Abstract
γδ T cells, a lineage of innate-like lymphocytes, are distinguished from conventional αβ T cells in their Ag recognition, cell activation requirements, and effector functions. γδ T cells have been implicated in the pathology of several human autoimmune and inflammatory diseases and their corresponding mouse models, but their specific roles in these diseases have not been elucidated. We report that γδ TCR+ cells, including both the CD27−CD44hi and CD27+CD44lo subsets, infiltrate islets of prediabetic NOD mice. Moreover, NOD CD27−CD44hi and CD27+CD44lo γδ T cells were preprogrammed to secrete IL-17, or IFN-γ upon activation. Adoptive transfer of type 1 diabetes (T1D) to T and B lymphocyte–deficient NOD recipients was greatly potentiated when γδ T cells, and specifically the CD27− γδ T cell subset, were included compared with transfer of αβ T cells alone. Ab-mediated blockade of IL-17 prevented T1D transfer in this setting. Moreover, introgression of genetic Tcrd deficiency onto the NOD background provided robust T1D protection, supporting a nonredundant, pathogenic role of γδ T cells in this model. The potent contributions of CD27− γδ T cells and IL-17 to islet inflammation and diabetes reported in this study suggest that these mechanisms may also underlie human T1D.
Introduction
T cell receptor γδ T cells are a highly conserved lineage of lymphocytes that use gene rearrangement to encode their defining Ag receptors (1) and provide nonredundant contributions to host protection against a broad spectrum of infectious diseases. In contrast to αβ T cells, γδ TCRs recognize Ag directly, without a requirement for processing and presentation by APCs (2–4). Moreover, whereas TCR engagement in the thymus is essential for conventional αβ T cell maturation, this event is necessary for development of some, but not all, TCRγδ+ thymocytes (5). These and other signatory properties have led to their classification as innate-like T cells, along with subsets of αβ T cells restricted by either the MHC class I–like molecule CD1d (invariant NKT [iNKT] cells) or MHC-related protein 1 (mucosal-associated invariant T [MAIT] cells) (6–9). Thus, γδ T cells can contribute to host protection by responding either to microbial nonpeptidic Ags or to endogenous self “stress Ags” upregulated at sites of inflammation. Most γδ T cells do not circulate according to the patterns of conventional αβ T cells, and they often respond much more rapidly than conventional lymphocytes, invoking the concept of lymphoid stress-surveillance (8). These unconventional activation requirements, their ready capacity to associate with tissues, and their broad functional potentials (10–12) suggest that γδ T cells may also act in settings of autoimmune disease (13).
Recent data point to disease-protective roles for CD1d-restricted iNKT and MR-1–restricted MAIT cells and, in contrast, a pathogenic contribution of γδ T cells in several inflammatory and autoimmune diseases, including type 1 diabetes (T1D). For example, earlier studies of both human T1D and the NOD mouse showed decreased iNKT cell frequencies and reduced ability to secrete cytokines such as IL-4 compared with healthy controls (14, 15). Introgression of the Cd1d-null mutation onto the NOD background disrupted iNKT cell function and further aggravated diabetes (16). A disease-suppressive role for MAIT cells was suggested in human multiple sclerosis (MS) (17) and was demonstrated in an experimental autoimmune encephalomyelitis (EAE) mouse model (18). In contrast to these proposed disease-protective functions, γδ TCR+ cells composed a high proportion of T cells in the lesions of MS patients (19), suggesting a pathogenic role for this T cell subset. Indeed, similar to findings in human MS, γδ T cells have been found at high frequency in the brains of mice with EAE (20). Moreover, γδ T cells are reportedly involved in other experimentally induced mouse models of autoimmune disease, including collagen-induced arthritis and colitis (5, 21–23). In this study, we provide evidence for a potent pathogenic role for γδ T cells in a spontaneous mouse model of T1D.
The NOD mouse has been extensively studied as a spontaneous model of human T1D, with which it shares both genetic risk variants and features of autoimmune pathogenesis (24, 25). However, γδ T cells have been reported to serve both protective and pathogenic roles in the NOD mouse model. γδ T cells purified from among the intraepithelial lymphocyte (IEL) population of NOD mice could prevent T1D when transferred to NOD recipients that had undergone neonatal thymectomy (26). Transfer of T1D protection by T cells preconditioned with aerosolized insulin depended on γδ T cells in the inoculum (27). Recently, γδ T cells were reported to protect NOD mice from T1D via a TGF-β–dependent mechanism (28). These studies support a T1D-suppressive function for γδ T cells in the NOD mouse. In contrast, γδ T cell clones isolated from the spleen and pancreatic lymph nodes (LNs) of NOD mice were reactive against insulin (29), predicting a pathogenic role for such cells. Additionally, in islet biopsies from human diabetic patients, γδ TCR sequences were predominant among the T cell clonotypes identified (30), suggesting that γδ T cells infiltrate the pancreatic islets in human T1D. Collectively, these data suggest that γδ T cells may participate in T1D pathogenesis.
In this study, we used a genetic approach, supported by in vivo immunological analyses, to identify a critical mechanism by which γδ T cells contribute to NOD mouse T1D pathogenesis. We provide evidence that IL-17 is an important mediator facilitating the contributions of the CD27− γδ T cell subset to islet inflammation and diabetes. Given the strong similarities between the NOD mouse model and human T1D, these data suggest a potential role of γδ T cells and IL-17 in the human disease.
