- Original Articles
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Changes in Human Mucosal γδ T Cell Repertoire and Function Associated with the Disease Process in Inflammatory Bowel Disease
Molecular Medicine volume 3, pages 183–203 (1997)
Although γδ T cells are a major component of the human intestinal mucosa, it is not clear what role they play in mucosal immunity or if they are involved in the disease process of inflammatory bowel disease (IBD).
Materials and Methods
Flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were used to identify quantitative and qualitative changes in the repertoire of γδ T cells present in surgical and/or biopsy samples of normal and inflamed colon from individual patients with ulcerative colitis (UC) or Crohn’s disease (CD). Cytokine production and the ability to adhere to and interact with colonic fibroblasts were used to compare the functional properties of γδ T cells isolated from the normal and diseased colonic mucosa.
Increased numbers of γδ T cells localized in areas of inflammation and tissue injury were found in the majority of patients, irrespective of the type of IBD present. This expansion was attributable to an increase in Vδ1+ cells expressing a Vδ1-(Dδ3)-Jδ1-encoded T cell receptor and was seen in patients with severe disease as well as those with newly diagnosed or less severe forms of IBD. Among T cells present in the inflamed mucosa of patients with CD, γδ T cells, particularly Vδ1+ cells, were a major source of the proinflammatory cytokine interferon-γ and could interact with colonic fibroblasts.
Our results demonstrate that the chronic inflammatory immune response characteristic of IBD is associated with distinct changes in the number, distribution, composition, and function of mucosal γδ T cells. Through the production of cytokines and physical interaction with other cells, γδ T cells can perform an immunoregulatory function and contribute to the pathophysiology of IBDs.
Inflammatory bowel disease (IBD) represents a complex group of idiopathic chronic inflammatory disorders of which Crohn’s disease (CD) and ulcerative colitis (UC) are the major forms. While there are several features that distinguish CD and UC, there are many epidemiological, clinical, radiologic, endoscopic, and histologic features that are shared between these two disorders.
Several immunologic and histopathologic features of UC and CD can be explained as a defect in mucosal immunoregulation, and as a direct or indirect consequence of persistent mucosal T cell activation (1–4). Experimental evidence for a direct role for T cells in IBD comes from various animal models, especially “gene knock-out” strains that spontaneously develop bowel lesions. In particular, the presence of intestinal inflammation in transgenic mice deficient in αβ T cells (5) or T cell-derived cytokines such as interleukin 2 (IL-2) (6) suggest that T cells are responsible for the homeostatic regulation of mucosal immune responses.
In the human intestine, γδ+ cells are a major component (30–40%) of the T cells in the intraepithelial spaces and the majority (>70%) express Vδ1-encoded receptors (7). In view of the predominance of Vδ1+ cells within the human intestinal mucosa and the possibility that they have similar effector functions to their murine counterparts, it seems likely that they may play an important role in the generation and/or regulation of inflammatory responses in the intestinal mucosa. The ability of γδ T cells to interact with and influence the activity of a variety of stromal cells (8–10), including epithelial cells (8,11), and the finding that the severity of IBD in T cell receptor (TCR)-αβ-deficient mice is significantly reduced when crossed with mice lacking γδ T cells (5) are all consistent with an immunoregulatory role for mucosal γδ T cells.
Whereas several groups of investigators have described an increase in the number of Vδ1+ intraepithelial (IE) γδ T cells within the intestine of patients with coeliac disease (12–16), the immunohistochemical analyses of mucosal γδ T cells in patients with IBD have generated contradictory results (16,17). Although one group has shown that the distribution and relative number of TCR-δ+ cells in sections of colonic mucosa obtained from IBD patients was similar to that in sections of normal colonic mucosa (16), the results of another study showed that there were fewer TCR-δ+ cells in the inflamed mucosa of patients with IBD, and particularly, those with UC (17). Thus, evidence for the involvement of γδ T cells in IBD remains inconclusive. To investigate further the possible involvement of mucosal γδ T cells in IBD we have used a combination of sensitive cellular and molecular techniques to determine if there are any qualitative or quantitative changes in γδ populations in the colonic mucosa of patients with CD and UC.
The results of our studies identify changes in the number, distribution, and repertoire of γδ T cells that are present within the lesions of the inflamed intestine in both UC and CD. The finding that, in contrast to γδ T cells present in the healthy, noninvolved mucosa, γδ cells in the inflamed mucosa constitutively produce the inflammatory cytokine interferon γ (IFN-γ) and selectively interact with colonic stromal (fibroblasts) cells suggest that mucosal γδ T cells can contribute to the development of the disease process in IBD.
Materials and Methods
Procurement and Processing of Patient Samples
Surgical resectioned segments of intact colon were obtained from patients with UC, CD, or diverticular disease (diverticulitis) at the time of elective surgery. Histologically normal, noninvolved, mucosa was obtained from the margins of surgical resectioned colon that were at least a distance of 15 cm from any lesions. Mucosal biopsies were obtained with the approval of the Human Investigations Committee of the University of Pennsylvania School of Medicine. The origin and nature of the patient samples used in this study are compiled in Tables 1 and 2. When available, peripheral blood mononuclear cells (PBMCs) from the same patient were used as control samples. Patient-derived samples were used for RNA extraction, reverse transcriptasepolymerase chain reaction (RT-PCR) analysis, in situ hybridization, and/or cell isolation for flow cytometric and functional analyses.
