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Lymphoid Precursors in Intestinal Cryptopatches Express CCR6 and Undergo Dysregulated Development in the Absence of CCR6 ¹

Andreas Lügering^*, Torsten Kucharzik^*,, Dulce Soler, Dominic Picarella, James T. Hudson, III^* and Ifor R. Williams^2,*,

^* Department of Pathology and Laboratory Medicine and Department of Dermatology, Emory University School of Medicine, Atlanta, GA 30322; Department of Medicine B, University of Munster, Munster, Germany; and Millennium Pharmaceuticals, Cambridge, MA 02139

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Small intestinal cryptopatches (CP) are the major anatomic site for extrathymic differentiation by precursors destined to become intestinal intraepithelial T lymphocytes (IEL). We found that mice deficient in CCR6 exhibited a 2.7-fold increase in the number of TCR IEL, but little or no expansion of TCR IEL. Among the TCR IEL subsets, the CD4^- CD8⁺ and CD4⁺ CD8⁺ subsets were preferentially expanded in CCR6 null mice. Because some CD8⁺ IEL can arise through extrathymic differentiation in CP, we investigated CCR6 expression by T lymphocyte precursors undergoing extrathymic differentiation in intestinal CP. In sections of CP, 50–60% of c-kit⁺ precursors were CCR6⁺. CD11c⁺ cells concentrated at the periphery of CP did not express CCR6. A subset of c-kit⁺, Lin^- cells in lamina propria suspensions was CCR6⁺, but CCR6 was absent from c-kit⁺ precursors in bone marrow. CCR6 was absent from the vast majority of mature IEL. CCR6 is present on lymphocyte precursors in cryptopatches, expressed transiently during extrathymic IEL development, and is required for homeostatic regulation of intestinal IEL.

	Introduction

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The epithelium of the small intestine contains abundant intestinal intraepithelial T lymphocytes (IEL) ³ that provide important regulatory and effector activities in the epithelial microenvironment (1). IEL are a heterogeneous assemblage of T cells expressing either an TCR or a TCR. Many features distinguish IEL from conventional peripheral T cells, including basal expression of some activation markers, spontaneous cytotoxic effector activity, and limited TCR diversity of the TCR IEL (2, 3, 4). In addition, most and TCR IEL express CD8 homodimers rather than the CD8 heterodimers typically present on peripheral CD8 T cells (5). Multiple lines of evidence support the idea that the T cells that normally occupy the IEL compartment consist of a mixture of cells that undergo differentiation at extrathymic sites with cells that require the thymus for development (6). Intestinal cryptopatches (CP) are the major intestinal site where T cell precursors are localized within the intestine. Transfer studies with isolated CP precursors support the hypothesis that CP are the predominant site for extrathymic development of T cells destined to become IEL (7, 8). However, analysis of recombinase-activating gene-2 promoter activity in a transgenic mouse model has implicated Peyer’s patches and the mesenteric lymph node, rather than CP, as the location of extrathymic T cell differentiation in athymic mice (9).

Correctly regulated migration of maturing T cell precursors to and from anatomic sites of development, including the thymus and intestinal CP, is critical for normal T cell development. The response of T cell precursors to local chemokine gradients through their chemokine receptors is a major mechanism by which T cell precursors move into sites of differentiation, traffic within these sites, and later exit to recirculate (10, 11). Consistent with this hypothesis is evidence that some chemokine receptors are selectively expressed by mature medullary thymocytes (CCR7 and CCR4), while others (CXCR4) show increased expression on immature cortical thymocytes. Additional chemokine receptors differentially expressed on thymocyte subsets are CCR8 (12) and CCR9 (13, 14). Some of these chemokine receptors are probably involved in specific migratory events involved in thymocyte selection and differentiation. Considerably less is currently known about chemokine receptors that might be involved in the formation of CP and the subsequent migration of the progeny of CP cells into the intestinal epithelium.