Materials and Methods
Mice
All mice used in this study were maintained in a specific pathogen-free facility at The Hospital for Sick Children. Spontaneous T1D incidence at age 6 mo in NOD/Jsd animals is 83% in females and 35% in males. NOD.B6;129P2-Tcrdtm1Mom/J (NOD.TCRδ-KO) mice were a gift from Dr. Robert Tigelaar (Yale University, New Haven, CT). All procedures performed on these mice were performed according to guidelines and animal use protocols of The Hospital for Sick Children Animal Care Committee.
DNA preparation and microsatellite and single nucleotide polymorphism genotyping
DNA was prepared from tail biopsies by digestion in tissue digest solution B (AutoGen, catalog no. AG00122) with 2 μg/ml proteinase K (Qiagen, catalog no. 19131). Single nucleotide polymorphism genotyping was performed using a mouse medium density linkage panel (Illumina, San Diego, CA) at the Centre for Applied Genomics at The Hospital for Sick Children. For high-resolution microsatellite genotyping, DNA was diluted 1:20 prior to introduction into PCR reactions. Novel microsatellite markers were generated as previously described (31). DNA was amplified for 35 cycles with the following conditions: 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C using a multiplex master mix (Qiagen, catalog no. 206143). The PCR product was diluted 1:20, and 1 μl was mixed with a 10 μl mixture of formamide and a 500 LIZ size standard (1 mL Hi-Di formamide per 7 μl size standard). Samples were run on the 3730xl DNA analyzer (Applied Biosystems). Alleles were sized, in comparison with standards, by viewing their electropherograms in GeneMapper (Applied Biosystems).
Spontaneous diabetes assessment
Blood and/or urine glucose levels were measured in mice biweekly. Animals were classified as diabetic when blood glucose was >16 mmol/l or urine glucose was >250 mg/dl. Statistical analyses on T1D life table data were performed using the Mantel–Cox log-rank test with Prism software (version 5.0b; GraphPad Software).
Insulitis assessment
Pancreata were dissected and immediately immersed in OCT media (Tissue-Tek, Torrance, CA), frozen at −78°C in 2-methylbutane over dry ice, and stored at −70°C. Preparation of frozen sections was performed with a Leica CM 3050 cryostat (Leica Canada). To maximize analysis of independent islet infiltrates, three 5-μm sections were cut at least 400 μm apart. Pancreatic sections were stained with Mayer’s H&E (Sigma-Aldrich, St. Louis, MO) to visualize leukocyte infiltration. Assessment of insulitis severity in pancreatic sections was performed as previously described (32). Briefly, islets were graded according to the following criteria: 0, no visible infiltrates; 1, peri-insulitis as indicated by perivascular and peri-islet infiltrates; 2, 25–50% of the islet interior occluded by leukocytes displaying invasive infiltrates; 3, 50–80% of the islet interior invaded by leukocytes; or 4, invasive insulitis involving >80% of the islet field.
Magnetic bead depletions
For purification of total spleen T cells, cell suspensions were stained with biotinylated rat Abs specific for CD49b (clone DX5; Becton Dickinson, San Jose, CA), CD105 (clone MJ7/18; Sunnybrook Research Institute), and microbeads specific for biotin, CD11c, CD11b, and CD19 (Miltenyi Biotec, Cologne, Germany). For purification of splenic αβ T cells, cell suspensions were stained as above, with the addition of a biotinylated hamster Ab against mouse TCRγ (clone GL3; Sunnybrook Research Institute). Labeled cells were depleted using an autoMACS Pro separator (Miltenyi Biotec). The purity of eluted cells was assessed by flow cytometry using hamster Abs against TCRβ (clone H57-597; Sunnybrook Research Institute) and TCRγ (clone GL3; Sunnybrook Research Institute).
Flow cytometry
Single-cell suspensions were prepared from thymus, spleen, pooled peripheral LNs, or the small intestine epithelium. Isolation of lymphocytes from intestinal epithelium was performed as previously described (33). Viable cell counts were determined by trypan blue exclusion, and cells were stained in staining media (1× HBSS [GIBCO-BRL, Gaithersburg, MD], 10 mM HEPES, 2% calf serum [Sigma-Aldrich]) with fluorochrome-conjugated Abs: TCRγ-PE (clone GL3; Sunnybrook Research Institute), TCRβ-FITC (clone H57-597; Sunnybrook Research Institute), CD19-FITC (clone 6D5; Sunnybrook Research Institute), CD44-allophycocyanin (clone IM781; eBioscience, San Diego, CA), CD27-biotin (clone LG.7F9; eBioscience), and avidin-allophycocyanin-Cy7 (Invitrogen, Carlsbad, CA). Flourescence was analyzed using a BD LSR II flow cytometer (Becton Dickinson). Dead cells were excluded using 1 μg/ml propidium iodide. At least 400 γδ TCR+ events were acquired for islet flow cytometry, and a minimum of 1000 γδ TCR events were acquired for analysis of other tissues.