Peripheral blood mononuclear cells were isolated from 5 to 10 ml of peripheral blood by density gradient centrifugation using Ficoll-Hypaque (Organo-Teknika, Durham, NC) and washed one to two times with PBS. Established human fibroblast cell lines of skin (CRL-1509), lung (CCD-11Lu), or colon (CCD-18Co) origin were obtained from the American Type Culture Collection (ATCC, Rockville, MD). A primary colonic fibroblast cell line was derived from the involved mucosal tissue fragments of a patient with ileocolonic CD (CD-11, Table 1) as described previously (18). This line was maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS) and penicillin/streptomycin/bartrim/amphotericin B. Prior to reaching confluency, cells were trypsinized and diluted 1:5 in complete media and plated into tissue culture-treated 20-mm petri dishes. Adherence assays were performed with primary fibroblasts that had been passaged twice.
IE and Lamina Propria (LP) T Cells
A modified version of a previously published procedure (19) was used to isolate IE and LP lymphocytes (L). Briefly, after removal of all associated mesentery, fat, and underlying muscularis, the mucosa was washed to remove mucus and cut into 2- to 3-cm pieces. IE lymphocytes were eluted by 3 to 4 sequential incubations with Hank’s balanced salt solution (HBSS) containing 0.1 mM EDTA at 37°C each for 15 min with constant magnetic stirring. The eluted cells were then subjected to two-step discontinuous Percoll (Pharmacia-LKB, Uppsala, Sweden) gradient centrifugation at 600 × g for 20 min using 70% and 40% Percoll. IEL were removed from the interface between the 70% and 40% Percoll layers. The remaining tissue fragments were incubated with HBSS containing collagenase type IV (Sigma, St. Louis, MO) at 37°C for 4–6 hr with constant stirring. The recovered LP cell suspension was then subjected to Ficoll-Hypaque density gradient centrifugation to isolate viable cells from which LP lymphocytes were obtained by Percoll gradient centrifugation as described above for IEL isolation. The yield of LP T cells (CD3+ cells) from the noninvolved mucosa and from the involved mucosa was calculated as the number of cells per gram of tissue used for isolation, and this yield was routinely comparable to that reported by others (20,21).
IMMUNOMAGNETIC SELECTION OF MUCOSAL IEL AND LP γδ+ T CELLS. A negative-immunomagnetic selection procedure (22) was used to obtain homogenous populations of TCR-γδ+ or TCR-αβ+ IEL and LP cells from the involved and noninvolved mucosa of colonic surgical specimens. Briefly, total LP and IEL cell samples were sequentially incubated with a mouse anti-human TCR-αβ (clone BW242/412; T Cell Diagnostics, Woburn, MA) or a mouse anti-human TCR-γδ antibody (11F2, Becton Dickinson) and a rabbit anti-mouse immunoglobulin G (IgG) antibody conjugated to supramagnetic particles (BioMag Beads, Perseptive Diagnostics, Cambridge, MA). The cells were then transferred to a 25-cm2 tissue culture flask and placed on a plate magnet and the “nonmagnetized” cells were removed. The purity of each T cell population was determined by flow cytometry, and cell viability, by trypan blue dye exclusion.
Aliquots (0.2−1.0 × 106) of bulk or fractionated IEL and LP mononuclear cells were stained with fluorochrome-conjugated mouse anti-human CD3- (clone S4.1; Caltag Labs., San Francisco, CA), CD8α- (3B5, Caltag), TCR-γδ- (11F2, Becton Dickinson), TCR-Vγ9- (T Cell Diagnostics), TCR-Vδ1- (TS8.2; T Cell Diagnostics), TCR-Vδ1-Jδ1- (δTCS-1; T Cell Diagnostics) and/or TCR-Vδ2- (23) specific antibodies, fixed and analyzed on a FACScan (Becton Dickinson) using Lysis II or CellQuest software as described previously (23). Fluorochrome-conjugated isotype-matched mouse antibodies or irrelevant specificity (Caltag) and anti-human CD45-specific antibodies (Dako Corporation, Carpinteria, CA) were used to control for nonspecific reactivity and as fluorochrome-compensation controls, respectively.
RT-PCR Analysis of the TCR-Vδ Repertoire
Total cellular RNA was prepared from intestinal tissue or PBMC by CsCl density gradient centrifugation (24). RNA pellets were extracted with acidic phenol once, with phenol:chloroform (1:1) once, and twice with chloroform prior to ethanol precipitation. Approximately 10 µg of total cellular RNA was used to synthesize cDNA using random hexanucleotides as described previously (25). A nested PCR assay (24) was used to analyze the Vδ-Jδ rearrangements expressed by γδ T cells in colonic tissue and blood mononuclear cell RNA/cDNA samples. Briefly, oligonucleotide primers specific for Vδ1, Vδ2, or Vδ3 in combination with a Cδ gene segment primer were used to amplify cDNA. The products of amplification were electrophoresed and TCR-PCR products gel purified and quantitated by DNA fluorimetry. Equivalent amounts of each cDNA were used as a template for a second amplification using the same forward Vδ primer and a reverse-primer specific for each of the four human Jδ genes. The products of amplification were electrophoresed on 1.8% agarose gels, Southern blotted, and hybridized with [γ32P]-ATP end-labeled Vδ1, Vδ2, or Vδ3 oligonucleotide probes containing sequence internal to the Vδ primers used for PCR. Individual Vδ1-Jδ1-4, Vδ2-Jδ1-4, and Vδ3-Jδ1-4 receptor cDNA clones were used to determine the optimum conditions for the proportional amplification of Vδ-Jδ genes with no cross-priming. For analysis of mucosal biopsy specimens, 5 to 6 individual pieces of tissue were pooled and total cellular RNA was isolated as described above. After generating cDNA, aliquots were analyzed for Vδ1-Jδ expression by PCR amplification and Southern blot analysis as described for the surgical samples.
