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The Journal of Immunology, 2006, 176: 6553-6559.
Copyright © 2006 by The American Association of Immunologists

TCR{gamma}{delta} Intraepithelial Lymphocytes Are Required for Self-Tolerance1

Natasha R. Locke, Sanda Stankovic, David P. Funda2 and Leonard C. Harrison3

Autoimmunity and Transplantation Division, Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Neonatal thymectomy (NTX) impairs T cell regulation and leads to organ-specific autoimmune disease in susceptible mouse strains. In the NOD mouse model of spontaneous type 1 diabetes, we observed that NTX dramatically accelerated autoimmune pancreatic beta cell destruction and diabetes. NTX had only a minor effect in NOD mice protected from diabetes by transgenic expression of the beta cell autoantigen proinsulin in APCs, inferring that accelerated diabetes after NTX is largely due to failure to regulate proinsulin-specific T cells. NTX markedly impaired the development of intraepithelial lymphocytes (IEL), the number of which was already reduced in euthymic NOD mice compared with control strains. IEL purified from euthymic NOD mice, specifically CD8{alpha}{alpha} TCR{gamma}{delta} IEL, when transferred into NTX-NOD mice, trafficked to the small intestinal epithelium and prevented diabetes. Transfer of prototypic CD4+CD25+ regulatory T cells also prevented diabetes in NTX-NOD mice; however, the induction of these cells by oral insulin in euthymic mice depended on the integrity of TCR{gamma}{delta} IEL. We conclude that TCR{gamma}{delta} IEL at the mucosal interface between self and nonself play a key role in maintaining peripheral tolerance both physiologically and during oral tolerance induction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Neonatal thymectomy (NTX)4 2–5 days after birth in susceptible mouse strains is associated with the development of organ-specific autoimmune disease (1, 2). Tung et al. (1) proposed that autoimmune disease after NTX results from a deficiency of regulatory T cells whose thymic-dependent maturation occurs in a narrow postnatal time window. The lymphopenia that follows NTX encompasses T cell populations involved in regulating immunity to self-Ags. For example, NTX leads to a deficiency of prototypic CD4+CD25+ regulatory T cells (Treg) (3), reconstitution of which prevents post-NTX autoimmune gastritis (3, 4). NTX also impairs development of regulatory NK T cells (5). In addition, a major T cell population whose development was earlier noted to be impaired by NTX (6) comprises intraepithelial lymphocytes (IEL); however, IEL have not previously been implicated in post-NTX autoimmune disease.

IEL are phenotypically heterogeneous and their developmental pathways have been contentious (7). Thymic-derived IEL express the {alpha}beta TCR and either the CD8 molecule comprised of {alpha}- and beta-chain heterodimers (CD8{alpha}beta) or the CD4 molecule. In the mouse, up to half of the intestinal IEL are distinguished from conventional thymic-derived T cells by expression of the CD8{alpha}{alpha} homodimer and include both TCR{alpha}beta and TCR{gamma}{delta} cells (7, 8). CD8{alpha}{alpha} IEL were proposed to have an extrathymic origin, being the progeny of bone marrow-derived stem cells that develop in novel lymphoid sites termed cryptopatches in the small and large intestinal mucosa (9), but this has been disputed (10). The maturation of CD8{alpha}{alpha} IEL is clearly thymic dependent because their number is substantially reduced in athymic nude mice (11) and after NTX (6). Thymic-derived IEL can be reconstituted with thymocytes or lymph node T cells (12, 13), but reconstitution of CD8{alpha}{alpha} IEL requires thymic stroma (6, 14, 15). Lineage tracing experiments (16) have recently indicated that CD8{alpha}{alpha} TCR{alpha}beta IEL are thymic derived, but the origin of CD8{alpha}{alpha} TCR{gamma}{delta} IEL, that develop in the absence of class I MHC molecules and recognize Ags presented by nonclassical MHC-like molecules on epithelial cells (17, 18), remains unclear.

