Myeloperoxidase (MPO) anti-neutrophil cytoplasmic Ab (ANCA)–associated vasculitis results from autoimmunity to MPO. IL-17A plays a critical role in generating this form of autoimmune injury but its cell of origin is uncertain. We addressed the hypothesis that IL-17A–producing γδ T cells are a nonredundant requisite in the development of MPO autoimmunity and glomerulonephritis (GN). We studied MPO-ANCA GN in wild type, αβ, or γδ T cell–deficient (C57BL/6, βTCR−/−, and δTCR−/− respectively) mice. Both T cell populations played important roles in the generation of autoimmunity to MPO and GN. Humoral autoimmunity was dependent on intact αβ T cells but was unaffected by γδ T cell deletion. Following MPO immunization, activated γδ T cells migrate to draining lymph nodes. Studies in δTCR−/− and transfer of γδ T cells to δTCR−/− mice show that γδ T cells facilitate the generation of anti-MPO autoimmunity and GN. δTCR−/− mice that received IL-17A−/− γδ T cells demonstrate that the development of anti-MPO autoimmunity and GN are dependent on γδ T cell IL-17A production. Finally, transfer of anti-MPO CD4+ T cell clones to naive δTCR−/− and wild type mice with planted glomerular MPO shows that γδ T cells are also necessary for recruitment of anti-MPO αβ CD4+ effector T cells. This study demonstrates that IL-17A produced by γδ T cells plays a critical role in the pathogenesis of MPO-ANCA GN by promoting the development of MPO-specific αβ T cells.

Anti-neutrophil cytoplasmic Ab (ANCA)–associated vasculitis (AAV) is a major cause of crescentic glomerulonephritis (GN) (1). It is now accepted that this is an autoimmune disease driven by nephritic immunity targeting the major neutrophil lysosomal enzymes, lysosome-associated membrane protein-2, proteinase 3, and myeloperoxidase (MPO). Much of our understanding of the immunopathogenesis of this disease comes from studies of murine models. These are mainly models of MPO-AAV and GN (24). Cytokines help drive and mediate this disease and a number of studies suggest an important role in disease pathogenesis for the cytokine IL-17A, produced by the Th17 subset of anti-MPO CD4+ T cells. The evidence comes from studies in IL-17A–deficient mice (5) or the use of neutralizing Abs (6). However, IL-17A expression is not limited to the adaptive immune system, and γδ T cells with a primary role in innate host defense are a major source of IL-17 (7, 8).

γδ T cells are potential contributors to AAV and GN. Recent evidence suggests that these cells can facilitate the induction of adaptive autoimmunity in several models of human diseases: autoimmune uveitis (9), colitis (10), psoriasis (11), and collagen arthritis (12). In addition, γδ T cells in the kidney can amplify immune injury in a model of anti–glomerular basement membrane (GBM) GN (13). γδ T cells are widely distributed in many tissues, particularly barrier regions, where they act as sentinels and first responders after microbial invasion (14). There is also a small resident population of γδ T cells in the kidney (13). γδ T cells have been shown to play an important role in the recruitment of neutrophils, mediated in part by the rapid production of IL-17A (15), a function that is potentially relevant in a disease where neutrophils are the major target of the underlying autoimmunity.

To address the role of γδ T cells, we used a murine model of MPO-ANCA GN. We studied disease in both β-chain TCR–deficient (βTCR−/−) and δ-chain TCR–deficient (δTCR−/−) mice. Both T cell populations were found to play nonredundant roles in the generation of autoimmunity to MPO and its associated GN. In addition, γδ T cells are required for the induction of αβ T cell anti-MPO autoimmunity by their production of IL-17A in secondary lymphoid organs. Furthermore, in proof-of-concept experiments via transferred nephritogenic αβ CD4+ T cell clones, we found that γδ T cells help recruit MPO-specific effector T cells. These experiments show that ANCA production is αβ T cell dependent and that anti-MPO αβ T cell autoimmunity and GN is γδ T cell (and IL-17A–producing γδ T cell) dependent.

C57BL/6 (wild type, WT), δTCR−/−, βTCR−/−, IL-17A−/− male mice (n = 6–10 per group) were used in the experiments. All genetically modified mice have been backcrossed to a C57BL/6 background and bred at Monash Medical Centre Animal Facility, Monash University, Australia. All mice were housed in specific pathogen-free conditions at Monash Medical Centre, and studies were approved by Monash University Animal Ethics Committee in accordance with the Australian National Health and Medical Research Council animal experimentation guidelines.

Experimental autoimmune MPO-ANCA GN model.

WT mice were immunized i.p. with 20 μg murine MPO in Freund’s complete adjuvant (FCA) (Sigma-Aldrich) and boosted s.c. with 10 μg murine MPO in Freund’s incomplete adjuvant (Sigma-Aldrich) on day 7. Murine MPO was purified from differentiated 32Dcl3 cells as described previously (16). Disease was initiated (triggered) by i.v. injection of 1.5 mg sheep anti-mouse GBM globulin (17, 18) consecutively on days 16 and 17. MPO autoimmunity and GN were assessed 3 d later (day 20).

γδ T cell transfer.

γδ T cells were isolated from spleen and lymph nodes (LN) from 6–8 wk old WT or IL-17A−/− mice using the magnetic bead isolation kits from Miltenyi Biotec (TCRγ/δ+ T cell Isolation Kit, 130-092-125; Bergisch Gladbach, Germany). δTCR−/− mice were reconstituted with 5 × 105 (>90% pure, CD3+γδTCR+ by FACS) γδ T cells i.v. and experimental autoimmune MPO-ANCA GN was induced 2 d following reconstitution.