The CCR6 chemokine receptor has a single chemokine ligand (macrophage inflammatory protein-3 (MIP-3)/CCL20) and can also bind human -defensin-1 and -2 (15, 16). Two independent laboratories have reported that mice lacking the CCR6 chemokine receptor exhibit a mucosal immune deficit characterized in part by expansion of small intestinal IEL, an associated increase in the number of lamina propria lymphocytes (LPL), and changes in the distribution of dendritic cells within Peyer’s patches (17, 18). The mechanism by which the absence of CCR6 leads to an increase in the number of IEL remains undefined. We have used a mouse model of CCR6 deficiency in which the CCR6 gene was disrupted by knockin of enhanced green fluorescent protein (EGFP) (19) to further characterize the IEL expansion occurring in CCR6-deficient mice. In the present study we report that the absence of CCR6 leads to selective expansion of TCR IEL subsets expressing a CD8 homodimer and an increased proportion of Thy-1⁺ cells among IEL. We also find that CCR6 is expressed by a subset of the c-kit⁺ lineage marker-negative (Lin^-) IEL precursor cells localized within CP, but is absent from the vast majority of mature IEL. These observations suggest that CCR6 influences the trafficking of intestinal IEL precursors and is crucial to the homeostatic regulation of IEL development.

	Materials and Methods

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Mice

The gene-targeting strategy used to generate CCR6 EGFP knockin mice was described in a previous report (19). The homozygous CCR6-deficient mice used for these studies were maintained on a mixed C57BL/6 and 129/Sv background. Wild-type control mice and heterozygous CCR6-deficient mice shared the same mixed background. Most wild-type and homozygous CCR6-deficient mice were bred from parents that were both either wild type or homozygous for the mutation. When genotyping of the individual offspring was required, a three-primer PCR method was used (19). Comparisons of CCR6-deficient and wild-type control mice were made using age-matched mice that were typically between 6 and 8 wk of age. All protocols for mouse experimentation were approved by the Emory University institutional animal care and use committee.

Preparation of IEL and LPL cell suspensions

Small intestinal IEL and LPL were prepared by a standard method (20) with minor modifications. Briefly, small intestinal tissue from which Peyer’s patches were removed was flushed with cold CMF solution (Ca²⁺ and Mg²⁺-free HBSS, 10 mM HEPES, 25 mM NaHCO₃, and 2% (v/v) FBS, pH 7.2) and cut into 0.5-cm pieces. The tissue was washed three times in 40 ml of cold CMF solution and transferred into 30 ml of CMF/FBS/EDTA solution (Ca²⁺ and Mg²⁺-free HBSS, 15 mM HEPES, 5 mM EDTA, 100 µg/ml gentamicin, and 10% (v/v) FBS, pH 7.2). After 30-min gentle shaking at 37°C, the sample was vortexed for 10 s, and cells from the supernatant were collected. This step was repeated twice, and all the supernatants representing the epithelial fraction were combined. To further purify the IEL, cells from the epithelial fraction were washed, resuspended in complete RPMI medium, filtered through nylon mesh, and resuspended in 44% Percoll solution (Amersham Pharmacia Biotech, Piscataway, NJ). The Percoll solution was underlain with 66% Percoll and centrifuged for 30 min at 600 x g. The IEL fraction was harvested from the 44/66% interface, washed, and counted. LPL suspensions were prepared from the EDTA-treated de-epithelialized intestinal tissue by further incubation with 100 U/ml collagenase and 5 U/ml DNase for 30 min at 37°C. The cells released into the supernatant were isolated from the interface of a 44/66% Percoll step gradient as described for the IEL fraction.