In Situ Hybridization
In situ hybridization was used to identify and localize populations of γδ T cells and IFN-γ-producing cells within frozen sections of intestine as described previously (22,26,27). Briefly, 1- to 2-cm2 pieces of tissue from involved or noninvolved areas along the length of the resected intestine were snap frozen in Tissue Tek (Miles, Elkhard, IN) and stored at −86°C. Six-10 µm sections were cut from tissue blocks using a cryostat, fixed in 4% paraformaldehyde in PBS, pH 7.4, for 20 min at room temperature, then stored in 70% ethanol at 4°C in airtight containers. 35S-labeled RNA probes (0.2 ng RNA/µl; specific activity >109 cpm/µg) complimentary to the coding (antisense) or noncoding (sense) sequences human IFN-γ (26) genes were used for detection of TCR- and IFN-γ-mRNA+ cells.
IFN-γ protein production was analyzed from bulk, nonfractionated T cells and purified αβ T cells, and γδ T cells isolated from segments of noninvolved or involved mucosa from two patients with ileocolonic CD and one with colonic CD. Two hundred thousand cells were cultured in triplicate in microtiter plate wells containing either media alone or wells coated with an anti-CD 3 antibody (OKT3). After 96 hr, the culture supernatant was harvested and assayed for IFN-γ using a commercial radioimmunoassay (RIA) kit (Centocor, Malvern, PA). The amount of IFN-γ present was determined by reference to a standard curve generated from samples containing known amounts of rIFN-γ assayed in parallel.
Fibroblast Adherence Assay
T cells (3 × 106) from peripheral blood and/or normal or involved colon from four patients with ileocolonic CD were allowed to adhere to confluent monolayers of established or primary fibroblast cell lines in 20-mm petri dishes at 37°C for 30 min. Nonadherent cells were removed by gentle washing with prewarmed media and adherent cells removed by brief (3–5 min) trypsin treatment. The number of adherent cells was determined and the T cell composition evaluated by flow cytometry. The degree of enrichment for γδ T cells after adherence to fibroblast monolayers was determined by the number of γδ T cells present in the input and adherent cells. The proportion of mucosal Vδ1+ γδ T cells able to adhere to fibroblasts was determined from the number of Vδ1+ cells present in the input and adherent cell populations.
In situ hybridization was used initially to visualize and localize TCR-γδ+ cells in frozen sections of normal and diseased colonic mucosa. Compared with sections of normal colonic mucosa, sections of diseased tissue (CD) contained many more TCR-δ-mRNA+ cells that were distributed throughout the mucosa and submucosa and within the vasculature and connective tissue of the muscularis mucosa and serosa (data not shown). This distribution was consistent with the transmural pathology of CD. In order to analyze the distribution of γδ T cells in the normal and inflamed colon in more detail, flow cytometry was used to analyze cells directly after their isolation from the colonic mucosa.
Changes in the Number, Distribution, and Composition of Isolated Mucosal TCR-γδ+ T Cells in Noninvolved and Involved Tissue from the Same Patient
Three-color flow cytometry was used to compare the number, distribution, and composition of γδ T cells in the IE space and LP of the noninvolved and involved mucosa of individual IBD patients. This approach was preferred to immunohistochemical detection of T cells in intestinal tissue since this technique is more tedious and labor intensive, it is difficult to perform, and it is a relatively insensitive means of detecting antigens such as the γδ TCR that are expressed at low levels (28,29). For our study, surgically resectioned segments of colon from a total of seven patients with colonic CD or UC were used for this study. It should be noted that in contrast to the small bowel which is rich in IELs, the colon has relatively few IELs. Peripheral blood samples from these patients were analyzed in parallel.
The cytofluorographs of the analysis of one UC patient sample (Fig. 1A) and the compiled analysis of all seven IBD patients (Fig. 1B) identify differences in the distribution, number, and composition of γδ T cells in the blood and intestine. In the blood, γδ T cells comprised a small proportion of T cells (3–4%), of which >95% were CD4−, CD8− (data not shown) and expressed a TCR encoded by the Vγ9 and Vδ2 gene segments (Fig. 1B). By contrast, in the colon, γδ T cells made up a larger proportion of the T cells present in the colonic mucosa, particularly in the IE space (20–30%). Less than 10% of the IEL population were CD19+, demonstrating that the IEL preparations contained low levels of contaminating LP lymphocytes. The 10–20% of CD3− cells present in the IEL preparations were CD45− (data not shown), which is consistent with them being contaminating epithelial cells.
