T cell receptor delta  repertoire in inflamed and noninflamed colon of patients with IBD analyzed by CDR3 spectratyping

Wolfgang Holtmeier1, Andreas Hennemann1, Ekkehard May2, Rainer Duchmann3, and Wolfgang F. Caspary1

1 Medizinische Klinik II, Division of Gastroenterology, Johann Wolfgang Goethe-Universität, 60590 Frankfurt am Main; 2 Department of Biology II, Ludwig-Maximilians-Universität München, 80539 München; and 3 Medizinische Klinik I, Universitätsklinikum Benjamin Franklin, 12200 Berlin, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

gamma /delta T cells might play an important role in autoimmune conditions like inflammatory bowel disease (IBD). In the present study, we characterized the T cell receptor (TCR)-delta repertoire by complementarity determining region 3 (CDR3) spectratyping in the inflamed and noninflamed mucosa and in the peripheral blood of subjects with Crohn's disease and ulcerative colitis. In contrast to previously published data about alpha /beta T cells, we rarely found oligoclonal expansions of gamma /delta T cells specific only for the inflamed mucosa. The same dominant gamma /delta T cell expansions were also present in the noninflamed colon. Furthermore, the peripheral gamma /delta TCR repertoire was oligoclonal but clearly distinct from that in the inflamed intestine. Thus our results do not support a role for antigen-specific gamma /delta T cells in IBD, and dominant gamma /delta T cells of the peripheral blood are not likely to be derived from the inflamed gut. However, in several patients, the TCR-delta -repertoire was highly diversified, whereas in others we observed a loss of dominant gamma /delta T cell clones when inflamed and noninflamed mucosa were compared. In conclusion, those changes indicate that gamma /delta T cells might play an important role in a subset of patients with IBD.

gamma/delta mucosal immunology; Crohn's disease; ulcerative colitis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INFLAMMATORY BOWEL DISEASE (IBD) comprises at least two human diseases of unknown etiology: Crohn's disease (CD) and ulcerative colitis (UC) (5). CD is characterized by a transmural inflammation and by skip lesions with macroscopically normal intervening mucosa among disease areas. The preferred localization of the disease is the terminal ileum, but the disease may affect any part of the gastrointestinal tract. One hallmark of CD is the granuloma formation in mucosal lesions. In contrast, the inflammatory lesions in UC are confined to the epithelium and the mucosa of the colon. Evidence that the immune system plays a major part in the pathophysiology of both diseases (5) derives from the ability to suppress these diseases by immunosuppressive therapy and from genetically modified animals with altered regulation of the immune system that develop severe chronic intestinal inflammation (15, 41).

Initial cellular events that take place at the outset of IBD are poorly understood. CD and UC resemble autoimmune diseases, and numerous publications support the notion that T cell-mediated mechanisms may play a pivotal role in the pathogenesis of the disease (41). For example, there is an increased number of activated Lamina propria lymphocytes in the inflamed mucosa of patients with CD (7, 11). These mucosal T cells might have lost the physiological hyporesponsiveness to enteric antigens (14). An important role of T cells is also supported by various animal models of IBD, showing that gut inflammation can be induced and transferred by T cells (36, 38, 42). In addition, normal microflora is necessary in the development of gut inflammation in all animal models (15), indicating that microbial antigens might activate autoreactive T cells. Thus T cells that specifically accumulate in mucosal lesions may represent a primary oligoclonal T cell response that triggers the onset of the disease. Clonally expanded alpha /beta T cells were described in the inflamed intestine of patients with CD and UC (9, 39, 43, 48, 49), and similar expansions could be also found in the peripheral blood (44). Although many studies have suggested a pathogenic role for alpha /beta T cells, little work has addressed gamma /delta T cells.

gamma /delta T cells are a minor T cell population in humans, and their functions are still largely unknown (reviewed in Refs. 25 and 33). It was suggested that they play an important role in regulating the mucosal immune response and are key mediators of autoimmune diseases (26). Intraepithelial gamma /delta T cells are thought to produce keratinocyte growth factor, which suggests a role in maintaining the integrity of the epithelium (3). However, two recent studies (16, 47) suggest that this might not be the case. We have previously shown that the human small and large intestine of healthy adults is populated by clonally expanded gamma /delta T cells that are widely distributed (10, 28, 31). Furthermore, the human skin, which forms another large surface to the external environment, is also composed of clonally expanded gamma /delta T cells (30). The nature of the ligands recognized by gamma /delta T cells has been, in part, elucidated and was shown to be nonproteinic and of low molecular mass (25). Furthermore antigen recognition can be immunoglobulin-like without the time-consuming protein processing step of major histocompatibility complex presentation (8). In addition, gamma /delta T cells are thought to respond to self antigens rather than to foreign antigens (1, 22, 24).

Several studies support the notion that gamma /delta T cells play a key role in the pathogenesis of IBD. Immunohistochemical studies (37) reported an increased number of gamma /delta T cells in the inflamed mucosa, although contradictory studies (6, 12, 53) were published as well. Furthermore, gamma /delta T cells were found to be frequent in T cell areas around lymphoid follicles and epithelioid granulomas of CD (19), and clonal gamma /delta T cell expansions were described in the inflamed mucosa of patients with CD (35). In addition, analysis of peripheral blood lymphocytes revealed an increased percentage of gamma /delta T cells expressing the T cell receptor DV1 (TCRDV1) gene segment (6, 20, 51). In normal subjects, TCRDV2 is preferentially expressed by peripheral gamma /delta T cells (4), whereas intestinal gamma /delta T cells express mainly TCRDV1 (13, 28). TCRDV1 is also the dominant V region in the inflamed mucosa of patients with IBD (37, 51). Thus circulating TCRDV1-expressing gamma /delta T cells might be derived from colonic gamma /delta T cells leaving the inflamed gut.