Abs and flow cytometry

The mAbs used to stain cell suspensions included Abs to CD3, CD4, CD8, CD8, CD11b, CD11c, CD19, CD45, TCR, TCR, c-kit, TER119, Thy-1.2, CCR6, and 5-bromo-2'-deoxyuridine (BrdU). The mAbs purchased from BD PharMingen were biotin-anti-CD3 (145-2C11), PE-anti-CD4 (RM4-5), PE-anti-CD8 (53-6.7), biotin-anti-CD11b (M1/70), PE-anti-CD11c (HL3), biotin-anti-CD19 (1D3), PE-anti-CD45 (30-F11), biotin-anti- TCR (H57-597), biotin-anti- TCR (GL3), PE-anti-c-kit (2B8), biotin-anti-Gr-1 (RB6-8C5), biotin-anti-Ter119, allophycocyanin anti-Thy-1.2 (53-2.1), and PE-conjugated anti-BrdU (3D4). Abs purchased from Caltag Laboratories (Burlingame, CA) were allophycocyanin-anti-CD4 (CT-CD4), allophycocyanin-anti-CD8 (5H10), FITC-anti-CD8 (CT-CD8b), and biotin-anti-c-kit (2B8). The lineage panel consisted of a combination of biotin-conjugated mAbs to CD3, CD11b, CD19, Gr-1, and TER-119 (all from BD PharMingen). Binding of biotinylated Abs was detected with streptavidin conjugated to PE (Immunotech, Westbrook, ME), PerCP (BD PharMingen), or allophycocyanin (Caltag). Two anti-CCR6 reagents were used: unconjugated 1C12 mAb (IgG2a rat anti-mouse mAb prepared at Millennium Pharmaceuticals, Cambridge, MA) (21) and PE-conjugated anti-CCR6 mAb (clone 140706) purchased from R&D Systems (Minneapolis, MN). Abs were diluted in PBS containing 0.2% BSA and 0.02% NaN₃ for 30 min on ice. Data on Ab-stained cell suspensions were acquired on a dual laser FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA), and the results were analyzed using CellQuest 3.3 (BD Biosciences). Cell populations were gated on the basis of forward and side scatter to allow selection of the viable lymphocytes.

BrdU labeling

Proliferating IEL in mice were labeled with BrdU by a series of five i.p. injections of 1.0 mg of BrdU in PBS at 6-h intervals. The mice were euthanized 1 h after the last injection, and small intestinal IEL were isolated as described above. After surface staining with Abs to TCR, TCR, CD8, or CD8 as described above, the IEL were fixed with 1% paraformaldehyde and 95% ethanol, followed by DNase treatment (22), before staining with PE-anti-BrdU.

Immunofluorescence staining

Mouse small intestine was prepared as a Swiss roll for longitudinal sections or as flat strips for horizontal sections (23) and embedded in OCT (Miles, Elkhart, IN). For detection of in situ EGFP fluorescence, mice were perfused with 3% paraformaldehyde, followed by 10% sucrose before embedding of the tissue (24). Five- to 10-µm frozen sections were cut with a cryostat and fixed in acetone. CCR6⁺ cells were detected using the 1C12 mAb, followed by biotinylated goat-anti-rat Ig (BD PharMingen) and the tyramide signal amplification (TSA) system with streptavidin-peroxidase and FITC-tyramide (PerkinElmer Life Sciences, Boston, MA). PE-anti-CD11c and allophycocyanin-Thy-1.2 mAbs from BD PharMingen were used in combination with TSA detection of CCR6. Biotin-anti-c-kit from Caltag in combination with TSA was used for detection of c-kit⁺ cells. Propidium iodide (Sigma-Aldrich, St. Louis, MO) and ToPro-3 (Molecular Probes, Eugene, OR) were used as nuclear counterstains in some experiments. Single-, double-, and triple-color fluorescence images were acquired using a LSM510 confocal microscope (Zeiss, New York, NY).

Radioligand binding assay

The binding of ¹²⁵I-labeled human MIP-3 (2200 Ci/mmol; NEN Life Science Products, Boston, MA) to spleen cells from wild-type and CCR6-deficient mice was measured using a method based on that described by Liao et al. (25). Briefly, the binding reaction was performed by incubating 0.1 nmol of the labeled chemokine with 1 x 10⁶ spleen cells at room temperature for 45 min in complete RPMI medium containing 10% FCS. The specific binding of ¹²⁵I-labeled MIP-3 was determined by subtracting the amount of binding observed after preincubation of the cells with a 500-fold molar excess of unlabeled recombinant human MIP-3 (R&D Systems). At the completion of the binding reaction, the cell suspension was spun through a cushion of 10% sucrose in PBS, and the pellet was cut off the end of the tube for gamma counting.