In the involved mucosa of all of the IBD samples analyzed, γδ T cells comprised a major T cell population in the LP of CD (mean, 43%; range, 23–63%) and UC (mean, 64%; range, 45–83%) patients. Assuming that the mucosal T cell isolation procedure did not result in the selective loss of γδ T cells (see Materials and Methods), it was possible to determine the relative number of γδ T cells present in the involved and noninvolved mucosa. The average yield of T cells (CD3+) recovered from the LP was 5.9 ± 0.4 × 106 cells/g from the noninvolved colonic mucosa and 2.4 ± 0.6 × 107 cells/g from the involved mucosa, which is comparable to the values reported previously (20,21). The proportion of TCR-αβ+ and TCR-γδ+ cells in the noninvolved mucosa was 74% ± 10% and 26% ± 9%, respectively (data not shown). Thus, noninvolved colonic mucosa samples contained an average of 1.6 ± 0.3 × 106 γδ T cells/g tissue. Since in the involved mucosa TCR-αβ and TCR-γδ represented 54% ± 15% and 46% ± 22%, respectively, this tissue contained on average 6.9 ± 0.3 × 106 γδ cells/g tissue, representing an almost 5-fold increase over that found in the normal mucosa.
Further characterization of the mucosal γδ T cells in the normal and inflamed colon using TCR-Vγ/δ-(Jδ)-specific antibodies revealed some interesting differences in the γδ T cell subsets present (Fig. 1B). In the noninvolved colonic mucosa, Vδ1+ γδ T cells comprised a major subset (43–52%) of IE γδ T cells. Of these cells, Vδ1-Jδ1-encoded TCR-δ chains predominated (63–76% of Vδ1+ cells). By contrast, in the LP, Vδ1+ γδ T cells represented a minor subset (6–10%) of γδ T cells; as seen among IEL, Vδ1+-Jδ1 was the predominant (43–58%) TCR-δ chain expressed by these cells. Thus, Vδ1-Jδ1-encoded receptors predominate among Vδ1+ γδ T cells in both the IE space and LP of the normal colonic mucosa. Vγ9/Vδ2+ γδ T cells that make up the largest γδ T cell subset in the blood (Fig. 1B) had a distribution in the normal colonic mucosa that was reciprocal to that of the Vδ1 subset. Whereas they represented a minor subset (13%) of TCR-γδ+ IEL, they were the major γδ T cell subset in the LP (67–79% of TCR-γδ+ cells).
In comparing the γδ T cell subset distribution in the involved and noninvolved mucosa, the most striking difference was the increase in the proportion of Vδ1+ γδ cells in the LP of the involved mucosa. In both the UC and CD patient groups, Vδ1+ cells represented the major (57–76%) population of γδ T cells, of which most (76–81%) expressed a Vδ1-Jδ1-encoded TCR. By contrast, there was no significant difference in the proportion of Vδ1-bearing γδ T cells in the IE space of the involved versus noninvolved mucosa. The inflammatory reaction that occurs in the intestinal mucosa of IBD patients is characterized, therefore, by changes in the distribution of γδ T cell populations and by the accumulation of Vδ1+ cells in the LP. Although we do not know if this increase in γδ T cells and Vδ1+ cells represents expansion of resident mucosal cells or cells infiltrating from the blood, differences in the phenotype of Vδ1+ cells in the LP (and IE spaces) of normal colonic mucosa (majority CD8+) versus involved mucosa (majority CD8−) suggests they are mainly of peripheral origin.
Bias in Expression of Vδ1-encoded Receptors by γδ T Cells in Surgical Resected Tissue Specimens of IBD Patients as Demonstrated by RT-PCR Analysis
Repertoire analysis using antibodies and flow cytometry is limited by the size of mucosal tissue samples, the number of cells that can be recovered from each sample, and by the availability of TCR-V(J)-specific antibodies. Also, since the Vδ1-Jδ1-specific antibody may have some reactivity with Jδ-encoded receptors other than Jδ1 (30), it was important to corroborate the results obtained by flow cytometry using another independent approach. A sensitive RT-PCR based method that we have previously used for analyzing TCR-γδ repertoire analysis in human fetal tissues (24,25) was used for this purpose. The assay uses oligonucleotide primers specific for Vδ1, Vδ2, or Vδ3, in combination with a Cδ gene segment primer to amplify cDNA generated from mRNA samples using reverse transcriptase. After gel electrophoresis the products of amplification were excised from the gel, and equivalent amounts used as a template for a second amplification using the same forward Vδ primer and a reverse-primer specific for each of the four human Jδ genes. In establishing this assay, individual Vδ1-Jδ1-4, Vδ2-Jδ1-4, and Vδ3-Jδ1-4 receptor cDNA clones were used to determine the optimum conditions for the proportional amplification of Vδ-Jδ genes; there was no cross-priming. The products of this second amplification were then electrophoresed on 1.8% agarose gels, Southern blotted, and hybridized with [γ32P]-ATP end-labeled Vδ1, Vδ2, or Vδ3 oligonucleotide probes containing sequence internal to the Vδ primers used for PCR.