The significance of these findings in subjects with IBD is unclear, because it is not known whether the same expanded gamma /delta T cell clones are also present in the noninflamed intestinal tissue or the peripheral blood. The finding of clonality within the inflamed colon does not allow the conclusion that they are disease related, because healthy subjects already express a highly oligoclonal TCR-delta repertoire (10, 28, 31). However, it might be possible to identify autoreactive gamma /delta T cell clones specifically expanded only within IBD lesions by comparing them with gamma /delta T cells from noninflamed mucosa. Because T cells are generally activated and clonally expanded through contact of their clonotypical TCR with an appropriate antigen, we evaluated the complementarity determining region 3 (CDR3) regions of TCR-delta transcripts of mucosal and peripheral lymphocytes from patients with CD and UC and compared them with those of inflammatory and healthy controls.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Sample collection and RT-PCR of TCR-delta transcripts. Mucosal biopsies from inflamed and noninflamed mucosa, 2-3 mm in size, were obtained by endoscopy from eight subjects with active CD (F-M) and three subjects with active UC (N-P). From subject L, we obtained additional colonic biopsies 5 mo before the exacerbation of CD. From subjects O and P, additional biopsies were available from moderately inflamed mucosa located between the noninflamed and highly inflamed colon. Biopsy specimens from IBD subjects were categorized into those from inflamed and noninflamed intestine according to endoscopic appearances and histology of adjacent samples. In addition, two inflammatory controls with diverticulitis (Q and R) and five normal controls (A-E) were included in the study. The distance between noninflamed and inflamed specimens was ~10-20 cm. From four healthy subjects (A-D), we (29) previously reported about the IgA and IgM variable heavy chain repertoire. The distance between the two colonic biopsies of healthy adults were 1 m (A-C) or 10 cm (D and E). Colonic biopsies were snap frozen immediately in liquid nitrogen. Peripheral blood specimens were obtained at the time of endoscopy, and peripheral blood mononuclear cells (PBMC) were isolated using a Ficoll density gradient (28, 31). The age range of studied subjects was 25-80 yr. All studies were approved by the University of Frankfurt ethical committee on human subjects.

On average, we obtained 7-15 µg of RNA from each biopsy; 1-2 µg were reverse transcribed into cDNA in a 20-µl reaction mix. PCR was performed with 2 µl of this reaction mix. Thus ~100 ng of cDNA was utilized in each PCR reaction. TCRDV1, -DV2, and -DV3 transcripts were amplified with Taq polymerase using Vdelta - and Cdelta -specific primers as previously described (28, 30, 31). After an initial hot start, amplification of TCRD rearrangements consisted of 37 cycles of 40 s at 94°C, 50 s at 61°C, and 1 min at 72°C, followed by a final extension for 10 min at 72°C. The expected PCR product length was 150-250 bp.

The primers used in this study were TCRDV1: CAG CCT TAC AGC TAG AAG ATT CAG C; TCRDV2: GCA CCA TCA GAG AGA GAT GAA GGG; TCRDV3: TCA CTT GGT GAT CTC TCC AGT AAG G; and TCRDC: AAA CGG ATG GTT TGG TAT GAG GC.

CDR3 spectratyping. For analysis of CDR3 lengths, 2-3 µl of each PCR mixture was added to formamide-containing loading buffer. PCR products were heat denatured for 2 min at 95°C. PCR products were then size separated on a 6% denaturing polyacrylamide gel and visualized by silver staining (Silver Sequence DNA staining reagents) as recommended by the manufacturer (Promega, Madison, WI). Bands were photographed by exposing polyacrylamide gels for 8-15 s to an automatic processor-compatible film (Silver Sequence). Demonstrating the reproducibility of our method, the CDR3 profiles of the two corresponding PBMC samples (Fig. 1, subjects A-D), which were analyzed independently, were almost 100% identical.


View larger version (118K):
[in this window]
[in a new window]
 
Fig. 1.   Complementarity determining region 3 (CDR3) length profiles of T cell receptor (TCR)-delta transcripts from the colon and peripheral blood of healthy adults. PCR-amplified TCR-delta transcripts from 5 healthy adults (A-E) were size separated on a denaturing gel. Both biopsies (colon I and II) and the two blood samples (peripheral blood mononuclear cells; PBMC I and II) were obtained at the same time. As shown, the overall CDR3 profiles from colon I and II (up to 1 m apart) were highly similar but not 100% identical, indicating that some variation exists among different colonic sites. Dominant bands not present in both colon sites (I and II) are indicated by an arrow. The peripheral TCR-delta repertoire was also oligoclonal but distinct from the colon.

Direct sequencing of individual CDR3 length bands. For direct sequencing of TCR-delta rearrangements, individual dominant bands were excised from the gels and incubated at room temperature in 50 µl sterile distilled H2O, after which 5-µl aliquots were reamplified for 30-35 cycles using the same primers and PCR conditions described above. Double-stranded PCR products were directly sequenced using the ABI automatic sequencer 310 and the ABI prism dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA Polymerase, FS (Perkin-Elmer, Weiterstadt, Germany), according to the conditions recommended by the manufacturer. For sequencing, the 3' primer, which was used for the PCR amplification, was taken.