A role for IEL in post-NTX autoimmune disease has not been addressed previously. However, several lines of evidence point to an immunoregulatory function of CD8{alpha}{alpha} TCR{gamma}{delta} IEL. First, in the NOD mouse, a model of autoimmune or type 1 diabetes, the high incidence of diabetes under germfree or specific pathogen-free conditions is reduced by microbial exposure and colonization of the intestine (19, 20, 21), which increases the numbers of IEL (21, 22) and promotes maturation of mucosal immune function (23). Second, exposure of the nasorespiratory mucosa of the NOD mouse to insulin, an autoantigen that drives T cells to destroy pancreatic beta cells in type 1 diabetes (24), induces regulatory, antidiabetogenic T cells with a CD8{alpha}{alpha} TCR{gamma}{delta} phenotype (25, 26). Third, oral mucosal tolerance is impaired in C57BL/6 mice treated with an Ab that inactivates {gamma}{delta} T cells (27, 28) and in TCR {delta} gene knockout mice (28). In this study, we use the NTX model in diabetes-prone NOD mice to reveal new evidence for the immunoregulatory role of CD8{alpha}{alpha} TCR{gamma}{delta} IEL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female mice were used in all experiments and were bred under specific pathogen-free conditions at the Walter and Eliza Hall Institute of Medical Research (WEHI). NOD/Lt and NOR mice were originally obtained from The Jackson Laboratory. NOD and NOR mice are ~90% genetically identical, including the MHC, but only NOD mice develop diabetes. NOD mice expressing the mouse proinsulin II transgene under the control of the I-E{kappa}{alpha} MHC class II promoter, herein called NOD-PI mice, were generated as previously described (29) and bred to homozygosity. Congenic CD45.1 and CD45.2 NOD mice were provided by G. Morahan (WEHI, Parkville, Victoria, Australia). beta2-microglobulin–/– NOD mice were provided by T. W. H. Kay (WEHI). Mice were killed by CO2 asphyxiation.

Neonatal thymectomy

The thymus was removed from NODLt/Jax pups by suction under cold anesthesia 3 days after birth as described previously (30). Euthymic littermate controls received a sham operation in which the thymus was left intact. Completeness of NTX was confirmed by visual examination at autopsy; a small number of mice (<5%) with thymic remnants were not included in experiments.

Diabetes assessment

Blood glucose was measured in retro-orbital venous blood samples with Advantage glucose test strips and an Advantage meter (Roche Diagnostics). Mice were considered diabetic if blood glucose was confirmed as >11 mM.

Insulitis assessment

The insulitis score, a measure of the degree of cellular infiltration of the pancreatic islets, was determined blindly by two investigators. A minimum of 15 islets was scored in serial (100 µm) Bouin’s-fixed, paraffin-embedded 6-µm sections of pancreas stained with H&E. The grading scale was: 0, no infiltration, islet intact; 1, <10 peri-islet lymphoid cells, islet intact; 2, 10–20 peri- and intraislet lymphoid cells, islet intact; 3, >20 peri- and intraislet lymphoid cells, <50% of islet replaced or destroyed; 4, massive lymphoid infiltrate with >50% of islet replaced or destroyed (31).

Antibodies

mAbs to {gamma}{delta} TCR (GL3; PE labeled), {alpha}betaTCR (H57-597; biotin or PE labeled), CD3 (145-2C11; FITC labeled), CD25 (PC61; PE labeled), CD45.2 (104; biotin labeled) and {alpha}E integrin CD103 (2E7; FITC labeled) were obtained from BD Pharmingen. Abs to CD8{alpha} (Ly-2; PE or FITC labeled), CD4 (CT-CD4; PE or FITC labeled) and streptavidin (PE labeled) were from Caltag Laboratories. Ab to CD8beta (Ly-3.1; PE labeled) was from Serotec. Cell culture supernatants of hybridomas recognizing CD3 (KT3), CD8 (53-6.7), CD4 (H129), anti-HSA (J11D) rat anti-beta-galactosidase (GL117.41), {gamma}{delta} TCR (GL3), and hamster anti-human Bcl-2 (6C8) were used for immunohistochemical staining.