This assay was first used to analyze samples of intestine from noninvolved and involved segments of tissue obtained from 20 patients with IBD for Vδ-Jδ receptor expression. Ten patients diagnosed with UC (1–16 in Table 1), seven with CD (17–28 in Table 1), and three patients with diverticular disease (diverticulitis, 29–31 in Table 1) were used as a source of involved and noninvolved mucosal mRNA. In addition, tissue from the normal margins of colonic mucosa from three colon cancer patients (32–34 in Table 1) were also used as noninvolved, “normal” controls. The results of this analysis shown in Fig. 2 identify changes in the pattern of Vδ1-Jδ expression in the involved mucosa compared to the noninvolved mucosa and blood of IBD patients. The Vδ-Jδ profile of the noninvolved colonic mucosa (* profiles in Fig. 2) was characterized by expression of receptors encoded by Vδ1-Jδ1, 2, and 3, and by very low levels or an absence of Vδ1-Jδ4-encoded receptor chains. This profile was distinct from that of the peripheral blood in which all four Jδ-gene segments were utilized by Vδ1-encoded receptors. By contrast, a single Vδ1-encoded receptor predominated in the involved colonic mucosa. Regardless of whether the involved intestinal tissue was derived from patients with UC, CD, or diverticulitis, Vδ1-Jδ1 receptor expression predominated and it was the only Vδ1-encoded receptor detected in 13/17 IBD patients and 3/3 patients with diverticular disease. Importantly, this finding agrees with the results obtained by flow cytometry in which Vδ1-Jδ1+ cells were the major Vδ1+ γδ T cell population in the involved colonic mucosa (Fig. 1), and it also demonstrates the utility of this procedure for TCR-γδ repertoire analysis.
DNA sequence analysis of randomly selected Vδ1-Cδ cDNAs from involved and noninvolved mucosal mRNA samples from a patient with UC did not identify any Vδ1 receptors that were unique to the inflamed colonic mucosa (data not shown). All of the Vδ1-TCR-cDNAs encoded functional, in-frame receptors consistent with the receptor cDNAs detected by RT-PCR analysis being derived from productively rearranged genes. The Vδ1-Cδ cDNAs from noninvolved mucosa and Vδ1-Cδ cDNAs from involved mucosa utilized the Jδ1 gene segment, which is consistent with the results from both flow cytometric and RT-PCR analyses. In addition, a single D-gene segment, Dδ3, was exclusively used by all of the Vδ1-receptor chains expressed in the noninvolved (33/33) and involved (22/22) mucosa.
Lack of Bias in the Expression of Vδ2- and Vδ3-encoded Receptors in Surgical Resected Tissue Specimens of Patients with IBD
We were interested in knowing whether the Vδ2 and Vδ3 receptors, which are similar to Vδ1-encoded receptors, were also biased in Jδ gene usage. Figure 3 shows the Vδ2/Vδ3 repertoire analysis of 10 IBD patients representative of a total of 20 patient samples analyzed. In the normal intestine, Vδ2 receptors primarily utilized Jδ1 and Jδ3 whereas Vδ3 receptors utilized all four Jδ gene segments. This profile of Vδ2-Jδ and Vδ3-Jδ expression did not change significantly in the noninvolved intestine or in tissue samples obtained from different regions of the inflamed intestine (data not shown). In 2/16 CD samples and 3/35 UC samples from the inflamed mucosa. Vδ2-Jδ1 was predominant or was the only receptor expressed. Likewise, in rare samples (i.e., UC-10) a bias in the VS3-JS expression was also evident. Taken together, our analysis of the TCR-Vδ repertoire in the colonic mucosa demonstrates that a single Vδ receptor, Vδ1-Jδ1, predominates in the inflamed mucosa of patients with CD and UC.
Bias in Expression of Vδ1-encoded Receptors by γδ T Cells in Biopsy Tissue Specimens
The RT-PCR assay was next applied to the analysis of colonic mucosal tissue biopsy samples to determine if changes in γδ T cell repertoire detected in patients with severe disease were present in patients with newly diagnosed and/or less severe forms of IBD (Table 2). Because of the small size of the mucosal tissue samples procured by sigmoidoscopy and colonoscopy, it is not normally possible to isolate sufficient numbers of cells for fluorescence-activated cell sorting (FACS) analysis. The samples obtained from eight UC patients (Table 2) were therefore processed for RNA isolation and RT-PCR analysis of TCR-Vδ-Jδ expression. The results of this analysis shown in Fig. 4 clearly identify Vδ1-Jδ1 receptor expression as being the predominant receptor expressed in all of the patient samples. In five of eight patient samples it was the only Vδ1-Jδ TCR-δ chain expressed. Interestingly, this receptor was also predominant in the mucosa of a newly diagnosed UC patient who had symptoms of disease for less than 2 weeks. The expression of other Vδ1-Jδ-encoded receptors in noninvolved samples strongly suggests that the predominance of Vδ1-Jδ1 receptor expression in the involved mucosa was not due to a PCR-related artifact.
In summary, our cellular and molecular analyses of γδ T cells in the colonic mucosa of patients with IBD has shown that these cells accumulate in large numbers at the site of inflammation and tissue injury and can be distinguished by expression of a TCR-δ chain encoded by Vδ1-Jδ1 gene segments. These changes were apparent in all the IBD patients we analyzed, irrespective of the form or stage and severity of disease. To begin to understand the significance of these changes in the mucosal γδ T cell repertoire for the disease process in IBD, subsequent experiments were designed to analyze the effector function of mucosal γδ T cells.