Sequence analysis and calculation of CDR3 lengths. Nucleotide sequences were analyzed using PC/Gene and OMIGA software (Oxford Molecular, Cambridge, UK). The lengths of the CDR3 regions of translated TCR-delta chains were calculated as previously described (28, 31, 45). This calculation is arbitrary, because the exact borders of the CDR3 region are not defined and other groups use different calculations. Here, CDR3 lengths were determined by the number of amino acids between the conserved cysteine C, which is encoded near the 3' end of TCRDV regions, and the conserved GXG triplet, which is encoded by all TCRDJ regions, and eight amino acids were subtracted (45) [e.g., the CDR3 region: CALGEQRNPLVNWTAQLFFGQG, which is 22 amino acids long, has a CDR3 length of 14 amino acids (22 - 8 = 14)].


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CDR3 length analysis of TCR-delta transcripts from the colon and PBMC of healthy subjects. In a first set of experiments, we analyzed the TCR-delta repertoire of the colonic mucosa and PBMC of five healthy adults (subjects A-E). As shown in Fig. 1, the TCRDV1, -DV2, and -DV3 repertoire in normal colon was oligoclonal, and highly similar CDR3 profiles with identical dominant bands were present at distant colon sites separated by a distance as far as 1 m. These findings indicate that dominant gamma /delta T cells are widely distributed throughout the colon. However, we occasionally observed expanded gamma /delta T cell clones, which were not present at both colon sites. For example, colon biopsy I of subject A had additional dominant TCRDV1 CDR3 length bands not present in the colon of biopsy II (see arrows in Fig. 1). These minor variations could not be assessed by our previous work where we characterized the TCR-delta repertoire by cloning and sequencing (10, 28, 31). Dominant bands were also present in the peripheral blood. However, the CDR3 profiles of the intestine and the peripheral blood were distinct from each other, indicating that different gamma /delta T cell subsets are present in each organ.

CDR3 length analysis of TCR-delta transcripts from the colon of subjects with CD. TCRDV1- and -DV2-specific CDR3 spectratyping from noninflamed and inflamed colonic mucosa of six subjects with active CD (F-K) revealed oligoclonal gamma /delta T cell expansions (Fig. 2). Minor differences between the CDR3 profiles of noninflamed and inflamed mucosa were within the range seen in healthy subjects when biopsies from different colonic sites were compared (Fig. 1). In contrast, the TCRDV3 profiles of inflamed and noninflamed mucosa were different in all but one patient (subject G). Because TCRDV3-specific PCR yields were very low in subjects F, I, and J, it is likely that TCRDV3 was rarely expressed. This is in accordance with antibody studies in patients with IBD that demonstrated that TCRDV1 and -DV2 are the major V regions expressed by gamma /delta T cells in the inflamed and noninflamed mucosa (37, 51). Thus it is possible that TCRDV3 transcripts were just above the detection level, which might have skewed the CDR3 profiles. This is supported by our data from subject J, where we obtained different TCRDV3 profiles from the two noninvolved biopsies. In each of those cases, PCR was repeated with a 10-fold higher amount of cDNA that did not result in stronger PCR products. However, in subjects H and K, good PCR yields were obtained and additional dominant bands were visible in the inflamed mucosa. Thus on the basis of the small number of patients analyzed, we do not know whether those differences are specific for Crohn's disease or are within the range seen in our healthy controls.


View larger version (121K):
[in this window]
[in a new window]
 
Fig. 2.   CDR3 length profiles of TCR-delta transcripts from the colon and peripheral blood of patients with active Crohn's disease (see also legend of Fig. 1). Similar CDR3 profiles of TCRDV1 and -DV2 transcripts were present in noninflamed (-) and inflamed (+) colonic mucosa of 6 subjects (F-K). CDR3 profiles of the PBMC samples were distinct from the colonic samples. No PBMC samples were available from subjects J and K. Dominant bands, containing TCR-delta transcripts from clonally expanded gamma /delta T cells, were excised from the gels and directly sequenced (Fig. 3). Those bands are indicated by the CDR3 length numbers at the side of the gels.

Identical TCR-delta transcripts are present in inflamed and noninflamed mucosa. To confirm that identical clonal expansions were present in inflamed and noninflamed tissue, dominant bands of identical CDR3 length from TCRDV1, -DV2, and -DV3 transcripts were isolated from the gels, reamplified, and directly sequenced. Sequence analysis confirmed identical TCR-delta rearrangements at both colon sites in 18 cases (Fig. 3). In subject K, different TCRDV3 transcripts with a CDR3 length of nine were isolated from involved and noninvolved mucosa.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 3.   TCR-delta nucleotide sequences of gamma /delta T cell clones expanded in the colon of patients with Crohn's disease. Corresponding pairs of dominant bands with an identical CDR3 length from noninflamed (-) and inflamed colon (+) of CD patients were excised from the gels (Fig. 2) and directly sequenced without cloning. The two "x's" in the fourth column indicate that the same TCR-delta sequence could be isolated from both colon sites. For example, the first sequence F5153 with a CDR3 length of 20 was derived from the noninflamed (-) and inflamed (+) colon of subject F. P nucleotides are underlined. All sequences except J5782 from subject J were in frame. These sequence data are available from EMBL/GenBank/DDBJ under accession numbers AF451695-AF451717.