Transfer of CD4+ T cell clones.

MPO CD4+ T cell clones were generated as described in detail in Ref. 19. To summarize, WT mice were immunized with 10 μg MPO409–428 (Mimotopes, Australia) in FCA, and draining LN were harvested 10 d later. LNs were cultured with 10 μg/ml MPO409–428 in supplemented RPMI 1640 media (10% FCS, 2 mM l-glutamine, 2-ME, 100 U/ml penicillin and 0.1 mg/ml streptomycin; Sigma-Aldrich). Well-isolated colonies of proliferating cells were identified and isolated by light microscopy and expanded together with erythrocyte-lysed MACS CD4+–depleted, mitomycin C–treated splenocytes (5 × 106 cells per ml with 50 μg/ml mitomycin C–treated splenocytes in PBS for 30 min at 37°C), 50 U/ml recombinant mouse IL-2 (eBioscience), and 50 μg/ml MPO409–428. Viable clones were expanded in 24-well plates with weekly media changes and fortnightly divisions. These MPO CD4+ T cell clones used expressed Vα3 and Vβ14, and secreted IFN-γ. As a control, OVA323–339 OT-II CD4+ T cell clones were expanded in vitro using the same method.

C57BL/6 (WT) and δTCR−/− mice received 2.5 × 107 MPO409–428 or OVA323–339 specific T cell clones. MPO was deposited in the glomeruli the following day using 3 mg sheep anti-mouse GBM Ab. Mice were humanely killed 4 d later to look at MPO-specific glomerular leukocyte effectors.

All data were analyzed with Graph Pad Version 6 (GraphPad Prism; GraphPad Software, San Diego, CA). Results are expressed as the mean ± SEM. Differences were considered to be statistically significant if p < 0.05. An unpaired t-test was used when comparing two groups and a one-way ANOVA test for experiments comparing more than two groups.

Urine was collected by housing mice in individual metabolic cages over the final 24 h of the experiment. Albuminuria was assessed by ELISA (Bethyl Laboratories, Montgomery, TX) and expressed as micrograms per 24 h.

Histological assessment of renal injury was performed on 3-μm-thick, formalin fixed, paraffin embedded, periodic acid–Schiff (PAS)–stained kidney sections. A minimum of 30 consecutive glomeruli per mouse were examined and results expressed as the percentage of segmental glomerular necrosis per glomerular cross-section. Glomerular segmental necrosis was defined by glomeruli with a proportion(s) of the glomerular area having loss of normal capillary loops replaced by PAS-positive extracellular matrix with only scattered nuclear remnants. Glomerular CD4+ T cells, macrophages (Mϕ), and neutrophils were assessed by an immunoperoxidase-staining technique on 6-μm-thick, periodate lysine paraformaldehyde fixed, OCT frozen kidney sections. The primary Abs used were GK1.5 for CD4+ T cells (anti-mouse CD4+; American Type Culture Collection, Manassas, VA), FA/11 for Mϕ (anti-mouse CD68 from Dr. G. L. Koch, Cambridge, U.K.), and RB6-8CS for neutrophils (anti–GR-1; DNAX, Palo Alto, CA). A minimum of 30 glomeruli were assessed and results expressed as cells per glomerular cross-section.

ELISA was used to detect circulating serum anti-MPO IgG titers using 100 μl per well, 1 μg/ml murine MPO, and HRP-conjugated sheep anti-mouse IgG (1:1000; Amersham Biosciences, Rydalmere, Australia). To assess MPO-specific dermal delayed-type hypersensitivity (DTH), mice were challenged by intradermal injection of 10 μg murine MPO in 30 μl saline in the right hind footpad (the contralateral footpad received saline). DTH was quantified 24 h later by measuring the difference between footpad thickness (∆mm) using a micrometer. MPO-specific cell proliferation was measured by culturing draining LN cells from MPO immunization sites at 5 × 105 cells per well in 96-well flat-bottom plates (Sarstedt, Newton, NC), restimulated with 10 μg/ml MPO, and incubated for 72 h. During the last 16 h of culture, 0.5 μCi of [3H]thymidine (Perkin Elmer, Waltham, MA) was added. [3H]Thymidine incorporation was measured as previously described (20). IFN-γ and IL-17A production was assessed by ELISPOT (Mouse IFN-γ ELISPOT kit and Mouse IL-17A ELISPOT kit; BD Biosciences) with draining LN cells seeded at 5 × 105 cells per well restimulated with 10 μg/ml of recombinant MPO for 18 h. IFN-γ– and IL-17A–producing cells were enumerated with an automated ELISPOT reader system.

γδ T cells, αβ T cells, IL-17A, and IFN-γ.

Isolated LN draining MPO immunization sites were seeded at 5 × 105 cells per well (96-well, clear, round-bottom microplates; Falcon) with recombinant MPO. After 18 h, 10 μg/ml Brefeldin A (Sigma-Aldrich) was added for a further 6 h. LN cells were first labeled with anti-mouse TCRδ APC (GL3; eBioscience), anti-mouse TCRβ Pacific Blue (H57-597; BioLegend, San Diego, CA) for 30 min at 4°C, and then fixed and permeabilized with fixation/permeabilization buffer as per the manufacturer’s protocol (eBioscience) before labeling with anti-mouse IL-17A (17b7, FITC; eBioscience) and anti-mouse IFN-γ (XMG1.2, PE; BD Biosciences). TCRδ+ cells were gated off the CD3+ gate after gating on lymphocytes on side versus forward scatter. IL-17A+ or IFN-γ+ gates were determined off the TCRδ or TCRβ gate. Voltages were optimized by single-color controls and gates drawn based on fluorescence minus one controls.