Mucosal γδ T Cells Are a Major Source of IFN-γ Production in IBD
Since IFN-γ production appears to be an important mediator of the inflammatory process and tissue injury in patients with IBD (31) and is involved in the pathogenesis of intestinal inflammation in animal models of IBD (32), we investigated whether γδ T cells in the intestinal mucosa of patients with IBD were a source of IFN-γ. In situ hybridization analysis of IFN-γ mRNA expression in tissue sections from involved or normal intestinal mucosal tissue (Fig. 5) showed that the IFN-γ gene was constitutively expressed at low levels by cells in the normal, noninvolved, mucosa. By contrast, in sections of involved colonic tissue from a CD patient (CD-1), the number of IFN-γ-mRNA+ cells was greatly increased (Fig. 5D). No hybridization signal was detected in sections probed with a control sense probe (Fig. 5A and C). The distribution of IFN-γ mRNA+ cells in diseased mucosa (Fig. 5D) was similar to that of TCR-δ+ mRNA-expressing cells (data not shown), suggesting T (γδ) cells as a likely source of IFN-γ production. In addition, the identification of IFN-γ mRNA+ cells in blood vessels in the submucosa (Fig. 5E) and muscle (Fig. 5F) was consistent with the transmural distribution of TCR-δ-mRNA+ cells (data not shown).
To identify the cellular source(s) of IFN-γ production in the colonic mucosa, IFN-γ-gene expression and protein secretion was analyzed in isolated populations of γδ and αβ LP T cells. These T cell populations were isolated from the mononuclear cell fraction of noninvolved and involved mucosa from three patients with CD using immunomagnetic selection. The isolated populations were routinely >95% CD3+ and the degree of contamination of the γδ fraction by αβ+ cells and the αβ fraction by γδ+ cells was routinely <10% (data not shown). As seen in our previous flow cytometric analyses of mucosal T cells (Fig. 1A), Vδ1+ cells were the major γδ T cell subset (67 ± 8%) in the involved mucosa compared with the noninvolved mucosa (12 ± 2%). The increase in the number of T cells in the LP of involved mucosa (see above) was paralleled by an increase in the frequency of cells that constitutively expressed IFN-γ-mRNA (Table 3). This was particularly apparent for γδ T cells that contained a higher proportion of mRNA+ cells than αβ T cells. The analysis of IFN-γ secretion was consistent with the mRNA analysis and demonstrated that γδ T cells, including Vδ1+ cells, were a major source of this proinflammatory cytokine in the involved mucosal CD samples analyzed.
Selective Interaction of γδ T Cells with Colonic Fibroblasts
In view of the accumulation of γδ T cells, particularly Vδ1+ cells, in the LP of the involved intestinal mucosa of CD patients and results from previous studies demonstrating that γδ T cells can interact with stromal cells in other tissues such as skin (9,33), we determined if mucosal γδ T cells, particularly Vδ1+ cells, from patients with IBD could adhere to and interact with colonic stromal cells, and fibroblasts in particular. To accomplish this we took advantage of the availability of established and well-characterized fibroblast cell lines of different mucosal tissue origin and the γδ T cell-skin fibroblast adherence assay described by White and co-workers (9). To date, the possibility that γδ T cells can interact with colonic stromal cells such as fibroblasts and how this interaction might be affected during IBD has not been investigated. Bulk populations of colonic T cells isolated from segments of normal and involved tissue of individual IBD patients were briefly incubated with confluent monolayers of fibroblasts and the composition and phenotype of adherent and nonadherent cells determined by flow cytometry.
As seen in Fig. 6A, mucosal γδ T cells from one of two patients with CD (CD8) were considerably enriched for after co-culture with colonic fibroblasts. Whereas co-culture with skin- or lung-derived fibroblasts produced between 1.5- and 3.5-fold enrichment of γδ T cells, culture with the colonic fibroblast cell line CCD-18Co resulted in an almost 10-fold enrichment of γδ T cells. A similar level of γδ T cell enrichment was also seen using another colonic fibroblast cell line (CCD-112Con) (data not shown), excluding the possibility that the interaction with the CCD-18Co cell line was due to some peculiar property of this cell line. Analysis of nonadherent T cells from cultures with colonic fibroblasts showed a reciprocal decrease in the γδ T cells (data not shown), excluding the trivial explanation that their enrichment was due to preferential survival during or after culture. The specificity of mucosal γδ T cell-fibroblast interaction was also demonstrated by the failure to see a similar enrichment of γδ T cells using peripheral blood from two of the same CD patients, or using fibroblasts derived from sources other than the colon. Enrichment of αβ T cells among adherent cells after co-culture with colonic fibroblasts, or fibroblasts of other tissue origin (data not shown), was 2-fold or less (Fig. 6B), which suggests that increased adhesiveness of γδ T cells reflects an inherent property of these cells rather than a function of the fibroblast monolayer. Slightly higher levels of γδ T cell enrichment were seen using lymphocytes from the involved versus noninvolved mucosa with three of the four patients with CD (Fig. 6B).