There is no evidence for "gut-like" gamma /delta T cells in the peripheral blood of subjects with Crohn's disease. From four patients (F-I) PBMC samples were obtained at the time of endoscopy. Similar to healthy controls (Fig. 1), most CDR3 profiles of peripheral TCR-delta transcripts were also restricted and clearly distinct from those of the intestine (Fig. 2). Thus dominant gamma /delta T cells are unlikely to be derived from the inflamed mucosa as suggested previously (6, 20, 51). From the PBMC sample of subject H, the dominant TCRDV3 band with a CDR3 length of nine was also analyzed, because it was identical in length with the dominant bands of the colon. However, sequence analysis revealed that it contained a distinct TCR-delta rearrangement (sequence H5200; Fig. 3), confirming that the repertoires of the blood and the inflamed intestine are different. Similar data were obtained from the patients with UC (see below).

The colonic and peripheral TCR-delta repertoire can be highly diverse. In two subjects with CD (L and M), we identified highly polyclonal CDR3 profiles of mucosal TCRDV1 transcripts (Fig. 4). From one of them (L), we were able to obtain tissue and PBMC during remission and at the onset of active disease 5 mo later. However, there was no significant change in the CDR3 profile over time and the repertoire remained polyclonal. Thus the TCR-delta repertoire can be very variable in CD, but we rarely identified dominant clones expanded only within the inflamed intestine.


View larger version (92K):
[in this window]
[in a new window]
 
Fig. 4.   Polyclonal CDR 3 length profiles of TCRDV1 transcripts in patients with Crohn's disease. From subject L, we obtained two colonic biopsies at remission (-) and two biopsies 5 mo later from noninflamed (-) and inflamed (+) colon. As shown, we obtained a polyclonal CDR3 profile of TCRDV1 transcripts (1/97) that remained unchanged until 5 mo later when active CD developed (6/97). Polyclonal CDR3 profiles were also seen in the colon and peripheral blood of subject M. Thus the TCR-delta repertoire can be highly diverse in patients with CD.

Distinct TCR-delta repertoires are present in the highly inflamed colon of subjects with UC. In subject N, the colonic CDR3 profile was polyclonal for TCRDV1 and -DV2 and oligoclonal for TCRDV3 (Fig. 5). We do note that the noninflamed colon contained clonal TCRDV1 expansions not visible in the polyclonal repertoire of the inflamed colon. The CDR3 profiles of peripheral TCRDV2 transcripts were polyclonal, whereas peripheral TCRDV1 and -DV3 repertoires were highly oligoclonal and distinct from the intestinal TCR-delta repertoire. In the second patient (O) we observed significant changes between normal and diseased colon. The CDR3 profiles of TCRDV1, -DV2, and -DV3 transcripts from the noninflamed and moderately inflamed tissue were highly oligoclonal and almost identical. Sequence analysis of dominant bands confirmed identical TCR-delta transcripts at different colon sites (Fig. 6A). However, this typical CDR3 pattern was lost in the highly inflamed areas and a polyclonal repertoire predominated. Possibly this could reflect the influx of peripheral gamma /delta T cells in the inflamed colon, because the TCRDV1 and -DV2 repertoires of PBMC were highly polyclonal as well. In contrast, there was a loss of dominant clones in the third, patient P, when the TCRDV1, -DV2, and -DV3 repertoires of highly inflamed and noninflamed mucosa were compared. Furthermore, sequence analysis of dominant TCRDV3 bands with a CDR3 length of 16 demonstrated that different TCRDV3 rearrangements were present in the inflamed and noninflamed colon (Fig. 6A).


View larger version (121K):
[in this window]
[in a new window]
 
Fig. 5.   CDR3 length profiles of TCR-delta transcripts from the colon and peripheral blood of three patients with ulcerative colitis (see also legend of Fig. 2). As shown, the CDR3 profiles of TCRDV1, -DV2, and -DV3 transcripts from subject O changed from a very oligoclonal repertoire in noninflamed (-) and moderately inflamed (±) mucosa to a very polyclonal repertoire within the highly inflamed mucosa (+). In contrast, there was no diversification of the repertoire in subject P and dominant bands that were present in non- or moderately inflamed mucosa were not found in the highly inflamed mucosa (e.g., DV3, CDR3 length 06). TCR-delta nucleotide sequences from selected CDR3 bands are shown in Fig. 6.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6.   TCR-delta nucleotide sequences of gamma /delta T cell clones expanded in the colon from patients with ulcerative colitis (UC) and diverticulitis. Dominant bands from colonic TCR-delta transcripts from subjects N, O, and P with UC (Fig. 5) (A) and subjects Q and R with diverticulitis (Fig. 7) (B) were excised from the gels and directly sequenced (see also legend of Fig. 3). Identical TCR-delta transcripts were recovered from noninflamed (-) and inflamed (+) mucosa. One exception was subject P, whose distinct TCRDV3 transcripts (CDR3 length 16) could be identified from noninflamed (-) and inflamed mucosa (± and +). Sequence O581 (TCRDV3) was out of frame. All other sequences were in frame. These sequence data are available from EMBL/GenBank/DDBJ under accession numbers AF451718-AF451730.

Identical dominant TCR-delta rearrangements are present in inflamed and noninflamed mucosa of patients with diverticulitis. We further studied the TCRDV1, -DV2, and -DV3 profiles of two inflammatory controls (Q and R) suffering from diverticulitis (Fig. 7). Similar to what we observed in the subjects with CD, we did not see a significant change in the CDR3 profiles of TCRDV1/transcripts between inflamed and noninflamed mucosa, and sequence analysis confirmed identical TCRDV1 transcripts at both sites (Fig. 6B). The TCR-delta repertoires of the peripheral blood and the intestine were distinct. The PCR yield of colonic TCRDV3 transcripts of subject Q was low, and we were unable to amplify any TCRDV3 transcripts from the noninflamed mucosa.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 7.   CDR3 length profiles of TCR-delta transcripts from the colon and peripheral blood of patients with diverticulitis. The CDR3 profiles of TCRDV1, -DV2, and -DV3 transcripts from subjects Q and R were highly similar in noninflamed (-) and inflamed (+) mucosa. Sequence analysis confirmed identical TCRDV1 transcripts at both colon sites (Fig. 6).