Cell stimulation and intracellular cytokine staining
Cell suspensions were prepared as described above and then resuspended at a concentration of 5 × 106/ml in DMEM with 10% FBS, 1% nonessential amino acids, 2 mM l-glutamine, 50 μM 2-ME, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate. Cells were incubated for 5 h at 37°C in the presence of 50 ng/ml PMA, 1 μg/ml ionomycin, and GolgiPlug protein transport inhibitor (1:1000 dilution; BD Biosciences). Cells were washed and then resuspended in staining media (1× HBSS [GIBCO-BRL], 10 mM HEPES, 2% calf serum [Sigma-Aldrich]) with fluorochrome-conjugated Abs against cell surface Ags: TCRγ-PE (clone GL3; Sunnybrook Research Institute), TCRβ-PE-Cy7 (clone H57-597; eBioscience), CD19-PE-Cy7 (clone 6D5; eBioscience), CD27-biotin (clone LG.7F9; eBioscience), fixable blue viability dye (Invitrogen), and avidin-allophycocyanin-Cy7 (Invitrogen). Cells were washed and then fixed and permeabilized according to the reagent manufacturer’s instructions (BD Biosciences Cytofix/Cytoperm kit, catalog no. 554714). Cells were then stained with fluorochrome-conjugated Abs against cytokines: IFN-γ-allophycocyanin (clone XMG1.2; BD Pharmingen) and IL-17A-FITC (clone eBio17B7; eBioscience).
Cell sorting
For sorting of CD27− γδ T cells, splenic cell suspensions were depleted of non-T cells (as above) and then stained with TCRγ-PE (clone GL3; Sunnybrook Research Institute), TCRβ-FITC (clone H57-597; Sunnybrook Research Institute), CD27-biotin (clone LG.7F9; eBioscience), avidin-allophycocyanin-Cy7 (Invitrogen), and 1 μg/ml propidium iodide. Sorting was performed on a MoFlo cell sorter (Dako Cytomation). Cells were gated on propidium iodide, forward versus side scatter, and two populations were collected: FITC+ (αβ T cells) and PE+allophycocyanin-Cy7− (CD27− γδ T cells). Purity of each of these cell populations was >96%.
T cell adoptive transfers
Either total NOD splenic T cells or NOD splenic αβ T cells were purified using magnetic bead depletion, as described above. T cells (107) were resuspended in 200 μl sterile PBS and then transferred to 4- to 5-wk-old female NOD.SCID recipients by tail vein i.v. injection. For transfer of αβ T cells and CD27− γδ T cells, these sorted populations were pooled and 107 cells were resuspended in 200 μl sterile PBS and transferred to 4- 5-wk-old female NOD.SCID recipients by i.v. tail vein injection. Highly purified, live CD27− γδ T cells were present in this inoculum at their orthotopic frequency and they numbered ∼80,000–100,000 cells among the 10734), beginning on the day of T cell transfer. This mAb was raised against Escherichia coli–derived recombinant murine (rm)IL-17 residues Thr22–Ala158 (National Center for Biotechnology Information accession no. Q62386http://www.rndsystems.com/Products/mab421/). The dosage and regimen of anti–IL-17 was based on a previous publication demonstrating this regimen to be effective at controlling Ag-specific Th17 cells in the EAE model (35). Treatment was continued until recipients became diabetic or until 100 d after adoptive transfer.
Islet preparations
Islets were prepared by published methods (36) with slight modifications. Briefly, a surgical clamp was placed over the ampulla to block the bile pathway to the duodenum. Three milliliters ice-cold HBSS (GIBCO-BRL) with 1 mM CaCl2, 10 mM HEPES, and 2 mg/ml collagenase type IV (Worthington Biochemical, Lakewood, NJ; CLS-4, lot no. 40C11795) was slowly perfused into the pancreas through a needle inserted into the common bile duct. The pancreas was dissected and placed in 2 ml HBSS with 1 mM CaCl2, 10 mM HEPES, and 2 mg/ml collagenase type IV and incubated at 37°C for 10 min. The suspension was shaken to disaggregate the tissue and then allowed to digest for another 2 min at 37°C. The tissue was washed by addition of 50 ml ice-cold staining media and centrifuged at 290 × g for 2 min. The supernatant was discarded and the wash was repeated. The islets were resuspended, passed through a 70-μm sterile cell strainer (BD Falcon 35-2350), and retained on the strainer. Then, the enriched islet preparation was resuspended and hand-picked under a dissecting microscope. The purified islets were incubated at 37°C for 5 min in enzyme-free cell dissociation buffer (Invitrogen, catalog no. 13151-014). The resulting single-cell suspension was used for Ab staining and flow cytometry analysis.
Results
γδ T cells potentiate αβ T cell–mediated transfer of T1D to NOD.SCID recipients
To elucidate potential pathogenic or protective roles of γδ T cells in T1D, we tested the impact of this cell subset on adoptive transfer of T1D to an immunodeficient, T1D-protected strain. Splenic total T cells, purified αβ T cells alone, or purified γδ T cells alone were transferred into T and B cell–deficient NOD.SCID recipients. Consistent with our own data and those of others (J. Danska, personal communication; Ref. 37), transfer of 107 total splenic T cells from 12- to 14-wk-old female prediabetic NOD mice resulted in 100% T1D incidence in NOD.SCID recipients by 100 d after transfer (Fig. 1, solid black trace). In contrast, removal of γδ T cells from the inocula rendered NOD αβ T cells less potent in transferring T1D to only 50% of recipients (Fig. 1, dashed trace; p < 0.0001 relative to total T cells, n ≥ 9 recipients/group), suggesting that γδ T cells contribute to disease immunopathogenesis. Notably, splenic γδ T cells alone were unable to transfer disease (Fig 1, gray trace; n = 9 recipients), underscoring the requirement for cooperation with αβ T cells to potentiate T1D in this adoptive transfer setting.
