The ability of CD4+ T cells to differentiate into pathogenic Th1 and Th17 or protective T regulatory cells plays a pivotal role in the pathogenesis of autoimmune diseases. Recent data suggest that CD4+ T cell subsets display a considerable plasticity. This plasticity seems to be a critical factor for their pathogenicity, but also for the potential transition of pathogenic effector T cells toward a more tolerogenic phenotype. The aim of the current study was to analyze the plasticity of Th17 cells in a mouse model of acute crescentic glomerulonephritis and in a mouse chronic model of lupus nephritis. By transferring in vitro generated, highly purified Th17 cells and by using IL-17A fate reporter mice, we demonstrate that Th17 cells fail to acquire substantial expression of the Th1 and Th2 signature cytokines IFN-γ and IL-13, respectively, or the T regulatory transcription factor Foxp3 throughout the course of renal inflammation. In an attempt to therapeutically break the stability of the Th17 phenotype in acute glomerulonephritis, we subjected nephritic mice to CD3-specific Ab treatment. Indeed, this treatment induced an immunoregulatory phenotype in Th17 cells, which was marked by high expression of IL-10 and attenuated renal tissue damage in acute glomerulonephritis. In summary, we show that Th17 cells display a minimum of plasticity in acute and chronic experimental glomerulonephritis and introduce anti-CD3 treatment as a tool to induce a regulatory phenotype in Th17 cells in the kidney that may be therapeutically exploited.

The CD4+ T cells play a central role in the pathogenesis of autoimmune diseases, including human and experimental crescentic glomerulonephritis. Classically, it was assumed that the cell-mediated immune reactions responsible for tissue injury are predominantly mediated by IFN-γ–producing Th1 cells (1, 2). However, the identification and functional characterization of Th17 cells have challenged this paradigm (3, 4). Indeed, numerous studies have clearly shown that Th17 cells drive autoimmune processes that were previously thought to be exclusively Th1 mediated, such as multiple sclerosis (5), rheumatoid arthritis (6), and psoriasis (7).

We and others recently demonstrated that the IL-17A/Th17 axis contributes to renal tissue injury in both experimental crescentic glomerulonephritis (i.e., nephrotoxic nephritis [NTN]) (810) and a model of pristane-induced lupus nephritis (11, 12). Time course analyses of the renal CD4+ T cell response in the NTN model revealed that Th17 cell numbers peak at ∼days 6–10 after nephritis induction and then decline, whereas the numbers of Th1 and T regulatory (Treg) cells continuously increase from days 6 to 10 onward (1316). This sequential infiltration of the different CD4+ T cell subsets into inflamed target organs with an early increase of Th17 cells that is followed by Th1 and Treg cell infiltration is a phenomenon that has also been reported in other inflammatory diseases (17, 18). Whether the gradual decline of Th17 cells observed during progression of renal autoimmunity is due to transdifferentiation into cells of another Th cell subtype, a result of apoptosis, or a consequence of emigration of Th17 cells is currently unclear.

In this context, recent studies indicating that CD4+ T cell subsets in other organs demonstrate a higher grade of plasticity than previously appreciated are of great interest (1821). In particular, Th17 and Treg cells might be highly flexible, which would allow these cells to switch to a different Th cell program and thereby adapt to various inflammatory settings during an immune response. However, whether the potential plasticity of Th17 cells in autoimmunity is a general finding, and whether this process is beneficial or detrimental remains to be fully elucidated.

The aim of the current study was to investigate the potential plasticity of Th17 cells in murine models of glomerulonephritis. We therefore did the following: 1) assessed the fate of adoptively transferred Th17 cells in nephritic Rag1-knockout mice; 2) studied the plasticity of in vivo differentiated Th17 cells in two models of glomerulonephritis (NTN and pristane-induced lupus nephritis) by using IL-17A fate reporter mice (Il17aCre × R26rYFP); and 3) modulated Th17 cell plasticity by CD3-specific Ab treatment to induce a regulatory phenotype of Th17 cells in crescentic glomerulonephritis (GN).

All mice in this study were on C57BL/6J background. Il-17aGFP/Ifn-γFP635/Foxp3mRFP and Il-10GFP reporter mice as well as IL-17ACre × R26rYFP mice were used, as described before (18, 2224). Rag1−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were raised under specific pathogen-free conditions in our animal facility at the University Medical Center Hamburg-Eppendorf. Animal experiments were performed according to national animal care and ethical guidelines, and were approved by local ethics committees.