Characteristics of mucosal TCR-delta transcripts from subjects with CD, UC, and diverticulitis. All sequences were highly complex, as shown by extensive trimming of the gene segments and multiple N region additions (Figs. 3 and 6). An overutilization of the TCRDJ3 gene segment was suggested before for TCRDV1 and -DV3 transcripts of the inflamed colon of subjects with CD (35). However, none of our TCRDV1 and -DV3 transcripts contained the DJ3 gene segment and the vast majority used DJ1. DJ3 was only found in conjunction with TCRDV2 transcripts. DJ3 is also frequently used by TCRDV2 transcripts in healthy subjects (28). Translation into corresponding amino acid sequences did not reveal any obvious motif shared among different subjects (Fig. 8).


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 8.   TCR-delta amino acid sequences from patients with Crohn's disease, UC, and diverticulitis. Amino acid sequences of translated TCR-delta transcripts from gamma /delta T cell clones expanded in the colon of subjects with Crohn's disease (Fig. 3) (A), UC (Fig. 6A) (B), and diverticulitis (Fig. 6B) (C).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies proposed an important role for gamma /delta T cells in the pathogenesis of IBD (6, 20, 51), and an increased number of gamma /delta T cells (19, 37) with clonal expansions was reported (35) in the inflamed mucosa. It was hypothesized that unidentified antigens, presumably from the intestinal microflora, might activate autoreactive mucosal gamma /delta T cells that initiate a destructive immune response. Autoreactive gamma /delta T cells were also thought to be responsible for other autoimmune diseases like multiple sclerosis where several groups (32, 50) identified oligoclonal expansions in brain lesions. Thus the identification of clonally expanded gamma /delta T cells in the inflamed intestine of subjects with IBD, as suggested before for alpha /beta T cells (9, 23, 39, 43, 48, 49), would support the notion that autoreactive gamma /delta T cells might damage the mucosa.

As shown herein, we rarely identified that gamma /delta T cell clones exclusively expanded only in the inflamed mucosa, and highly similar CDR3 profiles and identical TCR-delta transcripts were present in the inflamed and noninflamed mucosa of most patients. Because the TCR-delta repertoire can also differ between two colonic sites of healthy subjects, we do not know whether those expansions, which were especially found within the TCRDV3 repertoires, are specific for IBD. However, we observed significant changes, such as the diversification of the TCR-delta repertoire or the "loss" of dominant clones, in a subset of patients. A diverse intestinal TCRDV2 repertoire was also described by others in patients with CD. (34). Whereas the diversification of the mucosal repertoire might have been caused by peripheral gamma /delta T cells infiltrating the inflamed mucosa, it is not clear why dominant clones, which are present in the noninvolved mucosa, are absent in the inflamed mucosa of other patients. Thus changes of the TCR-delta repertoire can be highly variable and are distinct from those described for alpha /beta T cells.

Lack of clonally expanded gamma /delta T cells, which are specific for the inflamed mucosa, does not exclude the possibility that gamma /delta T cells play a significant role in the destructive nature of the immune response. First, gamma /delta T cells might be activated by stress-induced self antigens (22) that could lead into tissue damage. It is possible that the gamma /delta TCR repertoire in normal mucosa is already shaped by self antigens under physiological conditions, and further activation through the same self antigens would only cause a proliferation of local gamma /delta T cells with no change of the TCR-delta repertoire. This is supported by our observation that we could not see any change in the repertoire between the inflamed and noninflamed mucosa of subjects with diverticulitis. Second, mucosal gamma /delta T cells might be just bystanders of an ongoing inflammation and their response could be triggered by CD3-independent mechanisms or inflammatory signals such as chemokines alone, which are secreted by other immune cells (46). Third, there might be only a few disease-specific autoreactive gamma /delta T cells within the inflamed mucosa that are very rare but control the vast majority of nonspecific cells (52). Finally, the possibility remains that pathogenic T cell clones are not restricted to inflamed areas but are present throughout the intestine.

Several groups independently described an increase of TCRDV1-expressing gamma /delta T cells in the peripheral blood of subjects with active IBD. Because TCRDV1 is predominantly expressed not only in healthy mucosa (13, 28, 40) but also in the diseased mucosa (37, 51), it was suggested that peripheral gamma /delta T cells are likely to be derived from the inflamed intestine (6, 20, 51). However, this scenario is unlikely, because our data demonstrate that gamma /delta T cell clones, which predominate in the inflamed intestine, are different from those that predominate in the peripheral blood.

