Activation of T-cell receptor-γδ⁺ cells in the intestinal epithelia of KN6 transgenic mice

Abstract

We analysed the properties of intraepithelial lymphocytes of small intestine (SI-IEL) in KN6-transgenic (Tg) mice expressing cDNA of T-cell receptor (TCR)-γδ specific for the T22^b molecule. While most splenic Tg TCR-γδ⁺ cells from KN6-Tg mice with H-2^d/d background (Tg^d/d mice) were Thy-1⁺ CD8α^– CD44^dull+ CD45RB⁺ CD69^–, Tg TCR-γδ⁺ cells in SI-IEL (Tg γδ-IEL) were heterogeneous in the expression of Thy-1, CD8α and CD44 molecules and predominantly CD45RB⁺ CD69⁺. Tg γδ-IEL exhibited a much reduced proliferative response to the antigen (irradiated H-2^b splenocytes) than splenic Tg TCR-γδ⁺ cells; the CD44⁺ subset, but not the CD44^– subset, in Tg γδ-IEL responded to the antigen. Furthermore, Tg γδ-IEL, but not splenic Tg TCR-γδ⁺ cells, displayed cytolytic activity whether they were prepared from conventional or germ-free KN6-Tg mice. Comparative analysis of young and aged KN6-Tg mice revealed that the proportion of CD44⁺ cells in Tg γδ-IEL increased but the proliferative response of Tg γδ-IEL to the antigen attenuated in association with ageing. Moreover, although Tg γδ-IEL from Tg^b/d mice contained a higher proportion of CD44⁺ cells than Tg^d/d mice, they did not respond to the antigen. These results demonstrate that Tg TCR-γδ⁺ cells lose the ability to recognize the antigen following activation in the intestinal epithelia.

Introduction

The T-cell antigen receptor (TCR) is composed of either αβ- or γδ-heterodimer. TCR-αβ⁺ and TCR-γδ⁺ cells are distinct in terms of their developmental pathway and anatomical location. TCR-αβ⁺ cells mainly differentiate in the thymus and migrate into the peripheral lymphoid tissues such as spleen, lymph nodes, and Peyer's patches. By contrast, TCR-γδ⁺ cells preferentially colonize the epithelial layer of skin, uterus, and intestine.¹ The fact that TCR-αβ⁺ and TCR-γδ⁺ cells distribute in different tissues suggests that they may play distinct physiological functions.

One intriguing issue about TCR-γδ⁺ cells is that the usage of Vγ chains is tissue dependent. In mouse, dendritic epidermal TCR-γδ⁺ cells, uterus TCR-γδ⁺ cells, and intestinal intraepithelial lymphocytes (TCR-γδ⁺ cells; γδ-IEL) mainly utilize Vγ5 chain, Vγ6 chain, and Vγ1 or Vγ7 chains, respectively.^2–4 Another important point is that γδ-IEL are predominantly CD62L^– CD103⁺ CD44⁺ CD45RB^dull+, but TCR-γδ⁺ cells in peripheral lymph nodes contain subsets exhibiting the surface phenotype of CD62L⁺ CD103^– CD44^– CD45RB⁺.⁵ Moreover, γδ-IEL are known to display cytolytic activity when incubated with Fc receptor-positive target cells in the presence of anti-TCR-δ monoclonal antibodies (mAb).⁶ These results demonstrate that TCR-γδ⁺ cells in the intestinal epithelia are activated. Induction of cytolytic activity of γδ-IEL is independent of intestinal micro-organisms but abolished under antigen-minimized conditions,^7,8 suggesting that the existence of food-derived materials in the gut lumen is critical for induction of cytolytic activity of γδ-IEL. However, it is largely unknown how the ability of TCR-γδ⁺ cells to recognize the antigen is modulated in the intestinal epithelia.

KN6-transgenic (Tg) mice have been generated by introducing functionally rearranged cDNA of TCR-γδ, expressed in thymocytes of C57BL/6 mice, which is specific for the non-classical major histocompatibility complex (MHC) class Ib molecule, T22^b.⁹ As the intestines of KN6-Tg mice are colonized by Tg TCR-γδ⁺ cells,¹⁰ analysis of KN6-Tg mice enables us to examine the influence of intestinal environment on the ability of TCR-γδ⁺ cells to recognize the antigen. Our results show that Tg TCR-γδ⁺ cells activated in the intestinal epithelia lose the ability to recognize the antigen.