Statistics

Results are generally expressed as the mean ± SEM unless noted otherwise. The statistical significance of differences between groups was evaluated by Student’s t test. A value of p < 0.05 was considered statistically significant.

	Results

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CCR6⁺ cells express EGFP in heterozygous knockin mice

The degree of concordance between EGFP expression and surface expression of CCR6 in mice heterozygous for an EGFP knockin mutation of CCR6 was assessed by flow cytometric analysis of spleen cells. The anti-CCR6 mAb 140706 bound to a subset of spleen cells from wild-type and heterozygous mice, but did not bind spleen cells from homozygous knockout mice (Fig. 1A). The majority of the CCR6⁺ splenocytes were B220⁺ B cells (data not shown). Equivalent results were obtained with the 1C12 anti-CCR6 mAb (data not shown). In heterozygous knockin mice, cells that bound anti-CCR6 mAb were also positive for the expression of the knocked in EGFP gene, validating that EGFP expression in these mice identifies cells with cell surface CCR6. The peak of EGFP fluorescence was shifted to the right in homozygous mice compared with that in heterozygous mice, reflecting the presence of a second copy of the knocked in EGFP reporter (Fig. 1B). Radioligand binding experiments using iodinated human MIP-3 confirmed that the loss of surface expression of CCR6 by spleen cells from homozygous mice was associated with a complete loss of specific MIP-3 binding (Fig. 1C).

View larger version (45K): [in this window] [in a new window]   FIGURE 1. EGFP expression, CCR6 expression, and MIP-3 binding by spleen cells from CCR6 EGFP knockin mice. A, Spleen cells from wild-type, CCR6+/-, and CCR6-/- mice were stained with PE-conjugated anti-CCR6 mAb (140706 clone) and analyzed by flow cytometry. In CCR6+/- mice, EGFP expression is limited to CCR6-expressing cells. B, Overlay of histograms comparing EGFP fluorescence in heterozygous and homozygous CCR6 knockin mice to the autofluorescence of wild-type cells. C, Wild-type and CCR6-/- spleen cells were incubated with 125I-labeled MIP-3 in the presence and the absence of a 500-fold molar excess of cold MIP-3. The percent specific binding (specific binding as a percentage of total binding) in spleen cells from three mice of each genotype is shown along with the mean percent specific binding (indicated by a horizontal bar).

TCR IEL expressing CD8 homodimers are preferentially expanded in CCR6-deficient mice

Previous studies of CCR6-deficient mice reported an increased number of small intestinal IEL expressing an TCR (17, 18). IEL from CCR6-deficient mice homozygous for the EGFP knockin mutation were analyzed by staining with Abs to TCR, TCR, CD4, CD8, and CD8 to determine which of the major subsets of TCR IEL accounted for the increased number of TCR IEL. Fig. 2A shows that the absolute number of TCR IEL in the CCR6 knockout mice was 2.7-fold greater than that in wild-type controls (p < 0.001), compared with a 1.1-fold increase in TCR IEL (not significant; p > 0.10). Fig. 2C shows that the CD4^- CD8⁺ (3.2-fold increase; p < 0.001) and CD4⁺ CD8⁺ TCR IEL (6.3-fold increase; p < 0.001) subsets were expanded to a greater extent than cells expressing CD4 alone (2.1-fold increase; p = 0.002) or a CD8 heterodimer (2.1-fold increase; p = 0.007).