Immunohistochemical and histochemical staining

A section of small intestine 6–8 cm from the pyloroduodenal junction was dissected, frozen in OCT embedding compound on dry ice, and stored at –70°C. Tissue sections (6 µm) were cut with a cryostat onto Superfrost Plus slides. Acetone-fixed sections were blocked with normal goat serum and avidin/biotin followed by a 1-h incubation with Abs. Abs were detected with biotinylated goat anti-rat Ig (BD Pharmingen) or anti-hamster IgG (Vector Laboratories), followed by HRP-conjugated streptavidin ABC kit (DakoCytomation). Between incubation steps, sections were washed in 0.05 M TBS (pH 7.4). Staining was developed with 3,3'-diaminobenzidine in TBS containing 0.03% peroxidase. Sections were counterstained with hematoxylin, dehydrated through alcohol, and mounted. Because of better preservation of morphology, for quantitative in situ analysis of IEL proximal jejunum was fixed in Bouin’s solution and paraffin-embedded sections were stained with H&E.

Flow cytometry

Isotype control Abs were used in all experiments. Cells were incubated with Abs in 100 µl of staining buffer (PBS (pH 7.4), containing 2% FCS) for 30 min on ice, washed three times and, if required, incubated with streptavidin-FITC for a further 20 min on ice and washed three times. Analysis was performed on FACScan cytofluometer using CellQuest software (BD Biosciences). Forward and side angle light scatter were used to exclude epithelial cells and aggregates. Propidium iodide was used to exclude dead cells.

Isolation of IEL

IEL were isolated from the mouse small intestine as described by Lefrancois (32), with minor modifications. Briefly, the small intestine was removed, flushed with ice-cold RPMI 1640 medium, and freed of fat, mesentry and Peyer’s patches. The tissue was then cut into small pieces ~0.5-cm long and incubated at 37°C with shaking for 20–30 min in calcium- and magnesium-free HBSS containing 1 mM EDTA and 1 mM DTT to dissociate IEL. The released cells were filtered through a nylon wool column and centrifuged in a density gradient of 45–70% Percoll (Pharmacia) to purify IEL from the upper layer of contaminating epithelial cells. Bronchial cells were obtained by placing a cannula into the trachea and lavaging four times with sterile, warm PBS. Nasal-associated lymphoid tissue (NALT) was isolated after removal of the mandible as previously described (33). Cell suspensions counted for trypan blue exclusion were stained with appropriate Abs, directly or indirectly labeled, and analyzed by two- or three-color flow cytometry (FACScan; BD Biosciences) using CellQuest software (BD Biosciences).

Transfer of IEL into NTX-NOD mice

IEL isolated from 6-wk-old CD 45.2 NOD mice were injected i.v. into 4-wk-old NTX-CD45.1 NOD mice. IEL from the small intestine, and spleen, mesenteric lymph node, and pancreatic lymph node cells, were analyzed 4 wk later for the presence of donor CD45.2 cells.

Purification of CD4+ T cells

Spleen cells were stained with CD4-FITC and CD25-PE at optimal concentrations for FACS staining, for 20 min at 4°C. After washing, cells were incubated with microbeads conjugated to anti-FITC Abs (Miltenyi Biotec) for 20 min at 4°C and target cells purified in an AutoMACS (Miltenyi Biotec). FITC-microbeads were removed from positively selected CD4 T cells by incubation with 50 U/ml DNase and a second round of selection performed using microbeads conjugated to anti-PE Abs.

Adoptive transfer of diabetes

Male NOD mice at 6–9 wk of age were irradiated (800 rad) and 4–6 h later received 2 x 107 spleen cells from recently diabetic female NOD mice, either alone or with splenic CD4+ populations from oral insulin-treated mice. Cells were injected into the tail vein in 200 µl of PBS.