Materials and methods

Mice

BALB/c and C57BL/6 mice were purchased from Shizuoka Laboratory Animal Centre (Hamamatsu, Japan). KN6-Tg mice of H-2^d/d background (Tg^d/d mice) were kindly provided by Dr S. Tonegawa (Massachusetts Institute of Technology, MA). Tg^d/d mice were maintained by crossing with BALB/c mice, and Tg^b/d mice were generated by intercrossing Tg^d/d mice and C57BL/6 mice. Germ-free (GF) Tg^d/d mice were prepared by caesarean section. Identification of the KN6-Tg TCR-γ chain was analysed by polymerase chain reaction (PCR).¹¹ Mice were killed and subjected to the analysis according to the legislation of animal experiments established by Yakult Central Institute.

Preparation of cells

Spleens were teased over gauze using Hank's balanced salt solution (HBSS). To obtain splenic T cells, a single-cell suspension of splenocytes was applied to a nylon column (0·6 g/mouse) and nylon-non-adherent cells were recovered.

Intraepithelial lymphocytes of small intestine (SI-IEL) were prepared as follows. After intestinal contents were flushed away with 10 mm HEPES (pH 7·2)/HBSS, the intestines were opened longitudinally and cut into pieces of 1–2 cm length. After the fragments were washed twice with 0·45 mm dithiothreitol/10 mm HEPES (pH 7·2)/HBSS and immersed in prewarmed 5% fetal calf serum (FCS)/25 mm HEPES (pH 7·2)/RPMI-1640, they were shaken at 37° for 45 min. The single cell suspension was passed through a glass-wool column (0·6 g/mouse) to remove aggregates, and then applied on 30% Percoll, followed by centrifugation at 650 g for 20 min. Cells in the pellet were suspended in 44% Percoll, layered on 70% Percoll, and centrifuged at 650 g for 20 min. Cells banding at the interface between 44% and 70% Percoll solutions were recovered as SI-IEL.

Purification of TCR-γδ⁺ cells and separation into CD44⁺ and CD44^– subsets

Tg TCR-γδ⁺ cells were purified from splenic T cells and SI-IEL by magnetic beads. Cells were incubated with a mixture of biotinylated anti-mouse immunoglobulins (Cappel Organon Teknika Co., Durham, NC) and biotinylated anti-TCR-β mAb (H57-597, PharMingen, San Diego, CA) on ice for 20 min. After extensive washing, they were incubated with streptavidin-conjugated microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) at 4° for 15 min. The cells were applied to a separation column (MS⁺ Miltenyi Biotec GmbH), and non-bound cells were recovered. The proportion of TCR-γδ⁺ cells was more than 97·5% in this preparation.

To prepare CD44⁺ and CD44^– cells from purified TCR-γδ⁺ cells, they were incubated with biotinylated anti-CD44 mAb (IM7, PharMingen) on ice for 20 min, followed by incubation with streptavidin-conjugated microbeads at 4° for 15 min. After the cells were applied to a separation column, bound and non-bound cells were recovered as CD44⁺ and CD44^– cells, respectively.

Immunofluorescence analysis

Cells were incubated with 20 µl of each antibody solution (10–20 µg/ml) on ice for 20 min and analyzed by an EPICS Elite flow cytometer (Coulter, Miami, FL). Antibodies used were the following: biotinylated anti-TCR-β, fluoroscein isothiocyanate (FITC)-conjugated anti-TCR-δ (GL3; PharMingen), FITC-conjugated or biotinylated anti-Vγ4 (UC3-10A6; PharMingen), biotinylated anti-Thy-1.2 (30-H12; PharMingen), biotinylated anti-CD8α (53-6.7; PharMingen), biotinylated anti-CD62L (MEL-14; Southern Biotechnology Associates, Inc., Birmingham, AL), anti-CD103 (2E7; ¹² kindly provided by Dr L. Lefrançois), biotinylated anti-CD44, biotinylated anti-CD45RB (16A; PharMingen), biotinylated anti-CD69 (H1.2F3; PharMingen). Biotinylated antibodies were detected by phycoerythrin-conjugated streptavidin (Caltag Laboratories, Burlingame, CA), and anti-CD103 antibody was detected by biotinylated anti-hamster immunoglobulin G (IgG; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and then by phycoerythrin-conjugated streptavidin.