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Expansion of TCR subsets in small intestinal IEL from CCR6-/- mice. A, IEL from a series of 5- to 8-wk-old wild-type (n = 18) and CCR6-/- mice (n = 19) were isolated, counted, and stained with mAb to TCR and TCR. The mean absolute number (±SEM) of total IEL, TCR IEL, and TCR IEL are shown. The expansion ratios (number of cells in CCR6-/- mice divided by the number of cells in wild-type mice) calculated for each subset are found above the columns. **, p < 0.001; *, 0.001 < p < 0.05 (by Student’s t test). B, Representative dot plots of TCR vs TCR staining of IEL from wild-type and CCR6-/- mice. C, IEL from the same two groups of wild-type and CCR6-/- mice were also stained with mAb to TCR, CD4, CD8, and CD8. The absolute number of cells in the four major subsets of TCR cells was calculated, and the means were compared. Expansion ratios and asterisks indicating the p value are explained in A. D, Representative dot plots comparing CD8 vs CD8 staining in TCR-gated IEL from wild-type and CCR6-/- mice.

Thy-1 is normally present on subsets of TCR and TCR IEL and is associated with cells that display an activated phenotype (26). Expansion of IEL in CCR6-deficient mice was associated with an increase in the ratio of Thy-1⁺ cells to Thy-1^- cells in both TCR and TCR IEL compared with wild-type control mice (Fig. 3A). The increased percentage of Thy-1 expression correlated with increased lytic activity of CCR6 null IEL in an anti-CD3 redirected lysis assay (data not shown). To determine whether the absence of CCR6 led to a change in the proliferative rate of IEL, CCR6-deficient and control mice were labeled with BrdU for 25 h before isolation of IEL. The average rate of BrdU incorporation by IEL was significantly greater (p = 0.022) in IEL from CCR6 null mice than in wild-type controls (Fig. 3B). This increase was not due solely to the higher frequency of TCR IEL, since the proliferative rate was also increased when the analysis of BrdU incorporation was restricted to TCR⁺ cells (data not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Increased frequency of Thy-1 expression and proliferation by IEL from CCR6-/- mice. A, IEL from 6-wk-old wild-type and CCR6-/- mice were stained with mAb to TCR and Thy-1, and the results are presented as a dot plot. The fractions of both TCR and TCR IEL expressing Thy-1 are increased in CCR6 null mice. The results are representative of four individual experiments. B, IEL were isolated from wild-type and CCR6-/- mice (n = 5 in both groups) labeled with BrdU for 25 h. The percentage of the cells in the IEL fraction from each mouse that stained with PE-anti-BrdU Ab was assessed by flow cytometry. The results are presented as the mean ± SEM. The p value for difference between the groups was 0.022.

CCR6 is present on subsets of peripheral CD4 and CD8 T cells, with preferential expression on CD4 T cells (19, 27). Using an mAb to CCR6 and the EGFP reporter in homozygous knockin mice, we assessed CCR6 expression on both IEL and LPL by flow cytometry (Fig. 4). Only 2.7% of CD3⁺ IEL from wild-type mice bound anti-CCR6 mAb; similarly, just 2.5% of CD3⁺ IEL from the homozygous EGFP knockin mice expressed EGFP. In contrast, 14.2% of CD3⁺ LPL bound anti-CCR6 mAb. CCR6⁺ cells were also detected among the CD3^- LPL. The absolute number of LPL recovered from CCR6 null mice was increased (data not shown), in agreement with a previous study (17).

View larger version (43K): [in this window] [in a new window]   FIGURE 4. CCR6 is found on a subset of small intestinal LPL, but on very few IEL. LPL and IEL from wild-type and CCR6-/- mice were stained with PE-anti-CCR6 and biotin-anti-CD3, followed by streptavidin-allophycocyanin. Separate dot plots of CCR6 vs CD3 and FL1 (EGFP) vs CD3 are shown.