Both αβ T and γδ T cells are required for efficient T cell–mediated T1D transfer to NOD.SCID recipients. Splenocytes were harvested from 12- to 14-wk-old NOD mice. B cells and APCs, and in some cases γδ TCR+ cells, were depleted by magnetic bead purification. Purification of γδ T cells only was performed by FACS sorting. Either 107 splenic total T cells (containing αβ T and γδ T cells), 107 splenic αβ T cells alone, or 200,000 γδ T cells alone were transferred to 4- to 5-wk-old female NOD.SCID recipients by tail vein i.v. injection. Recipients of NOD total T cells (αβ T plus γδ T cells, black solid trace) all developed T1D within 100 d. In contrast, recipients of NOD αβ T cells alone (dashed trace) were protected from T1D compared with recipients of both T cell subsets (p < 0.0001). γδ T cells alone were not able to transfer T1D in this setting (p = 0.0141 compared with NOD αβ T cells alone; no recipients diabetic). The p values are Mantel–Cox log-rank test comparisons of survival curves (n ≥ 9 recipients/group).
CD27−CD44hi γδ T cells infiltrate the islets preceding T1D
A pathogenic role for γδT cells might be reflected by their presence at the site of β cell destruction. Hence, islets of Langerhans were purified, enzymatically dissociated, and infiltrating leukocytes were assessed by flow cytometry (Fig. 2A, 2C). γδ T cells were detected in NOD islets at two time points (8 and 12 wk of age) preceding T1D onset (Fig. 2A). Interestingly, the percentage of intraislet γδ T cells within the CD19−αβ TCR− population was greater at 12 wk compared with 8 wk of age (p < 0.05; Fig. 2B). Although TCR-mediated thymic selection does not constrain the γδ TCR repertoire, it does preprogram γδ T cell effector function (5, 38). Two subsets of γδ T cells can be distinguished by expression of the TNFR family member CD27. CD27− γδ T cell progenitors mature without cognate ligand engagement into CD44hi IL-17–secreting cells, whereas γδ progenitors that bind ligand mature into CD27+ cells with lower CD44 levels and primarily secrete IFN-γ (8, 12), phenotypes that remain stable over time in the periphery (5, 12). To define the frequencies of these functionally distinct subsets in various anatomical compartments, γδ T cells were assessed for CD27 and CD44 expression by flow cytometry in spleen, multiple peripheral LNs, and in highly purified islets from prediabetic 8- and 12-wk-old NOD mice (Fig. 2C–E). At both prediabetic time points assessed, most γδ T cells in all peripheral LNs tested were CD27+CD44lo, with a much lower frequency of CD27−CD44hi γδ T cells (Fig. 2C–E). In 8-wk-old NOD mice, the splenic γδ T cell pool also had a low frequency of CD27−CD44hi compared with CD27+CD44lo cells. The relative frequencies of CD27−CD44hi and CD27+CD44lo γδ T subsets in 8-wk-old NOD mice were similar to those observed in age-matched non–autoimmune-prone CD1 strain mice (Supplemental Fig. 2). In contrast, CD27−CD44hi γδ T cells were enriched in the spleens of 12-wk-old NOD mice relative to other peripheral LNs (p < 0.01; Fig. 2C, 2E). This finding was consistent with our observations that splenic T cells from 12-wk-old donors displayed enhanced diabetogenicity relative to equal numbers of LN T cells when transferred into NOD.SCID recipients (Supplemental Table I). In both 8- and 12-wk-old NOD mice, islet-infiltrating γδ TCR+ cells included both the CD27−CD44hi and CD27+CD44lo subsets (Fig. 2C), suggesting that the effector functions of either or both cell types might contribute to islet pathology. However, we noticed that within the γδ TCR+ fraction, there was a greater frequency of CD27−CD44hi T cells in the islets than in any LN compartments examined (p < 0.01; Fig. 2D, 2E). Moreover, CD27−CD44hi γδ T cells were present at time points corresponding to the early stages of insulitis. These data showed that γδ T cells with potential to produce IL-17 accumulate in the islets prior to T1D onset and thus might contribute to pathogenesis in this model.
CD27- γδ TCR+ cells infiltrate NOD islets during T1D pathogenesis. (A) Flow cytometry–based enumeration of γδ T cells in the pancreatic islets of 8- and 12-wk-old NOD mice. Pancreata were perfused with collagenase and the islets were prepared (see Materials and Methods). Cells of interest were gated based on excluding propidium iodide (live cells), followed by forward and side scatter profiles. Stains for both αβ TCR and CD19 were included to exclude B cells and αβ T cells from subsequent analysis. Within the CD19− αβ TCR− population, an intraislet γδ TCR+ population was identified in both 8- and 12-wk-old NOD mice. Representative plots were chosen from n ≥ 5 biological replicates. (B) The frequency of γδ TCR+ cells among non–B, non–αβ T cells is shown for islet samples from 8- and 12-wk-old NOD mice. The frequency of intraislet γδ T cells is significantly higher among 12-wk-old NOD mice, compared with 8-wk-old NOD mice (p < 0.05, two-tailed nonparametric t test, n ≥ 5 biological replicates). (C) Within the γδ TCR+ population, the expression of CD27 and CD44 was used to discriminate two subsets: the CD27+CD44low (putatively IFN-γ–secreting) and the CD27−CD44hi (putatively IL-17–secreting) γδ T cells. FACS profiling of these subsets was performed in spleen, various LNs, and islets from 8- and 12-wk-old NOD mice. Representative plots were chosen from n ≥ 5 biological replicates. (D) Statistical analysis of multiple lymphoid compartments reveals that CD27−CD44hi γδ T cells were present at high frequency in the islets of 8-wk-old NOD mice, compared with all other lymphoid compartments in 8-wk-old NOD mice (p < 0.01, two-tailed nonparametric t test, n ≥ 5 biological replicates). (E) In samples from 12-wk-old NOD mice, CD27−CD44hi γδ T cells were present at elevated frequencies in the spleen and islets, compared with all LNs tested (p < 0.01, two-tailed nonparametric t test, n = 8 biological replicates).