Measurement of proliferative response

Responder cells were suspended in 10% FCS/5 × 10⁻⁵ m 2-mercaptoethanol (2-ME)/RPMI-1640. For measurement of antigen-triggered response, cells were cultured for 3 days in the presence of 3000 rad-irradiated BALB/c or C57BL/6 splenocytes (5 × 10⁵ cells/well) without or with human recombinant interleukin-2 (rIL-2) at 25 U/ml (Shionogi Co., Osaka, Japan) using round-bottomed 96-well microtitre plates. To determine anti-Vγ4 mAb-triggered response, cells were cultured for 3 days in flat-bottomed 96-well microtitre plates precoated with hamster IgG or anti-Vγ4 mAb. After cells were pulsed with 0·5 µCi of [³H]thymidine for the last 8 hr, radioactivity incorporated into cells was measured with a β-emission scintillation counter.

Assay of redirected cytolytic activity

Splenic T cells or SI-IEL were mixed with ⁵¹Cr-labelled P815 cells at various effector-to-target ratios and incubated for 6 hr in the absence or presence of anti-TCR-δ mAb. Radioactivity released into the supernatant was measured with a γ-counter, and percentage of specific lysis was calculated as 100 × (experimental release − spontaneous release)/(detergent-induced re-lease − spontaneous release).

Measurement of interferon-γ (IFN-γ) production

Purified splenic TCR-γδ⁺ cells or γδ-IEL were cultured at 2 × 10⁵ cells/well using flat-bottomed 96-well microtitre plates precoated with hamster IgG or anti-Vγ4 mAb for 3 days. Culture supernatants were collected at intervals and the concentration of IFN-γ was measured with a Cytoscreen Immunoassay Kit (BioSource International, Carmarillo, CA).

Statistical analysis

Significance of difference between groups was evaluated by unpaired Student's t-test.

Results

Development of Tg TCR-γδ⁺ cells in KN6-Tg mice

There were very few TCR-γδ⁺ cells in the spleen (0·6± 0.3%) and covered 28·8 ± 10·1% of SI-IEL in non-Tg mice (BALB/c mice). By contrast, the relative proportion of TCR-γδ⁺ cells was 43·0 ± 13·3% of splenocytes and 95·2 ± 2·5% of SI-IEL in KN6-Tg mice of BALB/c background. TCR-γδ⁺ cells in the spleen and intestinal epithelia of KN6-Tg mice were co-stained by anti-Vγ4 mAb and anticlonotypic mAb (5C10¹³), but TCR-γδ⁺ cells in the intestinal epithelia of non-Tg mice contained Vγ4⁺ cells of at most 5% and were not co-stained by 5C10 mAb.

Most splenic Tg TCR-γδ⁺ cells were Thy-1⁺ CD8α^– CD62L⁺ and only a subpopulation of them were CD103⁺. By contrast, Tg TCR-γδ⁺ cells in the intestinal epithelia (Tg γδ-IEL) were heterogeneous in the expression of Thy-1 and CD8α molecules, but almost all of Tg γδ-IEL were CD103⁺ and very few of them expressed CD62L. While splenic Tg TCRγδ⁺ cells were predominantly CD44^dull+ CD45RB⁺ CD69^–, Tg γδ-IEL were composed of CD44^– and CD44⁺ populations, and almost all of them expressed CD45RB and CD69 molecules (Fig. 1). These results demonstrate that splenic Tg TCR-γδ⁺ cells and Tg γδ-IEL are phenotypically distinct populations despite expressing the same TCR-γδ.

Figure 1.

Immunofluorescence analysis of splenic Tg TCR-γδ⁺ cells and Tg γδ-IEL in KN6-Tg mice of BALB/c background. Cells were stained with mAbs specific for Thy-1, CD8α, CD62L, CD103, CD44, CD45RB or CD69 plus FITC-conjugated anti-Vγ4 mAb. After gating Vγ4⁺ cells, expression of each molecule was examined.