NTN was induced in 8- to 10-wk-old male mice by i.p. injection of 0.5 ml nephrotoxic sheep serum per mouse, as previously described (10). In adoptive cell transfer experiments, cells were administered i.v. 1 d prior to induction of glomerulonephritis i.v. Lupus nephritis was induced by i.p. injection of 0.5 ml pristane (Sigma-Aldrich, St. Louis, MO) (11).

Urinary albumin excretion was determined by standard ELISA analysis (Mice-Albumin Kit; Bethyl). Urinary creatinine was measured using standard laboratory methods. Anti-CD3 experiments were performed, as previously described (25). In brief, 10 μg of either hamster anti-mouse CD3 (clone 145-2C11) or isotype control Ab was injected i.p. (both BioLegend, San Diego, CA) at the time points indicated.

Experimental autoimmune encephalomyelitis (EAE) was induced by immunization of female mice (12–14 wk) s.c. with 200 μg myelin oligodendrocyte glycoprotein peptide fragment 35–55 (NeoMPS, San Diego, CA) in CFA (BD Biosciences, Franklin Lakes, NJ) containing 4 mg/ml Mycobacterium tuberculosis (H37Ra; BD Biosciences), as described previously (26). We injected 200 ng pertussis toxin (Merck Millipore, Darmstadt, Germany) i.v. on the day of immunization and 48 h later. Mice were scored for clinical signs by the following system: 0, no clinical deficits; 1, tail weakness; 2, hind limb paresis; 3, partial hind limb paralysis; 3.5, full hind limb paralysis; 4, full hind limb paralysis and forelimb paresis; and 5, premorbid or dead (26). Mice were analyzed at day 14 after EAE induction.

For splenocyte cultures, a total of 4 × 106 cells/ml was incubated for 60 h in the presence of sheep IgG. Supernatants were collected, and IL-17A and IFN-γ were measured by ELISA (BioLegend) or by cytometric bead array (BD Biosciences) (27), according to the manufacturer’s instructions.

For polarization of naive CD4+ cells, splenocytes were isolated from naive IL-17aGFP/IFN-γ-FP635 reporter mice using the MACS CD4+ T Cell Isolation Kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany), according to the manufacturer's protocol. A total of 5 × 105 cells/well was plated in anti-CD3 Ab-coated 96-well plates (2 μg/ml; eBioscience, San Diego, CA) and incubated for 65 h with soluble anti-CD28 at 5 μg/ml. Th17 polarization was achieved in the presence of anti–IL-4 (10 μg/ml), anti–IFN-γ (12 μg/ml), IL-6 (50 ng/ml), TGF-β (1 ng/ml), IL-1β (20 ng/ml), and IL-23 (5 ng/ml) (all from BioLegend).

Total RNA of the renal cortex was prepared according to standard laboratory methods. Real-time RT-PCR was performed on a StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA), as previously described (10).

Glomerular crescent formation was assessed in a blinded fashion in 30 glomeruli per mouse in periodic acid–Schiff (PAS)–stained paraffin sections (28). For the assessment of tubulointerstitial injury, low original magnification field photographs (×200) of four nonoverlapping cortical areas from PAS-stained kidney sections were taken per mouse. The interstitial area was then determined by superimposing the photographs with a grid containing 40 points with subsequent counting of points located in the interstitial area (excluding glomeruli, blood vessels, and tubules) in a blinded fashion. The percentage of points in the interstitial area corresponds to the tubulointerstitial injury score (13, 28). The glomerular tuft area was measured in glomeruli from five nonoverlapping cortical areas per mouse from PAS-stained kidney sections using ZEN lite 2011 software (Zeiss, Jena, Germany). The average glomerular tuft area per individual mouse was analyzed.

For immunohistochemical staining, paraffin-embedded sections were deparaffinized, rehydrated, and stained with the following Abs: GR-1 (1:50, Ly6 G/C, NIMP-R14; Hycult Biotech) and Foxp3 (FJK-16s; eBioscience). Ag retrieval was performed by incubation with proteinase type XXIV (5 mg/ml; Sigma-Aldrich) for 15 min at 37°C. Tissue sections were developed with the Vectastain ABC-AP kit (Vector Laboratories, Burlingame, CA). To quantify tubulointerstitial GR1+ cells, at least 20 low power fields (original magnification ×200) were counted.