Some IEL are the progeny of lymphocytes that undergo extrathymic differentiation within aggregates of c-kit⁺ Lin^- cells localized to intestinal CP. Histological evaluation of small intestinal tissue from CCR6-deficient mice demonstrated that CCR6^-/- CP were normal in size (Fig. 5, A and B). In both wild-type and CCR6^-/- CP, c-kit⁺ lymphocyte precursors were preferentially localized to the central portion of the CP and surrounded by a mantle of CD11c⁺ cells (Fig. 5, C and D). To determine whether any cells within CP expressed CCR6, horizontal sections through the crypt area of perfusion-fixed homozygous CCR6 EGFP knockin mice and controls were evaluated for EGFP expression. Only the CP from the knockin mice contained fluorescent cells making EGFP (Fig. 5, E and F). To confirm this finding, similar horizontal sections from wild-type small intestine were stained with anti-CCR6 mAb. Most of the CCR6⁺ cells observed were within round lymphoid aggregates located between crypts with the distribution and morphological features of CP (Fig. 5G).

View larger version (77K): [in this window] [in a new window]   FIGURE 5. A subset of T cell precursors in small intestinal CP express CCR6. Representative CP from wild-type (A) and CCR6 null (B) mice on paraffin-embedded longitudinal sections of small intestine were stained with H&E. CP in longitudinal frozen sections of small intestine from wild-type (C) and CCR6 null (D) mice were stained for CD11c (red) and c-kit (green) using biotinylated anti-c-kit and TSA with FITC-tyramide, followed by PE-anti-CD11c. Immunoreactivity for c-kit is present on T cell precursors in the CP and on interstitial cells of Cajal in the muscularis propria. Horizontal sections of perfusion-fixed, wild-type (E) and CCR6 null (F) small intestine were cut and stained with ToPro-3 (gray) as a nuclear counterstain. EGFP expression (green) is found only on cells in the CCR6 null cryptopatch. A horizontal section of wild-type small intestine (G) was stained for CCR6 (green) using the 1C12 mAb, followed by TSA with FITC-tyramide. Propidium iodide (blue) was added as a nuclear counterstain. The field illustrated contains two CP consisting of clusters of CCR6+ cells. The size bar in the fluorescence micrographs is 50 µm long.

View larger version (90K): [in this window] [in a new window]   FIGURE 6. The CCR6+ cells in small intestinal CP are T cell precursors. Frozen sections of wild-type (A, C, and E) and CCR6-/- small intestine (B, D, and F) were triple stained with anti-CCR6 mAb 1C12 (green) using TSA with FITC-tyramide, followed by allophycocyanin-anti-Thy-1 and PE-anti-CD11c. A series of three dual-color images of a single representative CP from wild-type and CCR6-/- mice are shown. A and B, Staining for CCR6 (green) and CD11c (red). C and D, Staining for CCR6 (green) and Thy-1 (blue) with double staining cells in light blue. E and F, Staining for Thy-1 (blue) and CD11c (red). The size bar in the fluorescence micrographs is 50 µm long.

Single-cell suspensions of small intestinal lamina propria include a population of c-kit⁺ Lin^- cells with the same immunophenotype as T cell precursors in CP (28). In the lamina propria of heterozygous CCR6 knockin mice, 20% of cells with the c-kit⁺ Lin^- CP phenotype cells were EGFP positive (Fig. 7A). To determine whether acquisition of CCR6 expression by c-kit⁺ Lin^- cells was a general feature of all c-kit⁺ Lin^- hemopoietic precursor cells, c-kit⁺ Lin^- bone marrow cells from heterozygous knockin mice were compared with c-kit⁺ Lin^- lamina propria cells. The c-kit⁺ Lin^- precursors in the bone marrow lack a detectable fraction of CCR6⁺ cells (Fig. 7B), indicating that CCR6 is selectively acquired in specific tissue microenvironments such as the intestinal CP.