Functional characterization of Tg TCR-γδ⁺ cells in spleen and intestinal epithelia of KN6-Tg mice

To compare the immunological competencies of splenic Tg TCR-γδ⁺ cells and Tg γδ-IEL, they were cultured in the presence of irradiated C57BL/6 (H-2^b) splenocytes or immobilized anti-Vγ4 mAb. Splenic Tg TCRγδ⁺ cells responded to H-2^b splenocytes more vigorously than Tg γδ-IEL, but the response to immobilized anti-Vγ4 mAb was not reduced in Tg γδ-IEL compared with splenic Tg TCR-γδ⁺ cells (Fig. 2). These results, together with analysis of surface phenotype, suggest that the intestinal epithelia activate Tg TCR-γδ⁺ cells and down-regulate their ability to recognize the antigen (H-2^b splenocytes), leaving their TCR-triggered proliferative capacity still active.

Figure 2.

Proliferative response of splenic Tg TCR-γδ⁺ cells and Tg γδ-IEL in KN6-Tg mice of BALB/c background. (a) Proliferation in response to irradiated H-2^d (○, ▵) or H-2^b (•, ▴) splenocytes. (b) Proliferation stimulated by immobilized hamster IgG (○, ▵) or anti-Vγ4 mAb (•, ▴). Circles represent the response of splenic Tg TCR-γδ⁺ cells, and triangles indicate the response of Tg γδ-IEL.

It is known that memory T cells express the CD44 molecule.^14,15 To explore whether Tg γδ-IEL contain activated memory T cells, we separated them into CD44⁺ and CD44^– subsets and examined their proliferative capacities. As a result, the CD44⁺ subset but not the CD44^– subset from Tg γδ-IEL responded to H-2^b splenocytes (Fig. 3). These results support that CD44⁺ Tg TCR-γδ⁺ cells in the intestinal epithelia are activated memory T cells. It should be noted that CD44^– as well as CD44⁺ Tg γδ-IEL proliferated in response to immobilized anti-Vγ4 mAb, showing that CD44^– Tg γδ-IEL maintain TCR-triggered proliferative capacity (Fig. 3).

Figure 3.

Proliferative response of CD44⁺ and CD44^– subsets in Tg γδ-IEL. CD44⁺ and CD44^– cells were prepared from Tg γδ-IEL in KN6-Tg mice of BALB/c background. (a) Staining profile by anti-CD44 mAb of total cells, CD44⁺ and CD44^– subsets of Tg γδ-IEL. (b) Proliferation of total cells (•), CD44⁺ subset (▪) and CD44^– subset (▴) of Tg γδ-IEL in response to irradiated H-2^b splenocytes. (c) Proliferation of total cells, CD44⁺ and CD44^– subsets of Tg γδ-IEL in response to immobilized anti-Vγ4 mAb.

In order to further investigate functional difference of splenic and intestinal intraepithelial Tg TCR-γδ⁺ cells, they were subjected to assay of redirected cytolytic activity in the presence of anti-TCR-δ mAb. SI-IEL from KN6-Tg mice consistently exhibited cytolytic activity, but splenic T cells from KN6-Tg mice did not show cytolytic activity; nevertheless they contained Tg TCR-γδ⁺ cells by 74%. However, splenic T cells from KN6-Tg mice became cytolytic after they were cultured in the presence of immobilized anti-TCR-δ mAb, indicating that splenic Tg TCR-γδ⁺ cells contain precursors of cytolytic T cells (Fig. 4). Moreover, cytolytic activity exhibited by SI-IEL from KN6-Tg mice raised under GF conditions was almost equivalent to that from KN6-Tg mice raised under conventional (CV) condition (Fig. 4). These results suggest that cytolytic activation of Tg γδ-IEL takes place independently of microbial colonization in the intestine, as previously reported.⁷

Figure 4.

Cytolytic activity of splenic Tg TCR-γδ⁺ cells and Tg γδ-IEL in KN6-Tg mice of BALB/c background. Splenic T cells (○, •) or SI-IEL (▵, ▴) were incubated with ⁵¹Cr-labelled P815 cells for 6 hr in the absence (open symbols) or the presence of anti-TCR-δ mAb (closed symbols). Effectors were cells freshly isolated from KN6-Tg mouse raised under CV condition (left) or GF condition (centre), and splenic T cells cultured for 5 days in the presence of immobilized anti-TCR-δ mAb (right).

