples of six healthy volunteers (four DQ2+ and two DQ2−) on a gluten-containing diet were examined. HLA typing for DQ2 (i.e., DQA1*05/DQB1*02) was determined either by serology (positive scoring for both DQ2 and DR3) or by genomic typing with OLERUP SSP kits for DQA1 and DQB1 (GenoVision/Qiagen, Hilden, Germany). Clinical details of participants are shown in Table 1. The diagnosis of celiac disease was based on combination of clinical signs and typical small intestinal histological finding of villous atrophy according to the modified Marsh criteria (34). For all celiac disease patients, the clinical status improved and histological abnormalities normalized after treatment with gluten-free diet. The six controls were gluten-intolerant persons who were on a gluten-free diet for 0.5–5 years. The diagnosis of celiac disease in these subjects was excluded because their intestinal biopsies were normal on a gluten-containing diet. These subjects had commenced a gluten-free diet because of personal conviction or by advice from their general practitioner. Study participants ate four slices of standard white bread (produced by Bakers AS) daily for 3 days, which corresponded to ≈160 g of bread, and then continued their usual gluten-free diet. On day 0 and day 6, blood was drawn, and response against gluten was assessed by direct staining of gluten-reactive T cells with tetramers and by ELISPOT assay. All participants gave written informed consent for the study. The study protocol was approved by the Regional Ethics Committee for Southern Norway.
Table 1.
| Participant no. | Category | Age | DQ2 expression | Gluten-free diet, years | Symptoms during challenge |
|---|---|---|---|---|---|
| p1 | Celiac | 29 | + | 2 | Nausea, diarrhea |
| p2 | Celiac | 52 | + | 2 | No symptoms |
| p3 | Celiac | 70 | + | 1.5 | Bloating |
| p4* | Celiac | 56 | + | 16 | Vomiting, bloating, pain |
| p5 | Celiac | 50 | + | 6 | Nausea |
| p6 | Celiac | 55 | + | 16 | Nausea |
| p7a† | Celiac | 33 | + | 32 | No symptoms |
| p7b† | Celiac | 33 | + | 32 | Bloating |
| p8 | Celiac | 62 | + | 7.5 | Fatigue, abdominal discomfort |
| p9 | Celiac | 39 | + | 4 | Nausea, abdominal discomfort |
| p10 | Control | 25 | + | 2.5 | Bloating, pain |
| p11 | Control | 42 | + | 3 | Bloating, nausea |
| p12 | Control | 37 | + | 0.5 | Bloating, pain, |
| p13 | Control | 40 | + | 1 | Bloating, pain |
| p14 | Control | 30 | – | 5 | Bloating, nausea diarrhea |
| p15 | Control | 26 | – | 1.5 | Fatigue, pain, diarrhea |
*Patient 4 participated in another study 4 months before this study, when she consumed 22.2 mg of a 14-mer synthetic peptide corresponding to the native DQ2-αII epitope (sequence PQPQLPYPQPQLPY) in a 3-day period.
†
Patient 7a and patient 7b are the same person; she underwent challenge twice, with 3.5 months between the challenges.
Preparation of DQ2 Tetramers.
Water-soluble DQ2 (DQA1*0501/DQB1*0201) molecules with covalently tethered peptides were produced in a baculovirus expression system (18). DQ2 molecules with three different peptides were produced; two with deamidated T cell epitopes of α-gliadin (i.e., the DQ2-αI tetramer and the DQ2-αII tetramer) and one with an HLA class I α-chain-derived peptide that is a dominating self ligand of DQ2 (35) (i.e., DQ2-control tetramer). Tetramers were made by conjugating the DQ2–peptide complexes with streptavidin PE (Molecular Probes, Eugene, OR). Gel-filtration analysis indicated that the conjugates generated by this protocol were not uniform in size (i.e., not only consisting of tetramers) (18). “Multimers” is thus a more appropriate term, but because such reagents typically are referred to as “tetramers,” we use the latter term. The specificity of the tetramers was assessed after each conjugation by staining two T cell clones TCC 380.E.2 (specific for the DQ2-αI epitope) and TCC 412.5.28 (specific for the DQ2-αII epitope) with all three DQ2 tetramers.
Separation of Cells.
PBMC were separated from 80–150 ml of citrated blood by density gradient centrifugation (Lymphoprep; Axis-Shield, Oslo, Norway). T cells were separated by using the CD4 Positive isolation kit (Dynal Biotech, Oslo, Norway), using DETACHaBEAD technology according to the manufacturer's instructions.
Flow Cytometry.