Statistics

Group data were compared with the two-tailed unpaired t test. Survival curves (Kaplan-Meier) were compared by log rank test using GraphPad Prism software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
NTX accelerates diabetes development in NOD mice

NTX markedly accelerated the onset and increased the incidence of diabetes in both female and male NOD mice. Following NTX (Fig. 1), ~60% of female and 40% of male NTX mice became diabetic by 100 days of age, compared with only 5 and 0% of sham NTX controls; at 300 days of age, diabetes incidence was 100% in females and over 80% in males. In parallel, NTX-NOD mice exhibited aggressive lymphoid infiltration of the pancreatic islets. As early as 70 days of age, the mean insulitis score in NTX-NOD mice (3.0 ± 0.70) was significantly greater (p < 0.01) than in sham NTX mice (2.1 ± 0.40). In control NOR mice, which share the diabetogenic MHC haplotype of NOD mice but do not develop diabetes, NTX did not provoke diabetes (Fig. 1), consistent with an absence of T cells with diabetogenic potential in these mice. In NOD-PI mice, which are protected from diabetes by transgenic expression of the beta cell autoantigen, proinsulin II, in APCs (29, 34), NTX had only a minor effect on diabetes development (Fig. 1). Protection from diabetes in NOD-PI mice is attributed to proinsulin-specific tolerance (29, 34), inferring a dominant role for proinsulin in driving beta-cell destruction. Therefore, the relatively minor effect of NTX in NOD-PI mice suggests that acceleration of diabetes after NTX in wild-type NOD mice is largely due to loss of regulation of proinsulin-specific T cells.


Figure 1
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  		FIGURE 1. NTX accelerates diabetes development in NOD mice. Diabetes incidence following NTX or sham-NTX in female (F) and male (M) NOD mice, female NOR mice, and female NOD mice transgenic for proinsulin expression in APCs (NOD-PI).

 
NTX impairs development of IEL

By 8 wk of age, the total spleen cell number in NTX-NOD mice was approximately half that of sham controls, with CD3, CD4 and CD8 T cells being decreased by up to 70% (data not shown). Histological analysis of the small intestine of NTX-NOD mice (Fig. 2) revealed a deficiency of CD3-expressing IEL; it was clearly evident that many CD8{alpha}-expressing IEL cells did not express CD3. In contrast, CD4 expression, mainly confined to cells in the lamina propria compartment, was not affected by NTX. The total number of IEL isolated from the small intestine after NTX was reduced by ~70% (Table I). The majority (>85%) of cells isolated expressed {alpha}E integrin, whereas <5% expressed CD4. Because virtually all IEL and only ~20% of lamina propria CD4 T cells express {alpha}E integrin (35), contamination by lamina propria T cells was therefore minimal. The absolute numbers of CD8{alpha}{alpha}, TCR{alpha}beta, and TCR{gamma}{delta} IEL were reduced by >90%, and classic thymic-derived CD8 and CD4 TCR{alpha}beta T cells by a lesser extent (Table I). CD3–CD8{alpha}{alpha}+ IEL predominated after NTX, doubling in absolute number and comprising 60% of total cells, indicating that NTX blocked the development of CD8{alpha}{alpha} IEL at the CD3– to CD3+ stage. TCR{alpha}beta CD8{alpha}beta IEL isolated from NTX mice (Table I) may represent cells that had exited the thymus before NTX and expanded in the periphery, given that their reduction was proportionally similar to that of peripheral CD8{alpha}beta T cells. NTX was associated with similar decreases in T cell subsets in bronchial and nasal mucosal tissues, notably almost total depletion of bronchial TCR{gamma}{delta} T cells (Table II).


Figure 2
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  		FIGURE 2. NTX impairs development of IEL. Serial frozen sections of jejunum of 6-wk-old female NOD mice that had undergone sham NTX or NTX examined by immunohistochemistry. Shown are representative sections stained with mAb to CD3, CD8{alpha}, or CD4 in brown and counterstained with hematoxylin (x200).