Finally, cytokine production by splenic Tg TCR-γδ⁺ cells and Tg γδ-IEL was analysed. As both splenic Tg TCR-γδ⁺ cells and Tg γδ-IEL expressed mRNA of IFN-γ but not of IL-4 (data not shown), their capacities to produce IFN-γ were compared. As shown in Fig. 5, splenic Tg TCR-γδ⁺ cells and Tg γδ-IEL similarly generated IFN-γ in response to immobilized anti-Vγ4 mAb. The above results demonstrate that Tg TCR-γδ⁺ cells are activated in the intestinal epithelia, with resulting loss of the ability to recognize the antigen and acquisition of cytolytic activity leaving the capacity to produce IFN-γ maintained.

Figure 5.

IFNγ production by splenic Tg TCR-γδ⁺ cells and Tg γδ-IEL in KN6-Tg mice of BALB/c background. Tg TCR-γδ⁺ cells purified from spleen (▵, ▴) or intestinal epithelia (○, •) were cultured in the presence of immobilized hamster IgG (open symbols) or anti-TCR-δ mAb (closed symbols), and concentration of IFN-γ in the supernatants was measured.

Ageing-associated change of Tg γδ-IEL in KN6-Tg mice

The above results suggest that the intestinal environment activates Tg TCR-γδ⁺ cells and deprives them of the ability to recognize the antigen. In order to further define the effect of intestinal environment on the accumulation of anergic Tg TCR-γδ⁺ cells, Tg γδ-IEL from young (6∼17-week-old) and aged (61∼92-week-old) KN6-Tg mice were examined. The proportion of Tg γδ-IEL in young and aged KN6-Tg mice was similar (young mice 99·7 ± 0·3% versus aged mice 99·3 ± 0·6%), while ageing induced the decrease of the absolute number of Tg γδ-IEL (young mice (28·0 ± 7·1) × 10⁶ cells versus aged mice (14·6 ± 6·6) × 10⁶ cells, P ≤ 0.05). Analysis of the subset constitution of Tg γδ-IEL from young and aged KN6-Tg mice revealed that the proportion of the CD44⁺ and CD45RB⁺ subsets increased in aged mice but there was no difference in the proportion of Thy-1⁺, CD8α⁺ or CD69⁺ subsets between young and aged mice (Fig. 6 and data not shown).

Figure 6.

Expression of CD44 molecule on γδ-IELs in young KN6-Tg, aged KN6-Tg, young non-Tg, and aged non-Tg mice of BALB/c background. (top) Cells were stained with anti-TCR-β mAb and anti-TCR-δ mAb. (bottom) Expression of the CD44 molecule was examined after gating Vγ4⁺ cells (KN6-Tg mice) or TCR-γδ⁺ cells (non-Tg mice).

Tg γδ-IEL from young KN6-Tg mice responded to H-2^b splenocytes and the addition of rIL-2 enhanced their proliferative response to H-2^b splenocytes. By contrast, Tg γδ-IEL from aged KN6-Tg mice exhibited poor response to H-2^b splenocytes and the addition of rIL-2 slightly augmented their proliferative response to the antigen. However, the response to rIL-2 alone was consistently higher in Tg γδ-IEL from aged mice. On the other hand, anti-Vγ4 mAb-triggered proliferation was almost comparable in Tg γδ-IEL from young and aged mice (Fig. 7). These results indicate that chronic stimulation in the intestinal environment renders Tg γδ-IEL severely hyporesponsive to the antigen.

Figure 7.

Proliferative response of Tg γδ-IELs from young and aged KN6-Tg mice of BALB/c background. Responder cells (2 × 10⁵ cells/well) were cultured as described in >Materials and methods. Three independent experiments gave similar results and a representative one is shown.

Development of Tg γδ-IEL in KN6-Tg mice of H-2^b/d background

To examine the effect of antigen on the development of Tg γδ-IEL in vivo, we generated KN6-Tg mice of H-2^b/d background (Tg^b/d mice). While a number of Tg TCR-γδ⁺ cells were present in the spleen of Tg^d/d mice (25·1 ± 12·1%), they were almost negligible in the spleen of Tg^b/d mice (1·0 ± 0·4%). On the other hand, a sizeable number of Tg γδ-IEL were present in Tg^b/d mice, although the relative proportion of Tg γδ-IEL in Tg^b/d mice (68·7 ± 14·4%) was significantly less than that of Tg^d/d mice (92·3 ± 2·0%).