Dendritic cell phenotype.

Dendritic cells (DCs) were identified as CD11chi cells on isolated draining LN cells as measured by flow cytometry. LN cells were stained for 30 min at 4°C with the following directly conjugated Ab hamster anti-mouse CD11c PE (HL3). For the detection of apoptotic cells, isolated LN cells were resuspended in 100 μl of Annexin-V labeling solution (Roche, Mannheim, Germany), which contained 10 μg/ml of propidium iodine, and incubated for 15 min at room temperature.

Phenotyping of tissue-infiltrating γδ T cells.

The expression of common Vγ or Vδ chains may be used to classify γδ T cells into subsets. We therefore used fluorochrome-conjugated monoclonal Abs to compare the phenotypic profiles of γδ T cells isolated from draining LNs and kidneys. Cells were incubated with a Live/Dead fixable Near IR Dead Cell stain (Life Technologies) and then stained with: anti-mouse CD3 PerCP/Cy5.5 (17A2; BioLegend); anti-mouse δTCR APC and FITC (GL3; eBioscience and BD Pharmingen respectively); anti-mouse Vγ1 APC (2.11; BioLegend); anti-mouse Vγ2 [Garman’s system: UC3-10A6; BioLegend (equivalent to Vγ4 designation of Heilig and Tonegawa system)]; anti-mouse Vγ3 BV510 [Garman’s system: 536; BD Biosciences (Heilig and Tonegawa: Vγ5 equivalent)]; anti-mouse Vδ4 PE (GL2; BioLegend); and anti-mouse Vδ6 BV421 (8F4H7B7; BD Biosciences). Lymphocytes were gated on forward versus side scatter and live cells gated using the Live/Dead fixable Near IR Dead Cell dye (Life Technologies). TCRδ+ expression was gated from the live CD3+ population. TCR surface expression of Vγ or Vδ is then determined from this gated population. Voltages were optimized by single-color controls and gates drawn based on fluorescence minus one controls.

Cells were analyzed on the FACSCanto II machine using FACSDiva software (BD Biosciences) and data analyzed using FlowJo (Tree Star, Palo Alto, CA).

Kidneys were digested with 5 mg/ml collagenase D (Roche Diagnostics, Indianapolis, IN) and 100 μg/ml DNase I (Roche Diagnostics) in HBSS (Sigma-Aldrich) for 30 min at 37°C. Cells were filtered using a 40 μm cell strainer (BD Biosciences), erythrocytes lysed, and CD45+ leukocytes isolated using MACs mouse CD45 microbeads (Miltenyi Biotec, Auburn, CA) (21).

C57BL/6 (WT) mice are susceptible to the development of MPO autoimmunity following s.c. immunization with murine MPO in FCA. GN can be induced in mice with established MPO autoimmunity when MPO is planted in the kidney using a subnephritogenic dose of anti-GBM globulin, resulting in an initial wave of glomerular neutrophil influx within the first 2–4 h and subsequent deposition of MPO. The requirement for αβ T cells in the generation of MPO autoimmunity mediating GN was assessed by comparing autoimmunity and GN between βTCR−/− and βTCR intact WT mice. WT mice developed titers of MPO-ANCA IgG but MPO-ANCA were undetectable in βTCR−/− mice (Fig. 1A). MPO-specific T cell autoimmunity was detected in βTCR intact WT mice by dermal DTH recall (MPO-induced footpad swelling, Fig. 1B), MPO-specific [3H]thymidine proliferation (Fig. 1C), IFN-γ– (Fig. 1D) and IL-17A– (Fig. 1E) producing cells from draining LN. The MPO autoimmunity induced in βTCR intact WT mice was capable of inducing significant GN characterized by significant albuminuria (Fig. 1F), prominent glomerular segmental necrosis (Fig. 1G), and the accumulation of effector leukocytes including CD4+ T cells, Mϕ, and neutrophils (polymorphonuclear leukocytes, PMN) (Fig. 1H). MPO-immunized βTCR−/− mice had a significant reduction in all of these parameters of glomerular disease. Taken together, these results suggest that humoral and cell-mediated anti-MPO autoimmunity are αβ T cell dependent.

FIGURE 1.

αβ T cells are vital in the pathogenesis of MPO-ANCA GN. (A) Serum MPO-ANCA IgG was significantly enhanced in MPO-immunized WT mice (C57BL/6, n = 7) compared with βTCR−/− mice (n = 6). (B) Systemic MPO-induced DTH footpad swelling was significantly reduced in mice deficient in αβ T cells. (C) Ex vivo cultures of LN cells draining MPO immunization sites restimulated with MPO resulted in reduced [3H]thymidine proliferation, and (D and E) decreased numbers of IFN-γ– and IL-17A–producing cells. (F) Albuminuria was attenuated in βTCR−/− mice as was (G) the proportion of glomeruli with segmental necrosis compared with WT mice. PAS stain micrographs were taken at original magnification ×400 (scale bars, 10 μm). Arrows highlight the section of glomeruli with necrosis. (H) Glomerular CD4+ T cells were not observed in βTCR−/− mice whereas WT mice with induced MPO-ANCA GN had significantly increased numbers of CD4+ T cells, Mϕs, and neutrophils. Error bars represent mean ± SEM with analysis by unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 1.