 

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  		Table I. IEL subsets isolated from sham NTX and NTX NOD mice (mean number ± SD x 10–3; n = 6)a

 

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  		Table II. T cell subsets in pooled bronchial lavage and NALT from 6-wk-old sham NTX and NTX NOD mice (mean number x 10–3)

 
Euthymic NOD mice have a selective deficiency of IEL

The impairment of IEL development associated with accelerated diabetes in NOD-NTX mice prompted us to examine IEL in euthymic NOD mice. NOD mice and MHC-identical but diabetes-resistant NOR mice were lymphopenic, with total spleen cell number in both being reduced by ~35% relative to BALB/c mice. This was reflected by lymphoid organ weights (Fig. 3, a–d), but flow cytometry revealed no differences in the proportions of major lymphocyte subsets (CD3+, CD4+, CD8+, CD19+) in thymus, spleen, and axillary lymph nodes between the three strains (data not shown). NOD mice, however, had significantly fewer IEL than either NOR or BALB/c mice (Fig. 3e), and NOR mice had significantly fewer IEL than BALB/c mice. In NOR mice, the reduction in IEL was in keeping with the lymphopenia, but in NOD mice with a similar degree of lymphopenia there were significantly fewer IEL.


Figure 3
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  		FIGURE 3. NOD mice are lymphopenic but have a selective deficiency of IEL. Organs were freshly removed and weighed to an accuracy of 0.1 mg. Serial Bouin’s fixed, paraffin-embedded sections of proximal jejunum taken from 6-wk-old female NOD, NOR, and BALB/c mice were stained with H&E. Using a calibrated eyepiece, the number of IEL was counted in 10 lengths (300 µm each) of intestinal epithelium for each mouse (six per group).

 
Donor CD8{alpha}{alpha} TCR{gamma}{delta} IEL reconstitute the intestinal mucosa and prevent diabetes in NTX-NOD mice

The selective deficiency of IEL in wild-type NOD mice and the impairment of IEL development with accelerated diabetes after NTX raised the question whether reconstitution of IEL in NTX-NOD mice could prevent diabetes. To identify IEL and enable their tracking in vivo, donor IEL were isolated from the small intestine of NOD mice congenic for CD45. In the first instance, IEL from 6-wk-old CD 45.2 NOD mice were injected i.v. into 3- to 4-wk-old CD 45.1 NTX-NOD mice. After 28 days, recipient mice were killed and intestinal IEL and cells from spleen and mesenteric and pancreatic lymph nodes were recovered and analyzed. Donor CD45.2 IEL, both TCR{alpha}beta and TCR{gamma}{delta}, were detected only in the intestine, not other organs (Fig. 4).


Figure 4
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  		FIGURE 4. IEL traffic to the small intestinal epithelium after injection into NOD-NTX mice. A total of 2 x 106 IEL isolated from 4- to 5-wk-old CD 45.2 NOD mice were injected i.v. into 3- to 4-wk-old NTX-CD45.1 NOD mice (n = 4/group). Recipient mice were killed after 28 days and small intestine, spleen, mesenteric lymph node, and pancreatic lymph node cells were analyzed by flow cytometry for the presence of donor CD45.2 cells. Donor cells were detected only in the small intestinal epithelium.

 
Transfer of 2 or 3 x 106 IEL i.v. from euthymic NOD mice into NTX-NOD mice at 10 or 28 days of age significantly delayed the onset of diabetes, and this effect was prevented by coinjecting mice i.p. with 200 µg of the TCR{gamma}{delta}-depleting mAb, GL3 (data not shown). To obtain pure CD8{alpha}{alpha} TCR{gamma}{delta} IEL for transfer, we took advantage of the fact that beta2-microglobulin–/– NOD mice lack TCR{alpha}b, CD8{alpha}{alpha}, and CD8{alpha}beta T cells (17). By flow cytometry, ≥96% of IEL isolated from these mice at 4–6 wk of age were CD8{alpha}{alpha} TCR{gamma}{delta} (data not shown). NTX-NOD mice at 4, 6, 8, and 10 wk of age were then injected i.v. with 1 x 106 CD8{alpha}{alpha} TCR{gamma}{delta} IEL obtained from 4 wk-old beta2-microglobulin–/– NOD mice. Mice that received CD8{alpha}{alpha} TCR{gamma}{delta} IEL had a significantly reduced incidence of diabetes (Fig. 5). This protective effect was not observed if donor IEL were first irradiated or if recipient mice were injected with GL3 anti-TCR{gamma}{delta} mAb. Thus, protection of NTX-NOD mice from diabetes was due to the transfer of CD8{alpha}{alpha} TCR{gamma}{delta} IEL and not to contaminating bacterial products or other T cells. In contrast, reconstitution of NTX-NOD mice with total spleen cells from 6-wk-old euthymic NOD mice increased rather then decreased diabetes incidence (Fig. 5).