The proportion of CD44⁺ cells in Tg γδ-IEL from Tg^b/d mice was markedly higher than that from Tg^d/d mice, while the proportions of the Thy-1⁺ and CD8α⁺ subsets in Tg γδ-IEL were comparable between Tg^d/d and Tg^b/d mice. As the relative ratio of CD44⁺ cells in γδ-IEL was almost comparable in non-Tg^d/d and non-Tg^b/d mice, we considered that increment of CD44⁺ cells in Tg γδ-IEL in Tg^b/d mice was caused by antigen-triggered activation (Fig. 8 and data not shown).

Figure 8.

Expression of the CD44 molecule on γδ-IELs in Tg^d/d, Tg^b/d, non-Tg^d/d, and non-Tg^b/d mice. (top) Cells were stained with anti-Vγ4 mAb and anti-TCR-δ mAb. (bottom) Expression of CD44 molecule was examined after gating Vγ4⁺ cells (Tg^d/d and Tg^b/d mice) or TCR-γδ⁺ cells (non-Tg^d/d and non-Tg^b/d mice).

γδ-IEL from Tg^d/d mice responded to H-2^b splenocytes, and the antigen-triggered proliferation of Tg^d/dγδ-IEL was augmented by the addition of rIL-2. By contrast, γδ-IEL from Tg^b/d mice did not respond to H-2^b splenocytes and the addition of rIL-2 did not rescue their proliferative response to H-2^b splenocytes. However, response to rIL-2 alone was higher in Tg^b/dγδ-IEL than Tg^d/dγδ-IEL. Moreover, immobilized anti-Vγ4 mAb hardly induced the proliferation of Tg^b/dγδ-IEL (Fig. 9). These results show that Tg^b/dγδ-IEL are activated but do not respond to the antigen.

Figure 9.

Proliferative response of Tg γδ-IELs in Tg^d/d and Tg^b/d mice. Responder cells (2 × 10⁵ cells/well) were cultured as described in Materials and methods. Four independent experiments gave similar results and a representative one is shown.

Discussion

In this report, we examined the effect of intestinal epithelia on the activation of KN6-Tg TCR-γδ⁺ cells. Comparative analysis of splenic Tg TCR-γδ⁺ cells and Tg γδ-IEL suggest that the intestinal epithelia activate Tg TCR-γδ⁺ cells and render them hyporesponsive to the antigen (H-2^b splenocytes), leaving the ability to respond to immobilized anti-Vγ4 mAb undisturbed. Furthermore, the ability of Tg γδ-IEL to recognize the antigen was deteriorated in association with ageing. These results demonstrate that chronic stimulation in the intestinal epithelia down-modulates the ability of Tg TCR-γδ⁺ cells to recognize the antigen. Proliferative response of SI-IEL to rIL-2 alone was higher in aged wild type mice than young wild type mice (unpublished data), indicating that ageing-associated activation may not be an event specific to Tg γδ-IEL.

It is of great importance to see how Tg TCR-γδ⁺ cells are activated in the intestinal epithelia of KN6-Tg mice of H-2^d background. One explanation is that KN6-Tg TCR-γδ⁺ cells are activated and tolerized in the intestinal epithelia in an antigen-independent manner. Linton et al. showed that splenic Tg TCRαβ⁺ cells in AND-Tg mice lose the ability to respond to the antigen (pigeon cytochrome c) in association with ageing even when they are maintained under pigeon cytochrome c-free conditions.¹⁶ Similar activation processes may work in the case of Tg γδ-IEL in KN6-Tg mice of H-2^d background. Another explanation is that endogenous TCR expressed on KN6-Tg TCR-γδ⁺ cells is triggered by the ligands in the intestine. Hurst et al. have reported that Tg TCR-αβ⁺ cells in the intestinal lamina propria express an activated/memory-like phenotype but they do express a naive-like phenotype in Tg × recombination-activating genes (RAG)-1^–/– mice, showing that signalling through endogenous TCR results in the activation of intestinal Tg TCR-αβ⁺ cells.¹⁷ However, this is possibly not the case in KN6-Tg mice, because the proportion of CD44⁺ cells among Tg γδ-IEL in KN6-Tg scid/scid mice lacking endogenous TCR is comparable to that in KN6-Tg mice (Dr H. Ishikawa, personal communication). Furthermore, ageing induced the accumulation of Tg γδ-IEL expressing TCR-γδ brightly (Fig. 6) while Tg γδ-IEL in KN6-Tg^b/d mice expressed the reduced intensity of TCRγδ than those in KN6-Tg^d/d mice (Fig. 8). Therefore, it is considered that hyporesponsiveness of Tg^d/dγδ-IEL induced by chronic stimulation in the intestinal epithelia is distinct from antigen-driven anergy of Tg γδ-IEL.