αβ T cells are vital in the pathogenesis of MPO-ANCA GN. (A) Serum MPO-ANCA IgG was significantly enhanced in MPO-immunized WT mice (C57BL/6, n = 7) compared with βTCR−/− mice (n = 6). (B) Systemic MPO-induced DTH footpad swelling was significantly reduced in mice deficient in αβ T cells. (C) Ex vivo cultures of LN cells draining MPO immunization sites restimulated with MPO resulted in reduced [3H]thymidine proliferation, and (D and E) decreased numbers of IFN-γ– and IL-17A–producing cells. (F) Albuminuria was attenuated in βTCR−/− mice as was (G) the proportion of glomeruli with segmental necrosis compared with WT mice. PAS stain micrographs were taken at original magnification ×400 (scale bars, 10 μm). Arrows highlight the section of glomeruli with necrosis. (H) Glomerular CD4+ T cells were not observed in βTCR−/− mice whereas WT mice with induced MPO-ANCA GN had significantly increased numbers of CD4+ T cells, Mϕs, and neutrophils. Error bars represent mean ± SEM with analysis by unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001.

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In WT mice with MPO-ANCA GN, there is a significant expansion of the proportion of CD3+δTCR+ cells in both draining LN and in the kidney compared with naive WT mice (Ctrl) (Supplemental Fig. 1A, 1B). These results suggested that γδ T cells may participate in the generation of anti-MPO autoimmunity and GN. We therefore compared the induction of autoimmune MPO-ANCA GN in mice deficient in γδ T cells (δTCR−/−) with δTCR-intact WT mice. Just as γδ T cells in the absence of αβ T cells are insufficient to induce autoimmune MPO-ANCA GN (as observed in βTCR−/− mice), we found that in the absence of γδ T cells, αβ T cells are not sufficient to mediate the full expression of disease in this model.

MPO-immunized δTCR−/− mice had no deficiency in humoral autoimmunity to MPO as MPO-ANCA autoantibody titers were the same as those observed in WT mice (Fig. 2A). However, δTCR−/− immunized mice were highly resistant to the development of systemic anti-MPO T cell autoimmunity. δTCR−/− mice had diminished dermal footpad DTH swelling after MPO challenge (Fig. 2B). Furthermore, MPO-specific restimulation of lymphocytes from draining LNs of δTCR−/− mice had fewer IFN-γ– and IL-17A–producing LN cells (Fig. 2C, 2D), and a trend toward less cellular proliferation (Fig. 2E) compared with WT mice. Glomerular effector leukocyte accumulation of CD4+ T cells, Mϕ, and PMNs were all similarly reduced in δTCR−/− mice (Fig. 2F). These reductions were associated with attenuated structural (Fig. 2G) and functional glomerular injury (Fig. 2H).

FIGURE 2.

γδ T cells are pathogenic in MPO-ANCA GN. (A) Serum MPO-ANCA IgG titers between WT (n = 8) and δTCR−/− (n = 7) mice did not differ. (B) δTCR−/− mice did not develop MPO-specific DTH footpad swelling compared with WT mice. (CE) Ex vivo MPO stimulated draining LN cells had significantly fewer IFN-γ– and IL-17A–producing cells, and was associated with a reduction in [3H]thymidine cellular proliferation in δTCR−/− mice. (F) Glomerular leukocyte accumulation of CD4+ T cells, Mϕs, and neutrophils was diminished in δTCR−/− mice. (G) PAS-stained kidney sections demonstrated that compared with WT mice, δTCR−/− mice had fewer glomeruli affected by segmental necrosis (arrow highlights the section of glomeruli with necrosis). Original magnification ×400 (scale bars, 10 μm). (H) Albuminuria was attenuated in δTCR−/− mice. Error bars represent mean ± SEM with analysis by unpaired t test. The experiment was repeated three times with similar results. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

γδ T cells are pathogenic in MPO-ANCA GN. (A) Serum MPO-ANCA IgG titers between WT (n = 8) and δTCR−/− (n = 7) mice did not differ. (B) δTCR−/− mice did not develop MPO-specific DTH footpad swelling compared with WT mice. (CE) Ex vivo MPO stimulated draining LN cells had significantly fewer IFN-γ– and IL-17A–producing cells, and was associated with a reduction in [3H]thymidine cellular proliferation in δTCR−/− mice. (F) Glomerular leukocyte accumulation of CD4+ T cells, Mϕs, and neutrophils was diminished in δTCR−/− mice. (G) PAS-stained kidney sections demonstrated that compared with WT mice, δTCR−/− mice had fewer glomeruli affected by segmental necrosis (arrow highlights the section of glomeruli with necrosis). Original magnification ×400 (scale bars, 10 μm). (H) Albuminuria was attenuated in δTCR−/− mice. Error bars represent mean ± SEM with analysis by unpaired t test. The experiment was repeated three times with similar results. *p < 0.05, **p < 0.01, ***p < 0.001.

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Using the Heilig and Tonegawa system of γδ T cell subset designation (22), we examined particular Vγ and Vδ usage in draining LNs and kidneys from MPO-ANCA GN mice that have been previously published to be important in other autoimmune and inflammatory models of human diseases (8, 12, 13). We noted site-specific differences in γδ T cell subset dominance with Vγ1- and Vγ4-bearing γδ T cell subsets dominant in draining LNs, whereas only Vδ6-bearing γδ T cells were primarily found in GN kidneys (Supplemental Fig. 1C). Furthermore, in disease, we found that only Vγ1 γδ T cell subset had expanded in both the draining LNs and kidneys compared with naive mice (Supplemental Figure 1C).