All slides were evaluated using an Axioskop light microscope (Zeiss) and photographed with an Axiocam HRc (Zeiss) using the ZEN lite 2011 software (Zeiss).

Previously described methods for leukocyte isolation from murine kidneys were used (9). In brief, kidneys were digested for 45 min at 37°C with 0.4 mg/ml collagenase D (Roche, Mannheim, Germany) and 0.01 mg/ml DNase I in RPMI 1640 medium (Life Technologies, Darmstadt, Germany) supplemented with 10% heat-inactivated FCS (Invitrogen, Carlsbad, CA), and finely minced using GentleMACS digester (Miltenyi Biotec, Bergisch-Gladbach, Germany). Subsequently, 37% Percoll gradient centrifugation was performed (GE Healthcare, Uppsala, Sweden).

Spleens were minced, sequentially filtered through 70-μm and 40-μm nylon meshes, and then washed with HBSS without Ca2+ and Mg2+ (Invitrogen). Cell viability was assessed by trypan blue staining before flow cytometry or cell transfer experiments.

To isolate leukocytes from CNS, mice were perfused via the vascular system with PBS by intracardial injection, and thereafter, brains and spinal cords were extracted. Brains and spinal cords were combined and digested for 45 min at 37°C with 0.4 mg/ml collagenase D (Roche) and 0.01 mg/ml DNase I in RPMI 1640 medium (Life Technologies) supplemented with 10% heat-inactivated FCS (Invitrogen), minced using GentleMACS digester (Miltenyi Biotec), and finely filtered through a 70-μm cell strainer (BD Biosciences). Subsequently, 37% Percoll gradient centrifugation was performed (GE Healthcare).

For FACS analysis, the following Abs were used: CD45 (30-F11), CD3 (17A2), CD4 (GK1.5), CD8 (53-6.7), CD11b (M1/70), IL-17A (TC11-18H10), IL-13 (eBio13A), and IFN-γ (XMG1.2) (BD Biosciences, eBioscience, or BioLegend) (14). Dead-cell staining was performed using the LIVE/DEAD Fixable Read Dead Stain Kit (Invitrogen). Analyses were performed on a BD Biosciences LSRII system with Diva software, and data were analyzed using the Flowjo software (Tree Star). For transfer experiments, cells were sorted with an Aria II system (BD Biosciences). For intracellular cytokine staining, cells were activated by incubation at 37°C, 5% CO2, for 4 h with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (1 μg/ml; Calbiochem-Merck, Darmstadt, Germany) in X-VIVO medium (Lonza AG, Walkersville, MD). After 30 min of incubation, brefeldin A (10 μg/ml; Sigma-Aldrich) was added.

Data were expressed as mean ± SEM. All statistical analyses were performed with the SPSS package and Graph Pad Prism 5 software. A p value <0.05 was considered to be statistically significant. The Student t test was used for comparison between two groups. In case of three or more groups, one-way ANOVA was used, followed by a post hoc analysis with Bonferroni’s test for multiple comparisons.

NTN is a well-characterized, T cell–mediated mouse model of crescentic glomerulonephritis, which is induced by the injection of sheep antiserum raised against cortical kidney components (10, 29). We first determined the pathogenic potential of Th17 cells in transfer experiments. To this end, we compared the effect of a transfer of a bulk population of in vitro generated Th17 cells (purity of 30%, Supplemental Fig. 1) with the transfer of Th0 cells on the course of experimental glomerulonephritis in Rag1-deficient mice. The transfer of Th17 cells resulted in aggravated renal damage, including glomerular crescent formation and tubulointerstitial injury (Supplemental Fig. 1A–C). In addition, renal neutrophil infiltration was increased after transfer of Th17 cells (Supplemental Fig. 1D). Therefore, our results demonstrated the increased pathogenicity of in vitro polarized Th17 cells in this model. Of note, immunohistochemistry showed no Foxp3+ cells in the kidney of mice after transfer of cells, indicating that Th0 and Th17 cells did not transdifferentiate into Foxp3+ Treg cells (Supplemental Fig. 1D). Interestingly, the reanalysis by flow cytometry revealed that only a minority of the transferred CD4+ T cells remained IL-17A positive (∼7%) in the inflamed kidney, whereas the majority produced IFN-γ (∼64%) (Supplemental Fig. 1E). However, technical limitations might restrict the significance of these results, because the transferred cell population included significant amounts of IL-17A–negative cells, which might be responsible for the emergence of IFN-γ–producing CD4+ T cells in nephritic Rag1−/− mice.