In summary, we have shown that there can be significant differences between the TCR-delta repertoire of the inflamed and noninflamed colon, indicating that gamma /delta T cells play a significant role in a subset of patients with IBD. The opposite findings, like the diversification of the TCR-delta repertoire or the loss of dominant clones, might be explained by the hypothesis that CD and UC are not just two diseases but are likely to consist of several different subgroups that all have a distinct pathophysiology (21). In animal models, different al terations of the immune system all resulted in a common final pathway: the inflamed intestine (15). Because our findings are distinct from those described for alpha /beta T cells, it is possible that gamma /delta T cells might not damage the mucosa but have important secondary roles in IBD like the downregulation of the inflammatory process or regeneration of the damaged mucosa (3). This is supported by studies that suggest that gamma /delta T cells induce oral tolerance (26), maintain T cell hyporesponsiveness of mucosal T cells (2, 18), and secrete cytokines promoting growth and differentiation of epithelial cells (3, 17). In addition, gamma /delta T cells were shown to have a protective role in rat 2,4,6-trinitrobenzene sulphonic acid colitis, because depletion caused increased mortality (27).


    ACKNOWLEDGEMENTS

This work was supported by Grant Ho1521/2-2 from the Deutsche Forschungsgemeinschaft (to W. Holtmeier) and Grant Du193/2-4 (to R. Duchmann).


    FOOTNOTES

This work was presented, in part, as a poster during the Digestive Disease Week in New Orleans, LA, May 1998.

Address for reprint requests and other correspondence: W. Holtmeier, Medizinische Klinik II, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany (E-mail: W.Holtmeier{at}em.uni-frankfurt.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 16, 2002;10.1152/ajpgi.00224.2001

Received 31 May 2001; accepted in final form 10 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bandeira, A, Mota-Santos T, Itohara S, Degermann S, Heusser C, Tonegawa S, and Coutinho A. Localization of gamma /delta T cells to the intestinal epithelium is independent of normal microbial colonization. J Exp Med 172: 239-244, 1990[Abstract].

2.   Barrett, TA, Tatsumi Y, and Bluestone JA. Tolerance of T cell receptor gamma /delta cells in the intestine. J Exp Med 177: 1755-1762, 1993[Abstract].

3.   Boismenu, R, and Havran WL. Modulation of epithelial cell growth by intraepithelial gamma /delta T cells. Science 266: 1253-1255, 1994[ISI][Medline].

4.   Borst, J, Wicherink A, van Dongen JJ, de Vries E, Comans-Bitter WM, Wassenaar F, and Van den Elsen P. Non-random expression of T cell receptor gamma  and delta  variable gene segments in functional T lymphocyte clones from human peripheral blood. Eur J Immunol 19: 1559-1568, 1989[ISI][Medline].

5.   Braegger, CP, and MacDonald TT. Immune mechanisms in chronic inflammatory bowel disease. Ann Allergy 72: 135-141, 1994[ISI][Medline].

6.   Bucht, A, Söderström K, Esin S, Grunewald J, Hagelberg S, Magnusson I, Wigzell H, Gronberg A, and Kiessling R. Analysis of gamma /delta V region usage in normal and diseased human intestinal biopsies and peripheral blood by polymerase chain reaction (PCR) and flow cytometry. Clin Exp Immunol 99: 57-64, 1995[ISI][Medline].

7.   Burgio, VL, Fais S, Boirivant M, Perrone A, and Pallone F. Peripheral monocyte and naive T-cell recruitment and activation in Crohn's disease. Gastroenterology 109: 1029-1038, 1995[ISI][Medline].

8.   Chien, YH, Jores R, and Crowley MP. Recognition by gamma /delta T cells. Annu Rev Immunol 14: 511-532, 1996[ISI][Medline].

9.   Chott, A, Probert CS, Gross GG, Blumberg RS, and Balk SP. A common TCR beta -chain expressed by CD8+ intestinal mucosa T cells in ulcerative colitis. J Immunol 156: 3024-3035, 1996[Abstract].

10.   Chowers, Y, Holtmeier W, Harwood J, Morzycka-Wroblewska E, and Kagnoff MF. The Vdelta 1 T cell receptor repertoire in human small intestine and colon. J Exp Med 180: 183-190, 1994[Abstract].

11.   Choy, MY, Walker-Smith JA, Williams CB, and MacDonald TT. Differential expression of CD25 (interleukin-2 receptor) on lamina propria T cells and macrophages in the intestinal lesions in Crohn's disease and ulcerative colitis. Gut 31: 1365-1370, 1990[Abstract].

12.   Cuvelier, CA, De Wever N, Mielants H, De Vos M, Veys EM, and Roels H. Expression of T cell receptors alpha /beta and gamma /delta in the ileal mucosa of patients with Crohn's disease and with spondylarthropathy. Clin Exp Immunol 90: 275-279, 1992[ISI][Medline].

13.   Deusch, K, Luling F, Reich K, Classen M, Wagner H, and Pfeffer K. A major fraction of human intraepithelial lymphocytes simultaneously expresses the gamma /delta T cell receptor, the CD8 accessory molecule and preferentially uses the Vdelta 1 gene segment. Eur J Immunol 21: 1053-1059, 1991[ISI][Medline].

14.   Duchmann, R, Kaiser I, Hermann E, Mayet W, Ewe K, and Meyer zum Büschenfelde KH. Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD). Clin Exp Immunol 102: 448-455, 1995[ISI][Medline].

15.   Elson, CO, Sartor RB, Tennyson GS, and Riddell RH. Experimental models of inflammatory bowel disease. Gastroenterology 109: 1344-1367, 1995[ISI][Medline].

16.   Fahrer, AM, Königshofer Y, Kerr EM, Ghandour G, Mack DH, Davis MM, and Chien YH. Attributes of gamma /delta intraepithelial lymphocytes as suggested by their transcriptional profile. Proc Natl Acad Sci USA 98: 10261-10266, 2001[Abstract/Free Full Text].