CD27−CD44hi γδ T cells are preprogrammed to secrete IL-17
A unique feature of γδ T cells is their expression of surface markers that are highly predictive of cytokine profile upon activation. In other strains of mice, CD27− γδ T cells are preprogrammed to produce IL-17 upon activation, whereas CD27+ γδ T cells are skewed toward IFN-γ production (12, 38). To confirm that this correlation between CD27 surface expression and cytokine potential was also observed in the NOD mouse, we assessed IL-17 and IFN-γ production in γδ T cells stimulated ex vivo. Cells were activated by exposure to PMA and ionomycin in the presence of a protein transport inhibitor and then assessed for CD27, IL-17A, and IFN-γ expression by flow cytometry. Without activation, these cells displayed no evidence of basal IL-17A or IFN-γ production (Fig. 3A). In contrast, activated NOD γδ T cells from the spleen, thymus, and pooled peripheral LNs all showed appreciable IL-17A+ and IFN-γ+ populations (Fig. 3A, upper row). CD27 expression was examined on IL-17+ (blue trace) and IFN-γ+ (red trace) γδ T cells (Fig. 3A, lower row). As expected, IL-17+ γδ T cells expressed low levels of CD27, whereas IFN-γ+ γδ T cells were CD27+ (Fig. 3A, lower row). These data confirmed that similar to other strains, NOD mouse CD27−CD44hi γδ T cells are preprogrammed to secrete IL-17 upon activation, and they suggested that the CD27−CD44hi γδ T cells observed in prediabetic NOD mouse islets (Fig. 2) may contribute pathogenic effects via IL-17.
CD27− γδ T cells mediate T1D transfer via an IL-17–dependent mechanism. (A) Cells from NOD spleen, thymus, or pooled peripheral LNs were cultured in the presence of 50 ng/ml PMA and 1 μg/ml ionomycin for 5 h. Cells were stained for surface markers (γδ TCR, αβ TCR, CD19, CD44, CD27, and a fixable viability dye) and then permeabilized and stained with Abs specific for IL-17 and IFN-γ. Contour plots in the upper row show production of IL-17 and IFN-γ by γδ T cells. Histograms in the lower row show CD27 expression on either IL-17+ γδ T cells (solid gray trace) or IFN-γ+ γδ T cells (black line, clear trace). Representative plots were chosen from n = 4 biological replicates. (B) Purified splenic T cells (107), containing αβ T cells and either total γδ T cells (gray traces) or purified CD27− γδ T cells only (black traces), were transferred from 12- to 14–wk-old NOD donors into 4- to 5-wk-old NOD.SCID recipients. CD27− γδ T cells were present in the inoculum at their orthtopic frequency (see Materials and Methods). On the same day, and twice a week thereafter, NOD.SCID recipients were injected i.p. with either anti–IL-17-neutralizing Ab or a rat IgG2a isotype control Ab. Ab injection was continued for 10 wk after T cell transfer and recipients were monitored for T1D. All recipients of the isotype control Ab progressed to T1D within the 100-d observation period. Recipients of the IL-17–neutralizing Ab were protected from T1D (total γδ T cells, p = 0.0006; CD27− γδ T cells, p < 0.0001; n ≥ 10 for each condition) compared with cohorts receiving isotype control Ab. The p values represent pairwise Mantel–Cox log-rank tests of survival curves.
IL-17 neutralization attenuated adoptive transfer of T1D by CD27− γδ T cells
In an adoptive transfer setting γδ T cells significantly potentiated the diabetogenic capacity of αβ T cells (Fig. 1). To test the role of the minority CD27−CD44hi γδ T cell subset, which accumulates in islets preceding T1D (Fig. 2D, 2E), we again used an adoptive transfer approach. T cell transfers into NOD.SCID recipients were performed with NOD splenic αβ T cells from 12- to 14-wk-old donors, supplemented with either highly purified total γδ T cells or with the CD27− γδ T cell subset supplied in the cell inoculum at their orthotopic frequencies. Interestingly, CD27− γδ T cells cooperated with αβ T cells to induce T1D in 100% of recipients, similar to the observations with total γδ T cells (Fig. 3B). These data do not preclude a possible role for CD27+ γδ T cells in T1D pathogenesis, but they indicated that the CD27− γδ T cell subset was sufficient to potentiate T1D transfer by αβ T cells. Because we had shown that CD27− γδ T cells were preprogrammed to produce IL-17 following activation (Fig. 3A), we asked whether their role in adoptive transfer of T1D was IL-17 dependent. To address this question, we performed adoptive transfers of αβ T cells supplemented with either total γδ T cells or only the CD27− γδ T cell fraction into recipients that were given biweekly injections of either IL-17–neutralizing Ab or an IgG2a isotype control Ab. Ab treatment was initiated on the day of T cell transfer and continued for 100 d. Recipients of splenic αβ T cells supplemented with total γδT cells and IL-17–neutralizing Ab were dramatically protected from T1D, relative to the appropriate isotype control cohort (p = 0.0006; Fig. 3B). Similarly, recipients of splenic αβ T cells supplemented with the CD27− γδ T cell subset together with IL-17–neutralizing Ab were protected from diabetes relative to their respective isotype control group (p < 0.0001; Fig. 3B). These data demonstrated that the CD27− γδ T cell subset can cooperate with αβ T cells to efficiently transfer T1D, and that functional blockade of IL-17 ablated the pathogenic contribution of this γδ T cell subset.