To determine the potential plasticity of Th17 cells in more detail, we isolated CD4+CD62L+ naive T cells from IL-17aGFP mice that express GFP under the control of the IL-17A promotor and polarized them into Th17 cells in vitro (23). Th17-differentiated cells were FACS sorted based on GFP expression to obtain a purity of >95% and were then transferred i.v. into Rag1-deficient recipients. Mice that had received Th17 cells showed signs of renal damage, as determined by glomerular crescent formation, tubulointerstitial injury (Fig. 1A, 1B), and proteinuria (Fig. 1C) at day 10 after induction of glomerulonephritis. In line with a predominant Th17 response, staining of renal tissue sections for the neutrophil marker GR-1 revealed massive neutrophil recruitment into the kidney in nephritic mice (Fig. 1D).

FIGURE 1.

Stability of Th17 cells in nephritic Rag1−/− mice. (A) PAS staining of renal cortex from Rag1/ mice at day 10 after transfer of 2 × 105 (n = 4) or 4 × 105 (n = 4) in vitro polarized Th17 cells after NTN induction. Original magnification ×400. (B) Crescent formation and tubulointerstitial injury in the different groups. (C) Urinary albumin-to-creatinine ratio at day 8 after NTN induction. (D) Immunohistochemical staining and quantification of neutrophils (top) and Treg cells (bottom) in renal cortex by staining of the granulocyte differentiation Ag (GR1)-positive cells and Foxp3-positive cells, respectively. Original magnification ×400. (E) Cytokine expression of FACS-sorted Th17 cells directly before transfer into Rag1−/− mice, and analysis of renal T cells at day 10 after NTN induction. The diagram shows the quantification of cytokine expression after transfer as quantified by flow cytometry. Bars represent mean ± SEM (*p < 0.05).

FIGURE 1.

Stability of Th17 cells in nephritic Rag1−/− mice. (A) PAS staining of renal cortex from Rag1/ mice at day 10 after transfer of 2 × 105 (n = 4) or 4 × 105 (n = 4) in vitro polarized Th17 cells after NTN induction. Original magnification ×400. (B) Crescent formation and tubulointerstitial injury in the different groups. (C) Urinary albumin-to-creatinine ratio at day 8 after NTN induction. (D) Immunohistochemical staining and quantification of neutrophils (top) and Treg cells (bottom) in renal cortex by staining of the granulocyte differentiation Ag (GR1)-positive cells and Foxp3-positive cells, respectively. Original magnification ×400. (E) Cytokine expression of FACS-sorted Th17 cells directly before transfer into Rag1−/− mice, and analysis of renal T cells at day 10 after NTN induction. The diagram shows the quantification of cytokine expression after transfer as quantified by flow cytometry. Bars represent mean ± SEM (*p < 0.05).

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To evaluate whether the transferred Th17 cells retain their cytokine pattern, we used a fluorescence reporter system (Il-17aGFP/Ifn-γFP635/Foxp3RFP) (24) to analyze cytokine expression of donor CD4+ T cells after restimulation with PMA and ionomycin. The analysis of CD4+ T cells before the transfer and after recovery from the inflamed kidney at day 10 showed that a major proportion of Th17 cells maintained IL-17A expression (∼40%), whereas only a minority initiated IFN-γ production (∼20%) (Fig. 1E). Another major proportion of renal CD4+ T cells became negative for IL-17A but also did not upregulate IFN-γ (∼40%) (Fig. 1E). Immunohistochemical staining for Foxp3 (Fig. 1D) and fluorescence analysis of the Foxp3-reporter (mRFP) (data not shown) revealed the absence of Treg cells in the kidney of Rag-1–deficient mice after transfer of Th17 cells.

Despite the transfer of a highly purified population of Th17 cells, we could not exclude that proliferation of contaminating non-Th17 cells in a lymphopenic environment might account for the considerable proportion of IL-17A–negative cells observed in the experiments delineated above. Therefore, we aimed at studying the fate of Th17 cells under more physiological conditions in immune-competent nephritic mice. To this end, we induced NTN in Il17aCre fate reporter mice (Il17aCre × R26RYFP) (18) and investigated parameters of tissue damage and cytokine expression of CD4+ T cells in the kidney at different time points. Glomerular crescents increased over time to >35% at day 20 (Fig. 2A, 2B). The percentage of Th17 fate-mapped (YFP+) cells of all CD4+ T cells peaked at day 10. In the course of NTN, YFP+ cells were the major population of CD4+ T cells expressing IL-17A (Fig. 2C). IL-17A expression by some YFP cells (non-Th17) (Fig. 2C) might be a result of new Th17 cells coming up that have not had time to switch on enough Cre.