Figure 5
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  		FIGURE 5. Reconstitution of NTX-NOD mice with CD8{alpha}{alpha} TCR{gamma}{delta} IEL prevents diabetes. Every 2 wk from 4- to 10-wk of age NTX-NOD mice (n = 12/group) received an i.p. injection of 1 x 106 IEL freshly isolated from 4-wk-old beta2-microglobulin–/– NOD mice. Three hours earlier, mice received 250 µg i.p. of either anti-TCR{gamma}{delta} mAb (GL3 clone) or hamster isotype control mAb (6C8 clone). Other groups received irradiated (3000 rad) IEL only or anti-TCR{gamma}{delta} mAb only. A separate group of NTX-NOD mice received a single injection at 10 days of age of 2 x 107 spleen cells from 6-wk-old NOD females. Blood glucose was measured every 3 wk from 70 days of age.

 
CD4+CD25+ T cells prevent diabetes in NTX-NOD mice but their induction by oral insulin requires TCR{gamma}{delta} T cells

Autoimmune disease in NTX mice has been attributed to a deficiency of prototypic thymic-derived CD4+CD25+ Treg (3, 4). In BALB/c mice, the development of autoimmune gastritis following NTX was prevented by transfer of CD4+CD25+ T cells from euthymic mice (4). NOD mice were initially reported to be deficient in CD4+CD25+ T cells (36), but it was shown subsequently that the proportion of CD4+CD25+ Treg in the thymus and in the periphery of NOD mice is normal (37). To determine whether CD4+CD25+ T cells could prevent diabetes after NTX, we transferred spleen cells from 4-wk-old euthymic NOD mice into NTX-NOD mice 10 days after birth. Mice that received total spleen cells rapidly developed diabetes. However, mice that received CD4+ spleen cells had a significantly reduced incidence of diabetes, and this protective effect was abolished by depletion of CD25+ cells (Fig. 6). Flow cytometry revealed that CD4+ cells and CD4+CD25+ cells were on average increased 2.6- and 2.9-fold, respectively, in the positively selected CD4+ population.


Figure 6
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  		FIGURE 6. Reconstitution of NTX-NOD mice with CD4+CD25+ T cells prevents diabetes. Female NTX-NOD mice (n = 12/group) were injected i.p. at 10 days of age with 2 x 107 spleen cells, or CD4+ cells positively selected from 2 x 107 spleen cells, or CD4+ cells depleted of CD25+ cells. Donor cells were from 4-wk-old euthymic female NOD mice. Blood glucose was measured every 3 wk from 50 days of age.