View larger version (53K): [in this window] [in a new window]   FIGURE 7. c-kit+, Lin- cells from small intestinal lamina propria express the CCR6 gene. A, Small intestinal lamina propria cells from wild-type and CCR6+/- mice were stained with PE-anti-c-kit and biotinylated lineage markers (anti-CD3, anti-CD19, anti-CD11b, anti-Gr-1, and anti-TER-119), followed by streptavidin-allophycocyanin. Overlays of histograms of FL1 expression for the marked c-kit+ Lin- subset is shown. B, Bone marrow cells from wild-type and CCR6+/- mice were stained with PE-c-kit and biotinylated lineage markers, followed by streptavidin-PerCP. Overlays of histograms of FL1 expression for the indicated c-kit+ Lin- subset show no expression of the knocked in EGFP gene in the heterozygous knockin mice.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The IEL subsets that are selectively increased in CCR6-deficient mice (i.e., CD8⁺ and Thy-1⁺ TCR IEL) resemble the IEL subsets that are preferentially expanded in germfree mice following exposure to the normal commensal flora found in a conventional housing environment (31). Previous studies have shown that germfree mice harbor a much smaller number of intestinal IEL, with absolute IEL counts of only 15% the level associated with conventionally housed specific pathogen-free mice (32). Most of this decrease in the IEL population is the result of a deficit in the number of TCR IEL. Microbial colonization results in the expansion of TCR IEL, but not TCR IEL, a process that is not prevented by thymectomy (31). Germfree mice also contain very few IEL that express Thy-1, and bacterial colonization of the gut of germfree mice results in the appearance of a normal fraction of IEL expressing Thy-1 (33). Taken together, these observations suggest that CCR6 is necessary for normal regulation of the expansion of extrathymic TCR IEL that occurs in response to bacterial colonization of the intestine. Comparison of CCR6-deficient mice and controls raised in a germfree environment will be necessary to further test the hypothesis that the observed expansion of IEL in conventionally housed, CCR6-deficient mice is dependent on the presence of resident bacterial flora in the intestine.

Although we observed that CCR6 deficiency was associated with selective expansion of TCR IEL and the CD8⁺ subsets of TCR IEL in particular, significant increases in TCR IEL subsets lacking CD8 were also observed. In addition, a higher percentage of both TCR IEL and TCR IEL were Thy-1⁺ in CCR6-deficient mice. These changes suggest that CCR6 deficiency may ultimately lead to disruption of the homeostatic regulation of proliferation and/or activation of all IEL subsets to some extent. A second potential mechanism, besides disordered trafficking of CCR6⁺ T cell precursors in CP, that may contribute to perturbed homeostasis of IEL in CCR6-deficient mice is impaired regulatory function of T cells within the intestinal microenvironment. Subsets of both CD4⁺ and CD4⁺CD25⁺ LPL express CCR6 (A. Lügering, unpublished observations), and defective regulation by CD4⁺ T cells of CD8⁺ T cells mediating contact hypersensitivity reactions was reported in CCR6-deficient mice (18).

Lymphocyte development in general is characterized by a series of discrete differentiation steps that occur within a series of distinct microenvironments within primary and secondary lymphoid tissues. For B or T lymphocytes to efficiently complete a normal differentiation pathway, their precursors take advantage of a chemokine-based guidance system that permits targeted migration from one microenvironment to the next (34). The discovery of multiple novel chemokines and chemokine receptors in recent years has led to the identification of chemokine ligand-receptor combinations that play pivotal roles in facilitating specific lymphocyte differentiation steps. For example, CXCR4 is critical for retention of B lymphocyte precursors in the bone marrow (35), while CCR7 is crucial for the efficient trafficking of naive T cells to the T cell areas in peripheral lymph nodes (36). Chemokines also appear to play a critical role in some of the earliest recognized steps that lead to the genesis of lymphoid organs such as peripheral lymph nodes and Peyer’s patches (37). The recruitment of immature T cell precursors originating in the bone marrow or thymus to intestinal CP capable of promoting differentiation of these precursors into mature IEL is also likely to involve a series of chemokine-mediated steps. However, very few chemokines and receptors have been implicated to date in the development of CP and IEL.