FIGURE 2.

Time kinetics of YFP and IL-17A expression in acute crescentic GN. (A) PAS staining of renal cortex from Il-17aCre × R26RYFP fate reporter mice at indicated time points after NTN induction. Original magnification ×400. (B) Crescent formation at indicated time points (n = 3–6 per group) after NTN induction. (C) Flow cytometry of renal CD3+CD4+ T cells for IL-17A fate (IL-17Afm/YFP) and IL-17A expression (anti–IL-17A mAb [mab]). (D) Quantification of renal YFP+ cells in relation to all renal CD4+ T cells. Bars represent mean ± SEM.

FIGURE 2.

Time kinetics of YFP and IL-17A expression in acute crescentic GN. (A) PAS staining of renal cortex from Il-17aCre × R26RYFP fate reporter mice at indicated time points after NTN induction. Original magnification ×400. (B) Crescent formation at indicated time points (n = 3–6 per group) after NTN induction. (C) Flow cytometry of renal CD3+CD4+ T cells for IL-17A fate (IL-17Afm/YFP) and IL-17A expression (anti–IL-17A mAb [mab]). (D) Quantification of renal YFP+ cells in relation to all renal CD4+ T cells. Bars represent mean ± SEM.

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To analyze the cytokine production of Th17 cells, we compared YFP+ (Fig. 3A, upper panel) with YFP cells (lower panel). YFP+ cells maintained IL-17A expression, but only a small subset of these cells expressed IFN-γ, and there was virtually no expression of the Th2 cytokine IL-13 in this population. YFP cells expressed high amounts of IFN-γ, and there was also a subset of IL-13–expressing YFP cells at later time points (Fig. 3). Taken together, this indicates that Th17 cells have a rather stable phenotype in acute experimental glomerulonephritis and that expression of IFN-γ and IL-13 by renal CD4+ T cells is restricted to non-Th17 cells.

FIGURE 3.

Stability of Th17 cells in acute crescentic GN. (A) Gating for renal IL-17A fate-mapped (IL-17Afm+/YFP+) and IL-17Afm (YFP) cells by flow cytometry. FACS analyses for IL-17A, IFN-γ, and IL-13 expression at days 0 and 10 after NTN expression. (B) Quantification of cytokine expression of IL-17Afm+ (red) and IL-17Afm cells (blue) in the course of NTN (days 0–20, n = 3–6 per group).

FIGURE 3.

Stability of Th17 cells in acute crescentic GN. (A) Gating for renal IL-17A fate-mapped (IL-17Afm+/YFP+) and IL-17Afm (YFP) cells by flow cytometry. FACS analyses for IL-17A, IFN-γ, and IL-13 expression at days 0 and 10 after NTN expression. (B) Quantification of cytokine expression of IL-17Afm+ (red) and IL-17Afm cells (blue) in the course of NTN (days 0–20, n = 3–6 per group).

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Importantly, we also induced EAE in Il17aCre fate reporter mice (Il17aCre × R26RYFP), a model in which Hirota et al. (18) observed a high degree of Th17 cell plasticity. EAE was induced by injection of myelin oligodendrocyte glycoprotein peptide fragment 35–55. Mice showed signs of disease with increasing EAE score starting at day 8 (Supplemental Fig. 2A). CD45+CD3+CD4+CD11bneg T cells from CNS (Supplemental Fig. 2B) and from the draining inguinal lymph node (Supplemental Fig. 2C) were analyzed at day 14 for IL-17A–positive cells. In agreement with the study of Hirota et al. (18) and in contrast to the situation in autoimmune kidney disease, intracerebral Th17 cells and Th17 cells from the draining lymph node showed indeed a high degree of plasticity to Th1 cells (Supplemental Fig. 2).