Genomic characterization of the NOD.TCRδ-KO strain
T1D in the NOD mouse is a polygenic trait resulting from the collective effects of many risk loci across the genome (31, 32, 39). To test whether γδ T cells make nonredundant contributions to T1D pathogenesis in the NOD mouse, we used a strain in which γδ T cell production was blocked by a Tcrd C region mutation, NOD.129P2-Tcrdtm1Mom/J (NOD.TCRδ-KO) (40). We genotyped the NOD.TCRδ-KO strain to exclude genomic contamination by other (non-Tcrd) diabetes-risk loci to T1D-related phenotypes. Genome-wide analysis of the NOD.TCRδ-KO strain revealed that the only region of the genome showing nonidentity between NOD and NOD.TCRδ-KO mice spanned positions 36200642 to 56445855 on chromosome 14 (see http://serrate.research.sickkids.ca:8081/Tcrd_Illumina_SNP_genotyping.xls) that carries the Tcra-d locus, the site of the TCRδ-targeted mutation (32, 41). The original TCRδ-KO mutation, made in 129 strain embryonic stem cells, was bred onto a C57BL/6 (B6) background (41) and then backcrossed to NOD for >10 generations (40). Thus it was possible that chromosome 14 could harbor B6- or 129-derived alleles in addition to the 129-derived Tcra-d locus. To further refine potential non–NOD-derived intervals on chromosome 14, we identified high-density microsatellite markers using the B6 reference genome within this region and genotyped DNA prepared from NOD, 129, B6, and NOD.TCRδ-KO mice to define the non-NOD genomic interval in NOD.TCRδ-KO as positions 35433125–57367153 (markers D14Gul503–D14Gul2331; Table I). We found that this interval did not include non-NOD alleles at two previously identified insulin-dependent diabetes (Idd) risk loci on chromosome 14, Idd8 (42, 43) and Idd12 (44). Therefore, the only non–NOD-derived portion of the genome in the NOD.TCRδ-KO mice was the chromosome 14 interval containing the Tcra-d locus.
Frequency of γδT cells displayed TCRδ gene dosage effect
The frequencies of γδ and αβ T cells were measured by flow cytometry in various peripheral compartments of NOD, NOD.TCRδ-KO, and (NOD.TCRδ-KO × NOD/Jsd)F1 mice carrying one wild-type and one TCRδ-KO allele (NOD.TCRδ-HET) (Fig. 4). Interestingly, the thymic CD4−CD8− population (thymic double-negative), pooled LN, spleen, and small intestinal IEL compartments of NOD.TCRδ-HET mice showed a γδ T cell frequency intermediate between that of NOD and NOD.TCRδ-KO mice in demonstrating a gene dosage effect of the Tcrd−/− mutation (Fig. 4, Supplemental Fig. 1). This result highlights an important feature of the γδ T cell lineages, where gene dosage determines the peripheral frequency of the γδ T cells.
NOD.TCRδ-HET mice had half the frequency of γδ T cells compared with wild-type NOD mice. Multicolor flow cytometry was performed to define frequencies of γδ T cells in CD4−CD8− thymocytes (thymic DN), pooled peripheral LNs, spleen, and small intestinal IELs. NOD.TCRδ-HET mice displayed γδ TCR+ cell frequencies intermediate between parental NOD and NOD.TCRδ-KO mice. Representative plots are from n = 3 per genotype.
TCRδ gene dosage correlates with T1D pathogenesis
Next, the impact of Tcrd gene dosage on the natural history of T1D was assessed by prospective longitudinal analysis of NOD, NOD.TCRδ-HET, and NOD.TCRδ-KO mouse cohorts during 250 d. As expected, NOD mice reached a high cumulative T1D incidence. In contrast, NOD.TCRδ-KO mice were dramatically protected from disease (p < 0.0001; Fig. 5A). Interestingly, NOD.TCRδ-HET mice showed an intermediate phenotype, more susceptible to T1D than NOD.TCRδ-KO (p = 0.0118) but protected relative to parental NOD mice (p = 0.0296; Fig. 5A). To asses potential consequences of genetic γδ T cell ablation on αβ T cell diabetogenicity, we transferred αβ T cells from NOD or NOD.TCRδ-KO mice to NOD.SCID recipients and found no evidence for impaired αβ T cell–mediated T1D transfer (Supplemental Table II). These data demonstrated a gene dose-dependent, nonredundant pathogenic role for γδ T cells in the NOD model of T1D. Work from our laboratory and from others has established that some Idd loci control distinct stages of insulitis progression in NOD mice (32, 45, 46). To identify the stage of insulitis most impacted by γδ T cells, longitudinal assessment of insulitis was performed in cohorts of nondiabetic NOD and NOD.TCRδ-KO mice at 80, 105, 120, and 240 d of age (Fig. 5B). NOD mice showed progressively more severe islet infiltration during this period (Fig. 5B). In contrast, insulitis severity in NOD.TCRδ-KO mice remained mild and stable after 80 d of age (Fig. 5B), and the mice were protected from severe insulitis at 120 and 240 d, relative to NOD mice (p < 0.05). Thus, the absence of γδ T cells attenuated progression to invasive insulitis, consistent with the idea that γδ T cells function as effectors in this disease model.