Analysis of the fibroblast-adherent γδ T cells showed that the majority of those isolated from the noninvolved (75% ± 8%) and involved (84% ± 9%) mucosa of four patients with CD (#7–10; Fig. 6B) were Vδ1+. By comparing the number of Vδ1+ cells in the input and adherent cell populations it was possible to determine what proportion of these cells in the noninvolved versus involved mucosa of the same patient were fibroblast-adherent (Fig. 7A). Whereas in the noninvolved mucosa fibroblast-adherent Vδ1 cells represented <40% (mean of 31% with a range of 24% to 39%) they represented almost 80% (mean of 78% with a range of 63% to 92%) of Vδ1+ cells in the involved mucosa. Thus, the accumulation of Vδ1+ γδ T cells in the inflamed mucosa of patients with IBD was associated with an increase in their ability to interact and adhere to colonic fibroblasts. Using a primary colonic fibroblast cell line derived from mucosal tissue sample from a patient with primary colonic CD (Fig. 7B), we have reproduced the results obtained using established colonic fibroblast cell lines. In particular, using either donor-matched or allogeneic LP lymphocytes from noninvolved or involved mucosa as input cells, a higher proportion of Vδ1+ cells from the involved mucosa (88% ± 6%) were fibroblast-adherent compared with those from the noninvolved mucosa (47% ± 5%).
We have compared the phenotypic and functional characteristics of γδ T cells within the normal and inflamed colonic mucosa of a large number of patients with IBD. Our results demonstrate that the chronic inflammatory immune response that occurs in the colon of IBD patients is associated with distinct changes in the number, distribution, composition, and function of mucosal γδ T cells. The size of the γδ T cell population in the diseased mucosa was significantly increased and could be attributed to an increase in Vδ1+ γδ T cells, among which, cells that expressed a TCRδ-chain encoded by Vδ1-Jδ1 gene segments predominated. These changes in mucosal γδ T cells were not restricted to individual patients or a particular group of patients with IBD but were present in the majority of UC, CD, and diverticulitis patient samples that we analyzed. The predominance of Vδ1-Jδ1 receptor-bearing cells was seen in patients with advanced forms of disease who required surgery and in biopsy samples obtained from patients with less severe forms of IBD, as well as in a patient that had clinical signs of disease for less than 2 weeks. Among T cells present in the inflamed mucosa of patients with CD, γδ T cells were a major source of the proinflammatory cytokine IFN-γ and were enriched for Vδ1+ γδ T cells that could selectively interact with colonic fibroblasts. Although it was not possible to determine if these changes in the properties of mucosal γδ cells were primary abnormalities of these disorders, they are consistent with, and suggest that they contribute to, the pathophysiology of IBD.
The results of our studies contrast with those obtained previously from the immunohistochemical analysis of γδ T cells in colonic tissue sections obtained from IBD patients in which a decrease (17) or no change (16) in the number of TCR-δ+ cells was reported. A likely explanation for the apparent discrepancy between these two studies and our own is the different techniques used. The immunohistochemical detection of T cells in intestinal tissue is difficult to perform and is a relatively insensitive means of detecting antigens such as the γδ TCR that are expressed at low levels (28,29). In addition, since the relative number of γδ T cells within the intestinal mucosa has been shown to vary greatly, not only between different sections of the same tissue but also between sections of the same site originating from different subjects (29), estimates of their relative number based upon this technique alone are unlikely to be accurate. Finally, since the degree of inflammatory activity may also significantly influence the distribution of γδ T cells in the diseased colon, variation among individual patients with IBD may also account for some of the contrasting results obtained from the different studies.
The increase in Vδ1-expressing γδ T cells in the LP of the inflamed colon of IBD patients may represent the expansion of resident and/or infiltrating cells from the blood. Since there are currently no phenotypic markers, including CD8, that can unambiguously discriminate between intestinal and blood-derived Vδ1 γδ T cells, it is not possible to definitively identify the origin of Vδ1+ cells within the LP of patients with IBD. However, several pieces of evidence suggest that a large number of these cells are derived from the peripheral blood. The phenotype of LP Vδ1+ (CD8−) cells in IBD patients is similar to that of those normally found in the blood (34) and resembles the phenotype of blood-derived γδ T cells that possess transendothelial migratory properties and accumulate within tissues at sites of inflammation (10). Interestingly, Chowers and colleagues (35) have recently shown that, on the basis of the presence of unique junctional sequences of TCRs expressed by intestinal γδ T cells, it is possible to distinguish between Vδ1+ cells normally resident in the blood and intestine.
Irrespective of the origin of the Vδ1+ γδ T cells that accumulate in the colonic mucosa of IBD patients, an important question that remains to be addressed is the nature of the stimulus that drives their expansion. The oligoclonal nature and limited structural diversity of the intestinal Vδ1 repertoire in the normal intestine (35) suggest that the array of ligands recognized by these receptors is limited. There are three possible sources of these ligands in the inflamed colon of IBD patients. The first is that they are enteric (bacterial and food) antigens that mucosal Vδ1+ γδ T cells could be exposed to as a result of them being able to cross the epithelial barrier of the intestinal wall, which is disrupted as part of the disease process in IBD. The recent observation that E. coli and P. aeruginosa, which normally inhabit the gastrointestinal tract, can stimulate Vδ1-bearing peripheral blood γδ T cells (36) is consistent with this possibility. Furthermore, the finding that gut T cells from patients with IBD proliferate vigorously to preparations of their own flora (37) suggests that a breakdown in the mechanisms that normally operate to maintain T cell hyporesponsiveness (“oral tolerance”) to enteric antigens is an important pathogenetic mechanism of IBD. A second possibility is that the antigens are endogenously derived and are expressed by stressed or damaged epithelial cells. The selective accumulation of Vδ1+ γδ T cells in the IE spaces of the intestine has long been interpreted as evidence for an immune surveillance role for these cells in which they recognize and react with a common antigen, such as heat shock proteins (HSP), expressed by epithelial cells as a result of infection, damage, or transformation (38). To date, however, recognition of self-antigens such as HSP by γδ T cells has only been demonstrated for cells that utilize a receptor encoded by the Vδ2 and Vγ9 gene segments (39,40). Extensive epithelial cell damage could, however, enable IEL such as Vδ1+ cells to “leak” into the LP, thus contributing to the increase in the number of Vδ1+ cells seen in the LP of IBD patients. The third possibility is that the activation of Vδ1-expressing γδ1 T cells is antigen-independent and does not involve the TCR/CD3 complex. The existence of alternative, antigen-independent pathways of mucosal T cell activation is suggested by the finding that LP T cells, unlike peripheral blood T cells, from normal and IBD patients exhibit vigorous proliferative responses via the CD2/CD28 signaling pathway (41–45). The presence in the inflamed mucosa of large numbers of activated T cells that produce cytokines (46–49) could also serve to activate, in a “bystander” manner, Vδ1+ cells (50), as well as to maintain a population of chronically activated T cells within the mucosa.