We next examined Th17 cell stability in a chronic model of lupus nephritis (pristane-induced lupus nephritis) using Il17aCre fate reporter mice. This model depends on the Th17 immune response, as shown before (11, 30). Six months after induction of the disease, PAS staining of renal tissue sections showed glomerular hypercellularity and glomerular hypertrophy, as a sign of immune-mediated kidney damage resembling certain aspects of human lupus nephritis (Fig. 4A, 4B). In line with a role of Th17 cells in this model of renal autoimmunity, flow cytometry revealed increased infiltration of Th17 cells into the kidney at the 6-mo time point (Fig. 4C). Most importantly, staining for intracellular cytokines showed IL-17A expression in ∼80% of renal CD4+YFP+ (IL-17Afm+) cells, but only in <2% of YFP (IL-17Afm) cells (Fig. 4D). In contrast, a high percentage of IL-17Afm cells expressed IFN-γ, but not IL-17A. IL-13 expression was found only in a small percentage of CD4+ T cells (Fig. 4E). Taken together, these analyses underscore the stability of Th17 cells in renal autoimmunity even in a chronic model that develops gradually over months.

FIGURE 4.

Stability of Th17 cells in pristane-induced lupus nephritis. (A) PAS staining of renal cortex from Il-17aCre × R26RYFP fate reporter mice 6 mo after induction of lupus nephritis by exposure to pristane (n = 11), and from age-matched controls (n = 10). Original magnification ×400. (B) Quantification of glomerular hypertrophy, as measured by glomerular tuft area. (C) Intracellular cytokine staining for IL-17A and analysis by flow cytometry of renal CD4+ T cells of mice with lupus nephritis and healthy control mice. The diagram shows the relative quantification of renal IL-17A fate mapped (IL-17Afm+/YFP+) (*p < 0.05). (D) Analysis of renal CD4+ IL-17Afm+ (YFP+) and IL-17Afm (YFP) cells for expression of IL-17A, IFN-γ, and IL-13 by flow cytometry. (E) Quantification of cytokine expression of IL-17Afm+ (red) and IL-17Afm cells (blue) (*p < 0.05, **p < 0.01).

FIGURE 4.

Stability of Th17 cells in pristane-induced lupus nephritis. (A) PAS staining of renal cortex from Il-17aCre × R26RYFP fate reporter mice 6 mo after induction of lupus nephritis by exposure to pristane (n = 11), and from age-matched controls (n = 10). Original magnification ×400. (B) Quantification of glomerular hypertrophy, as measured by glomerular tuft area. (C) Intracellular cytokine staining for IL-17A and analysis by flow cytometry of renal CD4+ T cells of mice with lupus nephritis and healthy control mice. The diagram shows the relative quantification of renal IL-17A fate mapped (IL-17Afm+/YFP+) (*p < 0.05). (D) Analysis of renal CD4+ IL-17Afm+ (YFP+) and IL-17Afm (YFP) cells for expression of IL-17A, IFN-γ, and IL-13 by flow cytometry. (E) Quantification of cytokine expression of IL-17Afm+ (red) and IL-17Afm cells (blue) (*p < 0.05, **p < 0.01).

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After having shown that Th17 cells are pathogenic in experimental glomerulonephritis and have a stable phenotype in renal autoimmune disease, we aimed at interfering with Th17 cell stability to induce a tolerogenic or regulatory phenotype in Th17 cells. We have previously shown that anti-CD3–specific Ab treatment induces a regulatory phenotype in Th17 cells in the intestine in vivo, which is characterized by high IL-10 production (25). We therefore aimed to test whether the phenotype of Th17 cells could be therapeutically manipulated in the kidney. To that end, we induced NTN and treated animals with anti-CD3 Abs (2 × 10 μg i.p.) in the course of the disease. To analyze the impact of anti-CD3 treatment on IL-10 expression in IL-17A+ cells, we induced NTN in IL-10 reporter mice (IL-10GFP) (15, 22). Mice were treated with anti-CD3 at day 8, and 4 h before analysis at day 10. Renal CD4+ T cells were analyzed for IL-17A, IFN-γ, and IL-13 expression after stimulation by intracellular Ab staining, and for IL-10 using the GFP reporter system. Flow cytometry showed IL-10 expression in 5% of renal IL-17A–expressing T cells from isotype-treated mice. After anti-CD3 treatment, >30% of renal IL-17A+ cells were positive for IL-10 (Fig. 5A, 5B). Analysis of IL-17A–negative cells also revealed significant induction of IL-10 in this population. However, the expression of IFN-γ and IL-13 was not influenced by anti-CD3 treatment (Fig. 5A, 5B).