17.   Finch, PW, Pricolo V, Wu A, and Finkelstein SD. Increased expression of keratinocyte growth factor messenger RNA associated with inflammatory bowel disease. Gastroenterology 110: 441-451, 1996[ISI][Medline].

18.   Fujihashi, K, Taguchi T, Aicher WK, McGhee JR, Bluestone JA, Eldridge JH, and Kiyono H. Immunoregulatory functions for murine intraepithelial lymphocytes: gamma /delta T cell receptor-positive (TCR+) T cells abrogate oral tolerance, while alpha /beta TCR+ T cells provide B cell help. J Exp Med 175: 695-707, 1992[Abstract].

19.   Fukushima, K, Masuda T, Ohtani H, Sasaki I, Funayama Y, Matsuno S, and Nagura H. Immunohistochemical characterization, distribution, and ultrastructure of lymphocytes bearing T-cell receptor gamma /delta in inflammatory bowel disease. Gastroenterology 101: 670-678, 1991[ISI][Medline].

20.   Giacomelli, R, Parzanese I, Frieri G, Passacantando A, Pizzuto F, Pimpo T, Cipriani P, Viscido A, Caprilli R, and Tonietti G. Increase of circulating gamma /delta T lymphocytes in the peripheral blood of patients affected by active inflammatory bowel disease. Clin Exp Immunol 98: 83-88, 1994[ISI][Medline].

21.   Gilberts, EC, Greenstein AJ, Katsel P, Harpaz N, and Greenstein RJ. Molecular evidence for two forms of Crohn's disease. Proc Natl Acad Sci USA 91: 12721-12724, 1994[Abstract/Free Full Text].

22.   Groh, V, Steinle A, Bauer S, and Spies T. Recognition of stress-induced MHC molecules by intestinal epithelial gamma /delta T cells. Science 279: 1737-1740, 1998[Abstract/Free Full Text].

23.   Gulwani-Akolkar, B, Akolkar PN, Minassian A, Pergolizzi R, McKinley MXMG, Fisher S, and Silver J. Selective expansion of specific T cell receptors in the inflamed colon of Crohn's disease. J Clin Invest 98: 1344-1354, 1996[Abstract/Free Full Text].

24.   Halary, F, Fournie JJ, and Bonneville M. Activation and control of self-reactive gamma /delta T cells. Microbes Infect 1: 247-253, 1999[ISI][Medline].

25.   Hayday, AC. gamma /delta Cells: a right time and a right place for a conserved third way of protection. Annu Rev Immunol 18: 975-1026, 2000[ISI][Medline].

26.   Hayday, AC, and Geng L. gamma /delta Cells regulate autoimmunity. Curr Opin Immunol 9: 884-889, 1997[ISI][Medline].

27.   Hoffmann, JC, Peters K, Henschke S, Herrmann B, Pfister K, Westermann J, and Zeitz M. Role of T lymphocytes in rat 2,4,6-trinitrobenzene sulphonic acid (TNBS) induced colitis: increased mortality after gamma /delta T cell depletion and no effect of alpha /beta T cell depletion. Gut 48: 489-495, 2001[Abstract/Free Full Text].

28.   Holtmeier, W, Chowers Y, Lumeng A, Morzycka-Wroblewska E, and Kagnoff MF. The delta  T cell receptor repertoire in human colon and peripheral blood is oligoclonal irrespective of V region usage. J Clin Invest 96: 1108-1117, 1995[ISI][Medline].

29.   Holtmeier, W, Hennemann A, and Caspary WF. IgA and IgM VH repertoires in human colon: evidence for clonally expanded B cells that are widely disseminated. Gastroenterology 119: 1253-1266, 2000[ISI][Medline].

30.   Holtmeier, W, Pfänder M, Hennemann A, Zollner TM, Kaufmann R, and Caspary WF. The TCR delta  repertoire in normal human skin is restricted and distinct from the TCR delta  repertoire in the peripheral blood. J Invest Dermatol 116: 275-280, 2001[Abstract/Free Full Text].

31.   Holtmeier, W, Witthöft T, Hennemann A, Winter HS, and Kagnoff MF. The TCR-delta repertoire in human intestine undergoes characteristic changes during fetal to adult development. J Immunol 158: 5632-5641, 1997[Abstract].

32.   Hvas, J, Oksenberg JR, Fernando R, Steinman L, and Bernard CC. gamma /delta T cell receptor repertoire in brain lesions of patients with multiple sclerosis. J Neuroimmunol 46: 225-234, 1993[ISI][Medline].

33.   Kagnoff, MF. Current concepts in mucosal immunity. III. Ontogeny and function of gamma /delta T cells in the intestine. Am J Physiol Gastrointest Liver Physiol 274: G455-G458, 1998[Abstract/Free Full Text].

34.   Kanazawa, H, Ishiguro Y, Munakata A, and Morita T. Multiple accumulation of Vdelta 2+ gamma /delta T-cell clonotypes in intestinal mucosa from patients with Crohn's disease. Dig Dis Sci 46: 410-416, 2001[Medline].

35.   Landau, SB, Probert CS, Stevens CA, Balk SP, and Blumberg RS. Over-utilization of the Jdelta 3 gene-segment in Crohn's disease. J Clin Lab Immunol 48: 33-44, 1996[Medline].

36.   Matsuda, JL, Gapin L, Sydora BC, Byrne F, Binder S, Kronenberg M, and Aranda R. Systemic activation and antigen-driven oligoclonal expansion of T cells in a mouse model of colitis. J Immunol 164: 2797-2806, 2000[Abstract/Free Full Text].