A standard method of assessing the role of γδ T cells is to adoptively transfer γδ T cells to δTCR−/− mice (i.e., reconstitution: WT γδ T cells→ δTCR−/− mice) 2 d prior to initiating disease. To demonstrate that WT γδ T cells persisted in δTCR−/− recipients, we transferred 5 × 105 WT γδ T cells 2 d prior to MPO immunization and mice were killed after a further 10 d, once they have developed strong anti-MPO T cell responses. At this timepoint, there was clear evidence for the persistence of transferred WT γδ T cells used to reconstitute δTCR−/− recipients (Supplemental Fig. 2A). The reconstitution of γδ T cells in δTCR−/− mice restores their capacity to develop anti-MPO autoimmunity and GN. Disease indices were significantly greater than those in a cohort of δTCR−/− mice (which received no cells) in which autoimmune MPO-ANCA GN was induced (albuminuria and glomerular segmental necrosis; Fig. 3A, 3B). Glomerular CD4+ T cell influx was significantly increased in δTCR−/− mice that received WT γδ T cells (Fig. 3C). Although there was a trend toward increased glomerular neutrophil and Mϕ influx, this did not reach statistical significance (Fig. 3D, 3E). Compared with δTCR−/− mice, transfer of γδ T cells to δTCR−/− mice had enhanced dermal MPO recall responses (Fig. 3F).

FIGURE 3.

δTCR−/− mice receiving WT γδ T cells (WT γδ T cells→δTCR−/−) restored the capacity to develop anti-MPO autoimmunity and GN. (A and B) Functional and structural renal injury was significantly augmented in WT γδ T cells→δTCR−/− (n = 4) compared with δTCR−/− (n = 7). (C) Increased numbers of glomerular CD4+ T cells were observed in WT γδ T cells→δTCR−/−. (D and E) No difference in glomerular Mϕ and neutrophil infiltration was observed between WT γδ T cells→δTCR−/− and δTCR−/− mice. (F) Systemic MPO autoimmunity measured by MPO-specific DTH footpad swelling was significantly elevated in WT γδ T cells→δTCR−/− compared with δTCR−/− mice. Error bars represent mean ± SEM with statistical analysis by unpaired t test. *p < 0.05, **p < 0.01.

FIGURE 3.

δTCR−/− mice receiving WT γδ T cells (WT γδ T cells→δTCR−/−) restored the capacity to develop anti-MPO autoimmunity and GN. (A and B) Functional and structural renal injury was significantly augmented in WT γδ T cells→δTCR−/− (n = 4) compared with δTCR−/− (n = 7). (C) Increased numbers of glomerular CD4+ T cells were observed in WT γδ T cells→δTCR−/−. (D and E) No difference in glomerular Mϕ and neutrophil infiltration was observed between WT γδ T cells→δTCR−/− and δTCR−/− mice. (F) Systemic MPO autoimmunity measured by MPO-specific DTH footpad swelling was significantly elevated in WT γδ T cells→δTCR−/− compared with δTCR−/− mice. Error bars represent mean ± SEM with statistical analysis by unpaired t test. *p < 0.05, **p < 0.01.

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γδ T cells have been linked to the initiation and enhancement of DC generation of autoimmunity (23). To assess the basis of γδ T cell help for anti-MPO αβ T cell generation of anti-MPO autoimmunity and its associated GN, we sought evidence for γδ T cell-mediated enhancement of anti-MPO autoimmunity in draining LN. We found that by 18 h there was an early increase in the number of CD3+ δTCR+ cells in MPO-immunized WT mice (Fig. 4A). In the absence of γδ T cells, there were fewer CD11c+ DCs (Fig. 4B) with more apoptotic DCs (CD11c+Annexin V+, Fig. 4C) compared with WT mice. The increase in CD3+δTCR+ cells in LNs was sustained at 10 d post–MPO immunization (Fig. 4D) and this corresponded with increased numbers of cytokine-producing γδ T cells in draining LN (IL-17A, Fig. 4E, and IFN-γ, Fig. 4F). Studies comparing WT and δTCR−/− mice during established anti-MPO autoimmunity (day 10), showed that in the absence of γδ T cells, the generation of MPO-specific (Fig. 4G), effector cytokine (IL-17A, Fig. 4H, and IFN-γ, Fig. 4I) producing αβ CD4+ T cells was significantly reduced. These observations are consistent with a role for TLR activated (by FCA immunization), cytokine-producing γδ T cells migrating to LNs where they positively interact with DCs to enhance adaptive αβ T cell autoimmunity.

FIGURE 4.