FIGURE 5.

CD3-specific Ab treatment induced an immunoregulatory phenotype of Th17 cells marked by IL-10 expression and ameliorated GN. (A and B) Cytokine expression of renal T cells in IL-10 acute reporter mice (Tiger) at day 10 after induction of NTN. Anti-CD3 (10 μg/mouse, n = 4) or isotype control Ab (n = 5) was applied i.p. 2 d and 4 h prior to the analysis. CD4+IL-17+ and CD4+IL-17 T cells were analyzed for IL-10, IFN-γ, and IL-13 expression, as indicated. (C and D) Nephritic Il-17aCre × R26RYFP fate reporter mice were treated with anti-CD3 (n = 4) or isotype control Ab (n = 4). Cytokine expression of renal CD4+ IL-17Afm+ (YFP+) and IL-17Afm (YFP) cells, as measured by flow cytometry and intracellular staining for IL-17A, IFN-γ, and IL-13 by mAb at day 10 after induction of glomerulonephritis. (D) The percentage of IL-17A+, IL-13+, and IFN-γ+ of IL-17Afm+ (upper panel) and IL-17Afm (lower panel) was quantified. (E and F) CD4+ T cells from fate reporter mice treated with anti-CD3 or isotype control Abs were analyzed for ROR-γt, T-bet, and Foxp3 expression. (G) The percentage of renal Th17 cells (IL-17Afm+) and (H) the number of CD4+ cells/kidney were quantified from the kidney of mice treated with anti-CD3 or isotype control Abs. (I) PAS staining of renal cortex from wild-type mice 10 d after NTN induction. Original magnification ×400. NTN mice were treated on days 6 and 8 with i.p. injection of anti-CD3 mAb (n = 6) or isotype control (n = 8). (J) Renal damage as measured by quantification of glomerular crescents and tubulointerstitial injury. Bars represent mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 5.

CD3-specific Ab treatment induced an immunoregulatory phenotype of Th17 cells marked by IL-10 expression and ameliorated GN. (A and B) Cytokine expression of renal T cells in IL-10 acute reporter mice (Tiger) at day 10 after induction of NTN. Anti-CD3 (10 μg/mouse, n = 4) or isotype control Ab (n = 5) was applied i.p. 2 d and 4 h prior to the analysis. CD4+IL-17+ and CD4+IL-17 T cells were analyzed for IL-10, IFN-γ, and IL-13 expression, as indicated. (C and D) Nephritic Il-17aCre × R26RYFP fate reporter mice were treated with anti-CD3 (n = 4) or isotype control Ab (n = 4). Cytokine expression of renal CD4+ IL-17Afm+ (YFP+) and IL-17Afm (YFP) cells, as measured by flow cytometry and intracellular staining for IL-17A, IFN-γ, and IL-13 by mAb at day 10 after induction of glomerulonephritis. (D) The percentage of IL-17A+, IL-13+, and IFN-γ+ of IL-17Afm+ (upper panel) and IL-17Afm (lower panel) was quantified. (E and F) CD4+ T cells from fate reporter mice treated with anti-CD3 or isotype control Abs were analyzed for ROR-γt, T-bet, and Foxp3 expression. (G) The percentage of renal Th17 cells (IL-17Afm+) and (H) the number of CD4+ cells/kidney were quantified from the kidney of mice treated with anti-CD3 or isotype control Abs. (I) PAS staining of renal cortex from wild-type mice 10 d after NTN induction. Original magnification ×400. NTN mice were treated on days 6 and 8 with i.p. injection of anti-CD3 mAb (n = 6) or isotype control (n = 8). (J) Renal damage as measured by quantification of glomerular crescents and tubulointerstitial injury. Bars represent mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

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Next, we used IL-17ACre fate reporter mice to investigate whether Th17 cells maintain IL-17A expression after anti-CD3 treatment. Therefore, fate reporter mice were treated with anti-CD3 at days 8 and 10 after induction of experimental glomerulonephritis. Still, Th17 cells (YFP+) maintained IL-17A expression after anti-CD3 treatment (Fig. 5C, 5D). IFN-γ and IL-13 were not induced in these cells by anti-CD3. In addition, anti-CD3 did not reduce the number of renal CD4+ T cells in the kidney significantly (Fig. 5H).