Involvement of intestinal bacteria in the activation of Tg γδ-IEL should not be overlooked. However, Tg γδ-IEL prepared from KN6-Tg mice exhibited cytolytic activity to a similar extent whether they were raised under CV or GF conditions, suggesting that microbial colonization is not critical for the development of Tg γδ-IEL. We have already shown, using three inbred strains of mice, that the constitution and cytolytic activity of γδ-IEL are comparable between CV and GF mice.⁷γδ-IEL from wild type mice predominantly utilize Vγ1 or Vγ7 chains but Tg γδ-IEL from KN6-Tg mice adopt the Vγ4 chain. Therefore, intestinal microflora-independent development of TCR-γδ⁺ cells is not connected with the usage of particular Vγ chains. On the other hand, the fact that cytolytic activity of γδ-IEL in antigen-minimized mice is sharply attenuated,⁸ suggests that chronic stimulation by food-derived materials in the gut lumen may result in the activation and hyporesponsiveness of γδ-IEL.

Comparative analysis of Tg γδ-IELs from Tg^d/d and Tg^b/d mice revealed that the number of Tg γδ-IEL decreases, the proportion of CD44⁺ subset in Tg γδ-IEL increases, and the ability of Tg γδ-IEL to recognize the antigen is deprived in the antigen-bearing mice. Antigen-driven activation of Tg γδ-IEL has already been investigated using G8-Tg mouse whose Tg TCR-γδ recognizes T10^b/T22^b molecule.^18,19 Our observation is consistent with the fact that G8-Tg mice of H-2^b/d background contain more CD44⁺ Tg γδ-IEL than G8-Tg mice of H-2^d/d background and G8-Tg^b/dγδ-IEL exhibit no proliferative response to H-2^b splenocytes. However, there is a difference in the antigen-driven change of KN6-Tg γδ-IEL and G8-Tg γδ-IEL. Based on the finding that the Thy-1 molecule is exclusively expressed on Tg γδ-IEL in G8-Tg^d/d mice but is barely expressed on Tg γδ-IEL in G8-Tg^b/d mice, Barrett et al. concluded that Thy-1 on G8-Tg γδ-IEL is down-regulated by antigenic stimulation.¹⁹ By contrast, the proportion of Thy-1⁺ KN6-Tg γδ-IEL was similar in KN6-Tg^d/d and KN6-Tg^b/d mice (data not shown), suggesting that Thy-1 is not an activation marker in KN6-Tg γδ-IEL. It is unclear why these differences should exist between KN6-Tg and G8-Tg mice. G8 TCRγδ⁺ clone has been derived from lymph node cells of athymic BALB/c (H-2^d) mice immunized by B10.BR (H-2^k) cells, and G8 TCR-γδ⁺ cells are eliminated in the thymus of G8-Tg^b/d mice.^20,21 On the other hand, KN6 TCR-γδ⁺ hybridoma has been originated from thymocytes of C57BL/6 (H-2^b) mice, and KN6 TCR-γδ⁺ cells are present but inactivated in the thymus of KN6-Tg^b/d mice.^22,23 These results hint that the origin of TCR-γδ may determine the differences detected between G8-Tg and KN6-Tg mice.

Anatomical location of γδ-IEL suggests that they play a significant role in the first line of defence. This hypothesis is strengthened by the finding that γδ-IEL respond to enteric infection of Eimeria vermiformis.²⁴ Moreover, γδ-IEL have been found to enhance the generation of intestinal epithelial cells (IEC), which may be important for the repair of damaged intestinal barrier.^25–27 Although γδ-IEL recognize stress-induced molecules expressed on IEC,²⁸ our present findings indicate that the ability of γδ-IEL to recognize the antigen may be lowered through chronic stimulation in the intestine. It remains to be elucidated whether γδ-IEL keep up intestinal integrity through recognizing IEC by TCR-γδ or through TCR-independent cell-to-cell interaction.

Glossary

Abbreviations

SI-IEL

intraepithelial lymphocytes of small intestine

Tg mouse

transgenic mouse

GF

germ-free

CV

conventional