γδ T cells bridge innate and adaptive anti-MPO immune responses. (A) 18 h post–MPO immunization, increase in the numbers of γδ T cells in LN draining MPO immunization sites was observed (MPO-imm, n = 6) compared with naive WT C57BL/6 mice (non-imm, n = 4). (B and C) CD11c+ DCs were reduced and proportions of apoptotic DCs were increased in δTCR−/− (n = 6) compared with MPO-immunized WT mice (n = 7). Then 10 d post–MPO immunization γδ and αβ T cell responses were measured. (DF) MPO-immunized WT mice have increased numbers of γδ T cells in draining LN 10 d post–MPO immunization (MPO-imm, n = 8) compared with naive mice (non-imm, n = 8) and is associated with increased numbers of MPO stimulated LN γδ T cells producing IL-17A and IFN-γ. (G) Compared with MPO-immunized C57BL/6 mice (n = 8), MPO-immunized δTCR−/− mice (n = 8) have reduced capacity to expand anti-MPO αβ T cells in draining LNs correlating with (H and I), diminished numbers of IL-17A– and IFN-γ–producing anti-MPO αβ T cells. Error bars represent mean ± SEM with statistical analysis by unpaired t test. The experiment was repeated twice with similar results. For multiple groups, one-way ANOVA was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4.

γδ T cells bridge innate and adaptive anti-MPO immune responses. (A) 18 h post–MPO immunization, increase in the numbers of γδ T cells in LN draining MPO immunization sites was observed (MPO-imm, n = 6) compared with naive WT C57BL/6 mice (non-imm, n = 4). (B and C) CD11c+ DCs were reduced and proportions of apoptotic DCs were increased in δTCR−/− (n = 6) compared with MPO-immunized WT mice (n = 7). Then 10 d post–MPO immunization γδ and αβ T cell responses were measured. (DF) MPO-immunized WT mice have increased numbers of γδ T cells in draining LN 10 d post–MPO immunization (MPO-imm, n = 8) compared with naive mice (non-imm, n = 8) and is associated with increased numbers of MPO stimulated LN γδ T cells producing IL-17A and IFN-γ. (G) Compared with MPO-immunized C57BL/6 mice (n = 8), MPO-immunized δTCR−/− mice (n = 8) have reduced capacity to expand anti-MPO αβ T cells in draining LNs correlating with (H and I), diminished numbers of IL-17A– and IFN-γ–producing anti-MPO αβ T cells. Error bars represent mean ± SEM with statistical analysis by unpaired t test. The experiment was repeated twice with similar results. For multiple groups, one-way ANOVA was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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γδ T cells are major producers of IL-17A in acute innate inflammation associated with the generation of both host defense and autoimmunity (14). We compared anti-MPO autoimmunity and GN in δTCR−/− mice that received intact WT γδ T cells or IL-17A−/− γδ T cells (WT γδ T→δTCR−/− or IL-17A−/− γδ T→δTCR−/−). Only IL-17A–intact WT γδ T cells could restore the major deficiency in anti-MPO autoimmunity in δTCR−/− mice. The capacity to generate MPO-specific CD4+ T cells as measured by dermal MPO-specific DTH (Fig. 5A) as well as the numbers of MPO-specific IFN-γ–producing (Fig. 5B) and IL-17A (Fig. 5C) LN cells were all increased only in WT γδT→δTCR−/− mice. When GN was triggered, WT γδ T→δTCR−/− mice developed significantly increased leukocyte glomerular influx (Fig. 5D) compared with δTCR−/− mice receiving IL-17A−/− γδ T cells, as well as enhanced glomerular segmental necrosis (Fig. 5E) and albuminuria (Fig. 5F). Serum MPO-ANCA autoantibody production was unaffected (Fig. 5G).

FIGURE 5.

IL-17A–producing γδ T cells are crucial in the development of anti-MPO autoimmunity and GN. (AC) δTCR−/− mice receiving IL-17A−/− γδ T cells (IL-17A−/− γδT→ δTCR−/−, n = 10) lack the capacity to generate systemic anti-MPO autoimmune response measured by MPO-specific DTH footpad swelling with reduced ex vivo MPO-stimulated IFN-γ– and IL-17A–producing LN cells compared with WT γδT→ δTCR−/−, n = 10. (D) Reduced glomerular leukocyte effector accumulation of CD4+ T cells, Mϕs, and neutrophils in IL-17A−/− γδT→ δTCR−/−. (E) Structural renal injury assessed on PAS-stained kidney sections and quantified by scoring glomerular segmental necrosis (arrow highlights the section of glomeruli with necrosis. Original magnification ×400, scale bar, 20 μm) was significantly attenuated in IL-17A−/− γδT→ δTCR−/− and (F) was associated with reduced albuminuria. (G) No difference in MPO-ANCA titers was observed between groups. Error bars represent mean ± SEM with statistical analysis by unpaired t test. The experiment was repeated twice with similar results. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5.

IL-17A–producing γδ T cells are crucial in the development of anti-MPO autoimmunity and GN. (AC) δTCR−/− mice receiving IL-17A−/− γδ T cells (IL-17A−/− γδT→ δTCR−/−, n = 10) lack the capacity to generate systemic anti-MPO autoimmune response measured by MPO-specific DTH footpad swelling with reduced ex vivo MPO-stimulated IFN-γ– and IL-17A–producing LN cells compared with WT γδT→ δTCR−/−, n = 10. (D) Reduced glomerular leukocyte effector accumulation of CD4+ T cells, Mϕs, and neutrophils in IL-17A−/− γδT→ δTCR−/−. (E) Structural renal injury assessed on PAS-stained kidney sections and quantified by scoring glomerular segmental necrosis (arrow highlights the section of glomeruli with necrosis. Original magnification ×400, scale bar, 20 μm) was significantly attenuated in IL-17A−/− γδT→ δTCR−/− and (F) was associated with reduced albuminuria. (G) No difference in MPO-ANCA titers was observed between groups. Error bars represent mean ± SEM with statistical analysis by unpaired t test. The experiment was repeated twice with similar results. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Close modal