 
CD4+CD25+ Treg were originally identified as a subset of thymocytes (3) but are also present in the periphery (4) and can be induced by oral Ag (38, 39). In NOD mice, oral insulin is known to induce CD4+ Treg that protect against diabetes (40). To determine whether TCR{gamma}{delta} T cells are required for oral insulin to induce CD4+ CD25+ Treg, female 6-wk-old NOD mice were given either isotype control or GL3 anti-TCR{gamma}{delta} mAb i.p. and then orally gavaged with insulin on 6 alternating days. A week later, spleen cells were isolated from the treated mice to determine their ability to suppress the adoptive transfer of diabetes, by cotransfer with diabetogenic spleen cells from recently diabetic NOD mice. By 42 days after transfer, 80% of mice that received only diabetogenic cells had become diabetic. The cotransfer of either CD4+CD25+ or CD4+CD25– spleen cells from mice given control Ab before oral insulin suppressed diabetes development (Fig. 7). This indicates that both the CD25+ and CD25– CD4+ populations contained regulatory cells induced by oral insulin. The regulatory activity of the CD4+CD25+, but not CD4+CD25–, cells was abolished by pretreating mice with GL3 mAb that inactivates {gamma}{delta} T cells (28). Thus, {gamma}{delta} T cells are required for the induction of CD4+CD25+ Treg by oral insulin.


Figure 7
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  		FIGURE 7. {gamma}{delta} T cells are required for induction of regulatory CD4+CD25+ cells by oral insulin. Female 4-wk-old NOD mice (n = 4/group) were injected i.p. with 250 mg of anti-TCR {gamma}{delta} (GL3) or hamster isotype control (6C8) mAb on day 0, then given 1 mg of insulin by oral gavage on 6 alternating days. A week later, splenic CD4+CD25+ and CD4+CD25– cells were separated by sequential automated MACS purification. CD4+CD25+ or CD4+CD25– cells from the equivalent of 2 x 107 spleen cells were cotransferred to irradiated male NOD mice (n = 10/group) with 2 x 107 diabetogenic spleen cells from recently diabetic female NOD mice. Blood glucose was measured every 3 wk from 50 days of age.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our findings support the hypothesis that NTX exacerbates autoimmune diabetes in NOD mice by a mechanism that involves failure to develop IEL, specifically CD8{alpha}{alpha} IEL, already deficient in NOD mice. NTX had a dramatic effect to accelerate diabetes development not only in female but also in male mice that have a low incidence of spontaneous diabetes. Previously, Dardenne et al. (41) had shown that thymectomy at weaning accelerated diabetes development in female, but not male, NOD mice. This difference may indicate that the maturation of thymus-dependent regulatory cells is sex hormone dependent and proceeds faster in male mice. NTX markedly impaired the maturation of IEL. The reduction in TCR-bearing IEL after NTX was accompanied by a 2-fold increase in CD3– CD8{alpha}{alpha}-expressing IEL, indicative of a block in T cell maturation before the acquisition of CD3. The key role of IEL in immune homeostasis was evidenced by the effect of reconstituting NTX-NOD mice with CD8{alpha}{alpha} TCR{gamma}{delta} IEL. These IEL trafficked to the small intestinal epithelium and suppressed diabetes development. To our knowledge, this is the first demonstration of an effect of IEL to protect against autoimmune disease.

Compared with wild-type NOD mice, the minor effect of NTX on diabetes incidence in NOD-PI mice, that are protected from diabetes by transgenic expression of proinsulin in APCs (29, 34), implies that TCR{gamma}{delta} IEL in NOD mice are required for regulating proinsulin-specific T cells. How this occurs and whether it is autoantigen specific requires further investigation. Acquisition of peripheral tolerance to proinsulin/insulin could occur physiologically in the neonate by exposure of the intestinal mucosa to insulin, a constituent of maternal milk (42). Transferred CD8{alpha}{alpha} TCR{gamma}{delta} IEL that protected NTX-NOD mice were from donors that had recently been exposed to maternal milk. Following nasorespiratory administration of insulin, CD8{alpha}{alpha} TCR{gamma}{delta} T cells expressing IL-10 appear in the NOD mouse spleen (25) and pancreatic lymph node (26). This suggests that the regulatory activity of TCR{gamma}{delta} IEL may not be unspecific but driven by exogenous Ags such as dietary insulin. Traditionally, IEL are considered to be sessile on the mucosal epithelium. However, a study in pigs (43) and our recent studies in mice (44) demonstrate that gut TCR{gamma}{delta} IEL contribute significantly to the pool of circulating TCR{gamma}{delta} T cells. Therefore, TCR{gamma}{delta} IEL exposed to insulin in the gut could conceivably exert a distal effect in pancreatic lymph nodes to suppress autoimmune diabetes, directly or indirectly by influencing another cell to traffic there.