37.   McVay, LD, Li B, Biancaniello R, Creighton MA, Bachwich D, Lichtenstein G, Rombeau JL, and Carding SR. Changes in human mucosal gamma /delta T cell repertoire and function associated with the disease process in inflammatory bowel disease. Mol Med 3: 183-203, 1997[ISI][Medline].

38.   Mizoguchi, A, Mizoguchi E, Tonegawa S, and Bhan AK. Alteration of a polyclonal to an oligoclonal immune response to cecal aerobic bacterial antigens in TCR-alpha mutant mice with inflammatory bowel disease. Int Immunol 8: 1387-1394, 1996[Abstract].

39.   Nakajima, A, Kodama T, Yazaki Y, Takazoe M, Saito N, Suzuki R, Nishino H, Yamamoto K, Silver J, and Matsuhashi N. Specific clonal T cell accumulation in intestinal lesions of Crohn's disease. J Immunol 157: 5683-5688, 1996[Abstract].

40.   Peyrat, MA, Davodeau F, Houde I, Romagne F, Necker A, Leget C, Cervoni JP, Cerf-Bensussan N, Vie H, and Bonneville M. Repertoire analysis of human peripheral blood lymphocytes using a human Vdelta 3 region-specific monoclonal antibody. Characterization of dual T cell receptor (TCR) delta -chain expressors and alpha /beta T cells expressing Vdelta 3J alpha Calpha -encoded TCR chains. J Immunol 155: 3060-3067, 1995[Abstract].

41.   Powrie, F. T cells in inflammatory bowel disease: protective and pathogenic roles. Immunity 3: 171-174, 1995[ISI][Medline].

42.   Powrie, F, Leach MW, Mauze S, Menon S, Caddle LB, and Coffman RL. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity 1: 553-562, 1994[ISI][Medline].

43.   Prindiville, TP, Cantrell MC, Matsumoto T, Brown WR, Ansari AA, Kotzin BL, and Gershwin ME. Analysis of function, specificity and T cell receptor expression of cloned mucosal T cell lines in Crohn's disease. J Autoimmun 9: 193-204, 1996[ISI][Medline].

44.   Probert, CS, Chott A, Turner JR, Saubermann LJ, Stevens AC, Bodinaku KX, Elson CO, Balk SP, and Blumberg RS. Persistent clonal expansions of peripheral blood CD4+ lymphocytes in chronic inflammatory bowel disease. J Immunol 157: 3183-3191, 1996[Abstract].

45.   Rock, EP, Sibbald PR, Davis MM, and Chien YH. CDR3 length in antigen-specific immune receptors. J Exp Med 179: 323-328, 1994[Abstract].

46.   Romagnani, P, Annunziato F, Baccari MC, and Parronchi P. T cells and cytokines in Crohn's disease. Curr Opin Immunol 9: 793-799, 1997[ISI][Medline].

47.   Salvati, VM, Bajaj-Elliott M, Poulsom R, Mazzarella G, Lundin KE, Nilsen EM, Troncone R, and MacDonald TT. Keratinocyte growth factor and coeliac disease. Gut 49: 176-181, 2001[Abstract/Free Full Text].

48.   Saubermann, LJ, Probert CS, Christ AD, Chott A, Turner JR, Stevens AC, Balk SP, and Blumberg RS. Evidence of T cell receptor beta -chain patterns in inflammatory and noninflammatory bowel disease states. Am J Physiol Gastrointest Liver Physiol 276: G613-G621, 1999[Abstract/Free Full Text].

49.   Shalon, L, Gulwani-Akolkar B, Fisher SE, Akolkar PN, Panja A, Mayer L, and Silver J. Evidence for an altered T-cell receptor repertoire in Crohn's disease. Autoimmunity 17: 301-307, 1994[ISI][Medline].

50.   Shimonkevitz, R, Colburn C, Burnham JA, Murray RS, and Kotzin BL. Clonal expansions of activated gamma /delta T cells in recent- onset multiple sclerosis. Proc Natl Acad Sci USA 90: 923-927, 1993[Abstract].

51.   Söderström, K, Bucht A, Halapi E, Gronberg A, Magnusson I, and Kiessling R. Increased frequency of abnormal gamma /delta T cells in blood of patients with inflammatory bowel diseases. J Immunol 156: 2331-2339, 1996[Abstract].

52.   Steinman, L. A few autoreactive cells in an autoimmune infiltrate control a vast population of nonspecific cells: a tale of smart bombs and the infantry. Proc Natl Acad Sci USA 93: 2253-2256, 1996[Abstract/Free Full Text].

53.   Trejdosiewicz, LK, Calabrese A, Smart CJ, Oakes DJ, Howdle PD, Crabtree JE, Losowsky MS, Lancaster F, and Boylston AW. gamma /delta T cell receptor-positive cells of the human gastrointestinal mucosa: occurrence and V region gene expression in Heliobacter pylori-associated gastritis, coeliac disease and inflammatory bowel disease. Clin Exp Immunol 84: 440-444, 1991[ISI][Medline].


Am J Physiol Gastrointest Liver Physiol 282(6):G1024-G1034
0193-1857/02 $5.00 Copyright © 2002 the American Physiological Society




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
282/6/G1024    most recent
00224.2001v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (5)
Google Scholar
Articles by Holtmeier, W.
Articles by Caspary, W. F.
Articles citing this Article
PubMed
PubMed Citation
Articles by Holtmeier, W.
Articles by Caspary, W. F.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online