With the availability of CD4+ T cell clones specific for the immunodominant MPO T cell epitope, MPO409–428 (19), we could determine the capacity of γδ T cells to recruit MPO-specific αβ CD4+ T cells to the kidney. A total of 2.5 × 107 MPO409–428-specific CD4+ T cell clones were transferred to WT or δTCR−/− mice (control mice received OVA323–339 OT-II CD4+ T cell clones). GN was triggered using a subnephritogenic dose of anti-GBM globulin. Mice were culled 4 d later to examine MPO-specific CD4+ T cell accumulation. In the absence of γδ T cells, glomerular MPO409–428-specific CD4+ T cell recruitment was similar to mice receiving OVA323–339-specific cells Fig. 6A, 6B). Additionally, the numbers of glomerular neutrophils were significantly increased in WT mice receiving MPO409–428 CD4+ T cell clones compared with δTCR−/− mice (Fig. 6C). These data demonstrate that MPO-specific adaptive immune effector responses direct the accumulation of glomerular neutrophils. However, although the numbers of glomerular Mϕs were reduced in δTCR−/− mice, this was not statistically significant (Fig. 6D). Overall, these results suggest that accumulation of glomerular anti-MPO CD4+ T cells is dependent on γδ T cells.

FIGURE 6.

Recruitment of MPO409–428-specific CD4+ T cell clones to glomeruli requires γδ T cells. MPO409–428-specific αβ CD4+ T cell clones (or control OVA323–339-specific T cell clones) were injected i.v. and MPO was deposited in glomeruli by injection of anti-GBM Ig. (A and B) After 4 d accumulation of CD4+ T cells could be seen in an Ag-specific manner, only in WT mice (n = 5). Glomerular CD4+ T cells numbers in γδ T cell–deficient mice (n = 5) were similar to mice receiving OVA323–339-specific cells (n = 5). Original magnification ×200 (scale bar, 60 μm). (C) Glomerular neutrophils and (D) Mϕ influx were also increased only in WT mice. The dotted line represents the respective mean glomerular leukocyte accumulation in mice receiving OVA323–339-specific cells. Error bars represent mean ± SEM with statistical analysis by unpaired t test, *p < 0.05.

FIGURE 6.

Recruitment of MPO409–428-specific CD4+ T cell clones to glomeruli requires γδ T cells. MPO409–428-specific αβ CD4+ T cell clones (or control OVA323–339-specific T cell clones) were injected i.v. and MPO was deposited in glomeruli by injection of anti-GBM Ig. (A and B) After 4 d accumulation of CD4+ T cells could be seen in an Ag-specific manner, only in WT mice (n = 5). Glomerular CD4+ T cells numbers in γδ T cell–deficient mice (n = 5) were similar to mice receiving OVA323–339-specific cells (n = 5). Original magnification ×200 (scale bar, 60 μm). (C) Glomerular neutrophils and (D) Mϕ influx were also increased only in WT mice. The dotted line represents the respective mean glomerular leukocyte accumulation in mice receiving OVA323–339-specific cells. Error bars represent mean ± SEM with statistical analysis by unpaired t test, *p < 0.05.

Close modal

The current studies examined the role of γδ T cells and αβ T cells in experimental autoimmune MPO-ANCA GN. We first confirmed the critical role for αβ T cells in the induction of MPO autoimmunity and GN. Although it is assumed that the generation of autoimmunity, both humoral and cellular, involves αβ T cells, this has not been directly assessed in MPO-ANCA–associated GN. The observation that anti-MPO autoimmunity was substantially attenuated in β-chain TCR−/− mice strongly suggests that this is an αβ T cell–dependent form of autoimmunity. Humoral immunity to MPO did not develop in mice lacking αβ T cells, showing MPO-ANCA production is T cell dependent. Many studies focus on the role of humoral immunity in this disease. Evidence from human (24) and animal studies (3) demonstrates that ANCA plays an essential role in this disease, and therefore is a major therapeutic target. However, the fact that the underlying immunopathology causing this disease is CD4+ T cell dependent also means that T cell–targeting therapeutics have a logical basis. We demonstrated that the full expression of αβ T cell anti-MPO autoimmunity and GN requires γδ T cells and their production of IL-17A. However, the development of MPO-ANCA autoantibodies was not γδ T cell dependent. These results are consistent with similar observations in two other murine GN models; in experimental anti-GBM GN, αβ T cell–deficient mice did not produce humoral immunity to the glomerular planted sheep anti-mouse GBM Ab (25), and in lupus-prone MRL/lpr mice, humoral immunity and GN were significantly impaired in αβ T cell–deficient mice (26). In both these models, γδ T cell–deficient mice showed no abnormality of humoral immunity.