Diabetes in NTX-NOD mice was also suppressed by transfer of splenic CD4+CD25+ T cells. This does not, however, constitute proof that a lack of CD4+CD25+ Treg is the primary or only cause of accelerated diabetes in NTX-NOD mice. The thymus, probably via stroma-derived factors, is also required for the maturation of CD8{alpha}{alpha} TCR{gamma}{delta} IEL (6, 14, 15). Others (27, 28) have shown that blockade of TCR{gamma}{delta} cells by treatment in vivo with the GL3 anti-{gamma}{delta} mAb prevents the induction and maintenance of oral tolerance, and we previously found (25, 26) that exposure of the nasorespiratory mucosa to insulin induced CD8{alpha}{alpha} TCR{gamma}{delta} cells that protected against diabetes. Thus, after finding that reconstitution with CD8{alpha}{alpha} TCR{gamma}{delta} IEL prevented diabetes in NTX-NOD mice, it was pertinent to ask whether {gamma}{delta} T cells are required for induction of CD4+CD25+ Treg by oral insulin. Our demonstration that treatment with GL3 anti-TCR{gamma}{delta} mAb prevents induction of CD4+CD25+ Treg by oral insulin does not constitute direct proof that the relevant {gamma}{delta} T cells are IEL; however, this seems likely because IEL comprise the majority of {gamma}{delta} T cells and the dependence of induced CD4+CD25+ Treg on {gamma}{delta} T cells was demonstrated by exposing the intestinal mucosa to insulin. How could this requirement for {gamma}{delta} T cells be explained? Recently, it was reported that human {gamma}{delta} T cells express MHC class II and other Ag-processing presenting molecules and can activate naive CD4 T cells (45), but there is no evidence that mouse {gamma}{delta} T cells have similar properties. Furthermore, it is unlikely that TCR{gamma}{delta} IEL resident on the luminal mucosal epithelium interact directly with conventional CD4+ T cells in the underlying lamina propria. In contrast, TCR{gamma}{delta} IEL may interact with lamina propria dendritic cells, which are known to insinuate the epithelium (46), to maintain the tolerogenic state of these cells. TCR{gamma}{delta} IEL clones abundantly express IL-10 and TGF-beta (47), cytokines that are known to confer tolerogenic properties on mucosal dendritic cells (48). Moreover, in mice transgenically expressing IL-10 in intestinal epithelial cells, the epithelial layer was substantially enriched for CD4+CD25+ T cells (49). Thus, {gamma}{delta} IEL could contribute to the milieu that favors tolerogenic mucosal dendritic cells, which in turn induce CD4+CD25+ Treg. In conclusion, we suggest that autoimmune disease following NTX is due not only to a lack of thymic-derived CD4+CD25+ Treg but also to the lack of thymic-dependent CD8{alpha}{alpha} TCR{gamma}{delta} IEL that are normally required for induction and maintenance of CD4+CD25+ Treg in the periphery.


   Acknowledgments
 
Margo Honeyman and Andrew Lew commented on the manuscript and Catherine McLean provided valuable secretarial assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by a Center Grant from the Juvenile Diabetes Research Foundation (JDRF) and by the National Health and Medical Research Council of Australia. D.P.F. was a JDRF Postdoctoral Fellow. Back

2 Current address: Academy of Sciences of the Czech Republic, Prague, Czech Republic. Back

3 Address correspondence and reprint requests to Dr. Leonard C. Harrison, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville 3050, Victoria, Australia. E-mail address: harrison{at}wehi.edu.au Back

4 Abbreviations used in this paper: NTX, neonatal thymectomy; IEL, intraepithelial lymphocyte; Treg, regulatory T cell; NALT, nasal-associated lymphoid tissue. Back

Received for publication November 18, 2005. Accepted for publication March 22, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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