Gene dosage at the TCRδ locus determines T1D susceptibility. (A) Longitudinal assessment of T1D was performed in cohorts of NOD, NOD.TCRδ-HET, and NOD.TCRδ-KO mice. In contrast to NOD females, NOD.TCRδ-KO mice were protected from diabetes (p < 0.0001), and NOD.TCRδ-HET mice displayed an intermediate phenotype different from both parental NOD (p = 0.0296) and NOD.TCRδ-KO (p = 0.0118) animals. The p values represent pairwise Mantel–Cox log-rank tests of survival curves (n > 22 per genotype). (B) Insulitis severity was assessed in the pancreata of nondiabetic female NOD and NOD.TCRδ-KO mice at the ages indicated (see Materials and Methods). NOD mice showed a progressive increase in insulitis severity from ages 80 to 240 d. In contrast, NOD.TCRδ-KO mice showed a low and constant level of insulitis throughout this time frame. Graph depicts mean and SEM (seven or more biological replicates per condition). *p < 0.05, two-tailed t test comparing NOD to NOD.TCRδ-KO within the indicated time point.
Discussion
In this study we provide evidence that γδ T cells are a key component of T1D pathogenesis in the NOD mouse model. The CD27−CD44hi phenotype of the γδ T cells defines those that make IL-17 upon activation. We identified CD27−CD44hi γδ T cells infiltrating the islets of prediabetic NOD mice, suggesting that they may participate directly or indirectly in islet destruction. Moreover, in an adoptive transfer model, we showed that CD27−CD44hi γδ T cells cooperate with αβ T cells to induce T1D, and that the pathogenic role of this IL-17–producing γδ T subset can be blocked by in vivo neutralization of IL-17. Using NOD.TCRδ-KO and NOD.TCRδ-HET mice, we show that the TcRδ locus displays a gene dosage effect in terms of the frequency of γδ T cells in thymic and peripheral lymphoid compartments. Finally, frequency of γδ T cells determines differential susceptibility of these NOD-background mice to spontaneous T1D.
Previous studies have considered the possible roles of γδ T cells and γδ T cell–produced IL-17 in the NOD mouse model of T1D. For example, IL-17–producing γδ T cells were reported to play a protective role in T1D by secreting TGF-β in an adoptive transfer setting (28). However, neither the CD27/CD44 surface phenotype nor IL-17 secreting capacity of the transferred γδ T cells was reported (28), and the splenocytes were isolated from young NOD mice at an age where we saw predominance of the CD27+CD44lo, IFN-γ–secreting subset (Figs. 2D, 3A). IL-17 blockade in the adoptive transfer setting was reported to have no effect on γδ T cell–dependent diabetes pathogenesis, but anti–IL-17 Ab treatment was given only during the first period of observation (28) and therefore differed from the approach presented in this study. In the present study, IL-17 action was opposed over the full-time course in which T1D development was scored, based on prior evidence that IL-17–producing cells participate in the effector phase rather than the initiation phase of T1D (34).
Another recent report described no effect on T1D among NOD mice in which lentiviral transgenesis was used to reduce IL-17 production (47). Although it was convincingly demonstrated that purified αβ TCR+CD4+ Th17 cells from these mice had reduced IL-17 production upon stimulation, IL-17 production was not reported for γδ T cells (47). Evidence from multiple studies suggests that Th17 αβ T cells and CD27−CD44hi γδ T cells produce IL-17 by different mechanisms, with different kinetics, and in response to distinct stimuli, and, additionally, γδ T cells are committed to their cytokine transcriptional program upon thymic egress (5) (reviewed in Ref. 48). It would be interesting to test how reduced IL-17 production by CD27−CD44hi γδ T cells impacts their ability to potentiate T1D. Finally, evidence for a tolerogenic role for γδ TCR+ IELs has been reported in the NOD mouse model (26). When stimulated, γδ TCR+ IELs secrete IFN-γ but not IL-17 (reviewed in Ref. 49), and thus the finding that IFN-γ–producing γδ T IELs are tolerogenic is compatible with the data presented in the present study that autoimmune pathogenesis is mediated by the IL-17–producing γδ T cell subset. Moreover, our demonstration that genetic γδ T cell deficiency strongly protects NOD mice from T1D reveals the dominant diabetogenic effect of γδ T cells in this model.
IL-17 has been implicated as an effector of other mouse models of autoimmune disease (reviewed in Refs. 9, 50), and treatment with exogenous IL-17 can induce autoimmunity in otherwise disease-resistant mice. For example, infection of B6 mice with an adenoviral IL-17A expression construct induced a model of Sjörgren’s syndrome (51). Injection of IL-17 into the joints induced proinflammatory TNF-α and IL-1β secretion, promoting rheumatoid arthritis (52). IL-17 produced by skin γδ T cells was shown to mediate epidermal inflammation in a model of psoriasis, which could be ameliorated by genetic deletion of Tcrd or Il17ra (53). Similarly, γδ T cell depletion or IL-17 neutralization ameliorated lung injury in a model of acute granulomatous disease (54).