It is now recognized that innate γδ T cells play a significant role in host defense and the generation of autoimmunity in many animal models of human autoimmune diseases (912). γδ T cells are distributed at sites of barrier defense. They act as sentinels detecting potential microbial invasion and are the first responders in host defense. γδ T cells detect and respond to danger signals. This response to danger signals by innate lymphocytes including γδ T cells is called lymphocyte stress-surveillance (27), which involves the production of activating cytokines IL-12, IL-23, and IL-33 produced by myeloid, epithelial, and stromal cells (28), as well as TLRs (29) and damage-associated molecular patterns (30). γδ T cells respond by amplifying inflammation through cytokine production, the most prominent of which is IL-17A (31), an acute proinflammatory cytokine capable of recruiting and activating neutrophils (32). γδ T cells share a number of critical differentiation molecules and cell surface markers with Th17 cells, including RORγt, IL-23R, and CCR6 (14). γδ T cell production of IL-17 is a major feature of these cells in innate immunity involving host defense and mediating disease (11, 33). However, there is growing evidence that these cells can migrate to secondary lymphoid organs and induce maturation of DCs and B cells, enhancing HLA-DR expression and IL-6 production (34, 35). γδ T cells and DCs also have positive reciprocal effects, enhancing DC maturation and survival (36, 37). These and other observations have led to the recognition that γδ T cells can bridge innate and adaptive immunity by their capacity to augment DC presentation of Ags to naive αβ CD4+ T cells, thereby triggering adaptive immunity and autoimmunity (38).

We found that following immunization of MPO in FCA, γδ T cells migrated to draining LN. The absence of γδ T cells was associated with fewer DCs and an enhanced proportion of apoptotic DCs, as well as fewer IL-17A– and IFN-γ–producing anti-MPO αβ T cells. These findings are consistent with a major role for γδ T cells in AAV in bridging innate and adaptive autoimmunity by supporting enhanced DC presentation of autoantigen. The transfer of WT γδ T cells to δTCR−/− mice significantly restores anti-MPO autoimmunity and GN, and demonstrates a significant requirement for γδ T cells in this model of autoimmune disease. Transfer of IL-17A−/− γδ T cells to δTCR−/− mice failed to enhance anti-MPO autoimmunity and GN, strongly suggesting that the proautoimmune effects of γδ T cells substantially results from their capacity to produce IL-17A. This is consistent with a major role for innate IL-17A in the development of MPO autoimmunity and supports the hypothesis that IL-17A–producing γδ T cells influence the generation of adaptive immunity by moving to draining LNs to support DC survival and capacity to help drive the intensity of DC activation to a level where tolerance can be overcome. The evidence of a reciprocal activation feedback loop between γδ T cells (by IL-17A production) and DCs (by IL-23 and IL-12 production) would support the proposition that IL-17A–enhanced DC activation may be one of the major mechanisms that explains the seemingly unexpected importance of γδ T cells in the development of anti-MPO autoimmunity. Although the detailed basis of loss of tolerance to MPO in human disease is unknown, there is reasonable evidence that inflammation and leukocyte activation are important. Perhaps the animal model with local MPO presentation together with TLR ligand–rich adjuvant s.c. in the skin, which has a high density of γδ T cells in mice, may accentuate the importance of γδ T cells in helping induce autoimmunity in this model.

There are other ways in which γδ T cells may enhance the development of autoimmune MPO-ANCA GN by their contributions systemically and in the target organ. Systemic effects of IL-17A produced by γδ T cells could enhance neutrophil mobilization and activation (39). This is highly relevant in disease where neutrophils are the major target of anti-MPO autoimmunity and are the disease mediator. ANCA binding to MPO-expressing neutrophils in the circulation is the key initiating event of this disease. ANCA binding to membrane translocated MPO on activated neutrophils results in Fc engagement and complement activation that facilitates neutrophil accumulation in glomeruli. In glomeruli, neutrophil degranulation, neutrophil extracellular trap formation, reactive oxygen species, and enzyme release mediate local injury but, in addition, deposit MPO extracellularly (40). Glomerular MPO recruits circulating MPO-specific CD4+ T cells to induce a local injurious DTH-like lesion (41).

The role of resident or infiltrating extrarenal immune γδ T cells in the target organ, the kidney, has not been assessed in autoimmune murine MPO-ANCA GN. There is a population of resident γδ T cells in the kidney (13) and studies of renal biopsies from patients with proliferative GN (42) show that the numbers of γδ T cells in nephritic kidneys are increased. We used anti-GBM globulin to recruit neutrophils to deposit MPO in the kidney of WT mice. CD4+ T cell clones (with a TCR binding the MPO-immunodominant nephritogenic peptide, MPO409–428) were transferred into γδ T cell–deficient and WT mice. The glomerular accumulation of MPO-specific T cells was dependent on the presence of γδ T cells. Interestingly, a previous study in an experimental model of nonautoimmune GN, autologous phase anti-GBM GN, demonstrated that γδ T cell–deficient mice were protected from the development of nephritis (13). It is therefore possible that the roles played by γδ T cells in secondary lymphoid organs is different from the diseased target organ as there are clear differences in TCR Vγ and Vδ usage, demonstrating distinct γδ T cell subset populations within these organs (Vγ1 and Vγ4 γδ T cells were dominant in draining LNs, whereas Vδ6 were dominant when Vγ4 and Vγ5 γδ T cells were virtually absent in the kidney). These results demonstrate a role for IL-17A–producing γδ T cells in the generation of nephritogenic MPO autoimmunity and show that γδ T cells facilitate the recruitment of the nephritogenic CD4+ T cells to the target organ.

The online version of this article contains supplemental material.

Abbreviations used in this article:

AAV

ANCA-associated vasculitis

ANCA

anti-neutrophil cytoplasmic Ab

DC

dendritic cell

DTH

delayed-type hypersensitivity

FCA

Freund’s complete adjuvant

GBM

glomerular basement membrane

GN

glomerulonephritis

LN

lymph node

macrophage

MPO

myeloperoxidase

PAS

periodic acid–Schiff

PMN

polymorphonuclear leukocyte

WT

wild type.

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The authors have no financial conflicts of interest.

Supplementary data