The γδ T cells that accumulate in the inflamed mucosa of patients with CD possess two functional characteristics that are relevant to the pathophysiology of this IBD. The first is that they are a major source of the proinflammatory cytokine IFN-γ, which has long been shown to mediate epithelial cell injury (51). Although the constitutive production of IFN-γ by colonic lymphocytes from IBD patients has been previously reported (44,49,51), our analysis identifies γδ T cells, including Vδ1+ cells, as a source of this cytokine in situ in the inflamed mucosa, and suggests a means by which they might contribute to the chronic inflammatory immune reaction and tissue injury in CD. The second characteristic is the selective adherence and interaction of mucosal γδ T cells, particularly Vδ1+ cells, with intestinal-derived fibroblasts. Although uncontrolled fibroblast proliferation, excessive collagen secretion, and fibrosis cause severe tissue damage and are important complications of CD (52,53), the processes regulating normal fibroblast activity are poorly understood. One way in which the activity of fibroblasts and other stromal cells could be regulated and coordinated is through direct interactions with other cells, such as Vδ1+ γδ T cells. The production by γδ T cells of tumor necrosis factor α (TNF-α), IL1-β, and transforming growth factor β (TGF-β) (54) that can promote fibroblast growth and collagen synthesis (55), and the observation that epithelial-associated γδ T cells in the skin can influence the activity of keratinocytes by the production of keratinocyte cell growth factor (8) are all consistent with this hypothesis. This immunoregulatory property of γδ T cells is further substantiated by the results from a variety of animal models of infectious disease in which the ability of γδ T cells to influence the activity of other cell populations and the outcome of inflammatory immune responses has been demonstrated (56–61). Since it has previously been shown that fibroblasts can prolong the survival of T cells in culture after the removal of IL-2 and antigen (62), fibroblast adherence may also provide a means of localizing or retaining activated, peripheral blood-derived, Vδ1+ cells in the inflamed colon. Although we do not yet have an explanation for the differences in γδ T cell subsets adhering to colonic fibroblasts, it could be related to quantitative and/or qualitative changes in the expression of adhesion/integrin molecules such as LFA-1 and VLA-5 by Vδ1+ cells (9,33,63) and their reciprocal receptors, ICAM-1 and fibronectin, respectively, by fibroblasts (9,33). The preferential association of Vδ1+ γδ T cells with fibroblasts provides a possible explanation for their accumulation in the lungs (64) and joints (65–69) in sarcoidosis and rheumatoid arthritis patients, respectively. Interestingly, inflammatory disorders of the lung, joints, skin, and eye are often present as extraintestinal manifestations of IBD (70), raising the possibility that Vδ1+ cells activated in the gut could migrate to other extraintestinal sites where, as a result of their ability to interact with fibroblasts or other connective tissue elements, they accumulate and can contribute to the inflammatory immune reaction and inflammation at these sites.
In summary, we have demonstrated that the chronic inflammatory reaction and tissue injury that occurs in the colon of patients with IBD is associated with distinct changes in the number, distribution, composition, and effector function of mucosal γδ T cells, particularly Vδ1-expressing cells. These findings suggest that these cells contribute to at least some of the immunological and pathological changes present in the inflamed mucosa of patients with IBD, and particularly CD.
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We are grateful to Ms. Linda Hurd for compiling pertinent data on IBD and normal patients in this study. We thank William G. Crowley for expert technical assistance. This work was supported by grants from the American Cancer Society (JFRA 399) and the National Institutes of Health (AI 31972) to S.R.C. L.D.M. was supported in part by a National Institutes of Health Training Grant (5-T32-CA-09140-20) and a Crohn’s and Colitis of America (CCFA) Fellowship.
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McVay, L.D., Li, B., Biancaniello, R. et al. Changes in Human Mucosal γδ T Cell Repertoire and Function Associated with the Disease Process in Inflammatory Bowel Disease. Mol Med 3, 183–203 (1997). https://doi.org/10.1007/BF03401672
- Diseased Colonic Mucosa
- Inflamed Colon
- Chronic Inflammatory Immune Response
- Colon Fibroblast Cell Line
- Cell Diagnostics