Expression profiling studies comparing gene expression by TCR IEL and TCR IEL has identified a small group of chemokine receptors that are expressed by both IEL subsets, including CCR9, CCR5, and CXCR3 (38). The patterns of expression of CCR9 and its single chemokine ligand TECK/CCL25 suggest that this chemokine-receptor pair participates in the targeting of lymphocytes to the intestinal epithelium. CCL25 is constitutively expressed by intestinal epithelial cells, but not by epithelial cells in most other organs. Almost all lymphocytes located within the small intestine express CCR9 (39). In vivo neutralization of CCL25 results in decreased localization of recently activated T cells to the small intestinal epithelium (40). However, two independent groups have reported that CCR9-deficient mice exhibit only a slight reduction in the number of TCR IEL, with no change in the absolute number of TCR IEL (41, 42). These modest changes in IEL subsets in CCR9 knockout mice suggest that there is some degree of redundancy in chemokine regulation of intestinal IEL homing.

Unlike CCR9, which is apparently expressed by both T cell precursors in CP and mature intestinal IEL (43), CCR6 is expressed by >50% of IEL precursors in CP, but only a few mature IEL. Comparison of c-kit⁺ Lin^- lymphocyte precursors in bone marrow and in the intestinal lamina propria demonstrated that CCR6 is restricted to the T cell precursors found in intestinal CP. The absence of CCR6 expression by double negative T cell precursors in the thymus (A. Lügering, unpublished observations) is further evidence that expression of CCR6 by immature T cell precursors is not a general phenomenon associated with all forms of T cell differentiation, but, instead, a specialized adaptation important for regulated development of the specialized T cell populations that inhabit the intestinal epithelium. While 50–60% of the c-kit⁺ Thy-1⁺ cells in CP appear to coexpress CCR6 based on dual immunofluorescence staining for c-kit and CCR6, a smaller percentage (15–20% on the average) of the c-kit⁺ Lin^- cells in the lamina propria fraction analyzed by flow cytometry expressed detectable levels of the knocked in EGFP reporter in heterozygous mice. The most likely explanation for this difference is the inclusion in the lamina propria cell preparation of a substantial number of c-kit⁺ Lin^- cells found outside the organized CP and expression of CCR6 by these cells at a much lower frequency compared with the c-kit⁺ Lin^- cells within the CP.

Our studies of CCR6 expression by T cell precursors in the intestine and the effects of CCR6 deficiency on IEL development suggest a hypothetical model shown in Fig. 8. This model postulates that loss of normal CCR6 expression by immature CP T cell precursors and/or T cells within the lamina propria impairs regulatory mechanisms that normally limit the extrathymic development of TCR IEL stimulated by bacterial products arising from the commensal intestinal flora.

View larger version (54K): [in this window] [in a new window]   FIGURE 8. Schematic diagram illustrating the selective expression of CCR6 (cells with shaded cytoplasm) on a subset of T cell precursors found in CP. CCR6 is absent from T cell precursors in the bone marrow and thymus and is also absent from the vast majority of mature IEL in the epithelium. CCR6 expression by T cell precursors in CP is hypothesized to be important in regulating their trafficking and the extent to which these T cell precursors expand and differentiate to produce mature IEL.

	Acknowledgments

 
We thank P. Jensen, G. Kersh, and R. Waikel for critical review of the manuscript, H. Rees for assistance with perfusion of mice, and J. Zimring for assistance with the redirected lysis assay.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health (AR44268 to I.R.W.), the Emory University Research Committee (to I.R.W.), and Deutsche Forschungsgemeinschaft (Lu816/1-1 to A.L. and Ku1328-1 to T.K.).

² Address correspondence and reprint requests to Dr. Ifor R. Williams, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Whitehead Building 105D, 615 Michael Street, Atlanta, GA 30322. E-mail address: irwilli{at}emory.edu

³ Abbreviations used in this paper: IEL, intraepithelial T lymphocytes; BrdU, 5-bromo-2'-deoxyuridine; CP, cryptopatch; EGFP, enhanced green fluorescent protein; Lin^-, lineage marker negative; LPL, lamina propria lymphocytes; MIP, macrophage inflammatory protein; TSA, tyramide signal amplification.

Received for publication February 4, 2003. Accepted for publication June 19, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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