Whereas TCRαβ+ Th17 cells primarily reside in the intestine (55), γδ cells are a major IL-17 source in multiple tissues, particularly during infection (56–58). In contrast to conventional αβ T cells, γδ TCRs recognize currently poorly defined ligands that do not require processing and presentation by specialized APCs. They can produce IL-17 without TCR ligation (reviewed in Ref. 59) and far more rapidly than TCRαβ+ Th17 cells (20) owing to developmental preprogramming of this function (8, 12). Interestingly, distinct from TCRαβ+ Th17 cells, activation of CD27− γδ T cells does not require cognate ligand engagement (60). CD27− γδ T cells respond poorly to TCR agonists under conditions that promote robust activation of the CD27+ γδ T cell subset and can respond to IL-23 without TCR engagement (12, 20). These unique characteristics of CD27− γδ T cells more closely resemble innate lymphocytes. Thus, in the NOD mouse model, the pathogenic role of CD27− γδ T cells may not require recognition of cognate γδ TCR ligand, but instead depend on amplification of the Ag-specific αβ T cell response in response to local cytokines.
One defined γδ TCR ligand shown to regulate autoimmunity in the NOD mouse is the activating receptor NKG2D, expressed in the pancreatic islets of prediabetic animals. Moreover, mAb blockade of NKG2D can prevent progression to overt T1D in this model (61). Interestingly, NKG2D is highly expressed by IL-17–producing γδ T cells (A. Hayday, unpublished observations). Upregulated expression of self-encoded “stress-ligands” for NKG2D can activate some murine γδ T cells in vivo and human γδ T cells in vitro (62–64). Such ligands might be evoked by macrophage or αβ T cell–mediated islet cell damage in NOD mice. Another study generated hybridomas from the NOD mouse spleen and pancreatic LNs that included γδ TCR+ clones responsive to purified islets and to a processed insulin peptide (29). These clones used diverse TCR γ- and δ-chain sequences, indicative of a polyclonal population, but the cytokine expression or diabetogenic potential of these islet-reactive γδ T cells was not reported. Further work will be required to resolve the relative contributions of γδ T cells activated by TCR engagement and those activated by TCR-independent signals in T1D development in the NOD mouse.
Human IL-17–producing γδ T cells have been difficult to isolate and characterize, owing to their low frequency in peripheral blood. However, recent work has shown that both murine and human CD27− γδ T cells express high levels of IL-7R and can be expanded in vitro with IL-7, allowing for examination of their functional capacity (65). This recent study also clarifies the link between CD27 expression on human γδ T cells and their cytokine potential. Specifically, 15–40% of CD27− γδ T cells isolated from cord blood and expanded with IL-7 produced IL-17 following PMA/ionomycin stimulation (65). Moreover, IL-17–producing γδ T cells were enriched in the skin lesions of psoriasis patients (53, 66). Taken together, these studies suggest that the correlation between CD27 expression, cytokine program, and contribution to inflammatory pathology is conserved between NOD mice and humans. A CD4−CD8− double negative IL-17–secreting T cell population, potentially including γδ T cells, is present in the blood and kidneys of patients with systemic lupus erythematosus (67). γδ T cell accumulations have also been reported in brain lesions and in the cerebrospinal fluid of MS patients (19, 68). Future studies are warranted to determine whether γδ Τ cells and their cytokine products contribute to the pathogenesis of human autoimmune diseases.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We acknowledge significant contributions of Robert Tigelaar and Julie Lewis (Yale University) to the early derivation of TCRδ-deficient NOD mouse strain and of Marie-Laure Michel (London Research Institute–Cancer Research UK) to data review. We thank Omid Gulban (The Hospital for Sick Children) for bioinformatics analysis and Andrei Malko (The Hospital for Sick Children) for technical assistance.
Footnotes
L.G. and A.H. began the introgression of the TCRδ deletion onto the NOD background and initiated husbandry and analysis of the mice; A.H. provided mice to J.G.M.M. and J.S.D. who performed rigorous backcrossing and genomic analyses; J.G.M.M., A.S.L.W., and S.M.-T. performed experiments and analyzed data; J.G.M.M. and J.S.D. designed experiments, analyzed data, and wrote the manuscript; and A.H. reviewed data and experimental design and edited the manuscript.
J.G.M.M. was supported by a Canadian Institutes of Health Research doctoral scholarship. A.H. acknowledges funding from the Wellcome Trust. This work was supported by grants to J.S.D. from the Canadian Institutes of Health Research and from Genome Canada with funds administered by the Ontario Genomics Institute.
The online version of this article contains supplemental material.
- Abbreviations used in this article:
- EAE
- experimental autoimmune encephalomyelitis
- Idd
- insulin-dependent diabetes
- IEL
- intraepithelial lymphocyte
- iNKT
- invariant NKT
- LN
- lymph node
- MAIT
- mucosal-associated invariant T
- MS
- multiple sclerosis
- rm
- recombinant murine
- T1D
- type 1 diabetes.
- Received January 7, 2013.
- Accepted March 28, 2013.
- Copyright © 2013 by The American Association of Immunologists, Inc.
References
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