For tetramer staining, CD4+ T cells separated from freshly isolated PBMC were used. Cells were washed and stained in 5% FCS/PBS as described previously (18). Briefly, 1 to 4 million CD4+ T cells were stained in a reaction volume of 100–200 μl with 30 μg/ml tetramer at 37°C for 30 min. Cells were then cooled on ice for 5 min. Conjugated mAbs against various surface markers were added, and staining was conducted at 4°C for an additional 30 min. Cells were then washed once and analyzed on a FACSCalibur instrument (BD Pharmingen, San Jose, CA). Usually 1 million but at least 0.5 million events were acquired for analyses. The antibodies used were CD45RA-FITC, CD45R0-PE-Cy5, CD45RO-APC, β7-integrin-APC, CD62L-FITC, CD62L-APC, CD27-FITC, CD28-PerCP, CD95-FITC, CD69-FITC, CD25-FITC, CD25-APC, CD11c-PE-Cy5, and CD64-PE-Cy5 from BD Pharmingen; CCR7-FITC from R & D Systems (Abingdon, U.K.); and CD4-FITC, CD3-PE, and CD14-APC from Diatec (Oslo, Norway). Isotype control antibodies were from BD Pharmingen.
Culturing of DQ2-αI Tetramer-Positive T Cells.
CD4+ T cells were prepared from cryopreserved PBMC and stained for CD14-APC and DQ2 tetramers. CD14− cells binding either the DQ2-αI or the DQ2-αII tetramers were sorted into a tube by using a FACSAria instrument (BD Pharmingen). Cloning of the sorted cells was done by seeding one to three cells into the wells of a Terasaki plate in 20 μl of complete medium containing 1 million per ml irradiated allogenic PBMC, 1 μg/ml PHA (Remel, Dartford, U.K.), 10 units/ml IL-2 (R & D Systems), and 2 ng/ml IL-15 (R & D Systems). Alternatively, polyclonal T cell lines were made by pipetting 50–500 cells into the wells of a 96-well plate in 100–200 μl of medium with the above-mentioned composition. After 8–10 days, the growing T cell clones and T cell lines were restimulated and expanded for another 8 days, followed by testing of specificity and cryopreservation.
ELISPOT Assays.
PBMC were resuspended in RPMI containing 15% heat-inactivated and pooled human serum. ELISPOT assays for single-cell secretion of IFN-γ were done as follows. Wells of 96-well plates (MAHAS4510; Millipore, Bedford, MA) were coated with 100 μl of 5 μg/ml IFN-γ mAb (MAB285; R & D Systems) overnight at 4°C and then blocked with RPMI containing 10% FCS at room temperature for at least 2 h. Subsequently, 4 × 105 PBMC and various peptides were added to the wells, incubated overnight at 37°C, and incubated with MilliQ water for 5 min to destroy adherent cells. Released IFN-γ was detected by a biotinylated anti-IFN-γ mAb (BAF285; R & D Systems) that was added for 18–24 h at 4°C. Plates were washed, and streptavidin AP (Southern Biotechnology Associates, Birmingham, AL) was added for 1–2 h at room temperature. Spots were developed by using the AP Conjugate Substrate kit (Bio-Rad, Hercules, CA) and counted electronically (KS ELISPOT 401; Zeiss, Hamburg, Germany). Plates were washed four times with PBS between each step. Responses to peptides were assessed in quadruplicate. Stimulation without antigen was used as negative control, and stimulation with 20 μg/ml Mycobacterium tuberculosis purified protein derivative (National Institute of Veterinary Medicine, Oslo, Norway) was included as a positive control in all assays. We used native and deamidated synthetic 33-mer peptide.
Abbreviations
- ELISPOT
- enzyme-linked immunospot
- PBMC
- peripheral blood mononuclear cells.
Acknowledgments
We thank Hans Christian Aass for help with cell sorting and Jorunn Bratlie and Marie K. Johannesen for excellent technical help. This work was supported by grants from Medinnova, the Research Council of Norway, and the Eastern Norway Regional Health Authority.
Supporting Information
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© 2007 by The National Academy of Sciences of the USA. Freely available online through the PNAS open access option.
Submission history
Received: September 29, 2006
Published online: February 20, 2007
Published in issue: February 20, 2007
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Acknowledgments
We thank Hans Christian Aass for help with cell sorting and Jorunn Bratlie and Marie K. Johannesen for excellent technical help. This work was supported by grants from Medinnova, the Research Council of Norway, and the Eastern Norway Regional Health Authority.
Notes
This article is a PNAS direct submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0608610104/DC1.
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The authors declare no conflict of interest.
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Tetramer visualization of gut-homing gluten-specific T cells in the peripheral blood of celiac disease patients, Proc. Natl. Acad. Sci. U.S.A.
104 (8) 2831-2836,
https://doi.org/10.1073/pnas.0608610104
(2007).
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