In addition, we analyzed the systemic effect of anti-CD3 treatment in mice with crescentic glomerulonephritis. We therefore measured in vitro cytokine production of sheep IgG-stimulated splenocytes from mice with anti-CD3 or isotype using cytometric bead array analyses. Interestingly, IL-10 was significantly released into the supernatants of splenocytes from anti-CD3–treated mice (Supplemental Fig. 3A), whereas Th1, Th2, and Th17 cytokines were not altered (Supplemental Fig. 3B, 3C).

To test whether anti-CD3 treatment induces Foxp3+ Treg cells in vivo, we also analyzed Foxp3 expression in nephritic IL-17ACre fate reporter mice (Fig. 5E, 5F). Our data revealed that anti-CD3 treatment did not induce Foxp3 expression in Th17 cells (YFP+).

To evaluate the impact of anti-CD3 treatment on renal tissue injury, PAS-stained kidney sections were analyzed at day 10 in mice after treatment with anti-CD3 (days 6 and 8). Interestingly, in mice treated with anti-CD3, renal tissue damage was reduced as compared with isotype controls (Fig. 5I, 5J), indicating that anti-CD3 treatment has beneficial effects in experimental glomerulonephritis.

CD4+ T cell responses can be subdivided into proinflammatory and anti-inflammatory reactions. Although Th1, Th2, and Th17 cells are well-characterized subsets that provide protection against exogenous harmful agents, but also drive autoimmune diseases, Treg cells avoid autoimmune reactions and overwhelming effector responses (31). These CD4+ T cell subsets have classically been viewed as terminally differentiated lineages with limited flexibility. Recent data suggest that Th17 cells show a high degree of plasticity in the brain and intestine, in particular upon inflammatory stimuli, where Th17 cells can produce IFN-γ and transdifferentiate into highly pathogenic Th1 cells (18, 21). However, the mechanisms driving plasticity or stability, as well as their relevance in autoimmunity, remain to be fully elucidated.

The pathogenic impact of Th1 and Th17 cells in human and experimental GN has recently been demonstrated (8, 9, 16, 32). Interestingly, during the course of murine experimental GN, we and others observed a shift from a Th17-dominated inflammation to a Th1-dominated immune response in the kidney (13, 33). The rapid decline in Th17 cell numbers observed between days 10 and 20 in the NTN model of GN raises the question what the fate of renal Th17 cells might be. We therefore transferred in vitro polarized Th17 cells into nephritic Rag1−/− mice. In line with recent studies, the transferred Th17 cells induced severe renal injury in terms of glomerular crescent formation and tubulointerstitial damage (10, 12, 34). Reanalysis of renal CD4+ T cells after transfer revealed a very poor stability of the Th17 phenotype and a shift to IFN-γ production. However, technical limitations restrict the significance of these results because a bulk Th17 population that included significant amounts of IL-17A–negative cells was transferred. To analyze whether the emergence of IFN-γ–producing T cells is caused by cellular or population plasticity, we used acute reporter mice that express GFP under the control of the IL-17A promotor (23) to obtain highly purified Th17 cells (>95%). After transfer of these cells into Rag1−/− mice, the majority of Th17 cells continued to produce IL-17A, whereas only a minor population produced IFN-γ, indicating minimal single cell plasticity of in vitro polarized Th17 cells in GN. It is noteworthy that immunohistochemistry revealed no Foxp3-positive cells in the kidney after transfer of polarized Th17 cells, excluding significant transdifferentiation of Th17 cells into Treg cells. This observation nicely extends our earlier data showing that renal and systemic Treg cells develop from neither transferred Foxp3-negative T effector cells (35) nor RORγt+ Th17 cells (36).

Cells transferred into lymphopenic Rag1−/− mice encounter a rather nonphysiological environment. We therefore aimed at investigating the plasticity of Th17 cells in immune-competent mice. To this end, we induced experimental glomerulonephritis (NTN) in fate reporter mice (Il-17aCre × R26rYFP) in which the activation of the IL-17A promotor results in the expression of Cre recombinase. Subsequently, Cre-mediated excision of a floxed stop codon upstream of the fluorescent protein YFP under control of a ubiquitously active promoter leads to permanent labeling of IL-17A–producing cells, regardless of their actual production of this cytokine at the time of analysis (18). Similar to the data obtained with intracellular cytokine staining after restimulation (13), time course analysis revealed that the percentage of Th17 fate-mapped cells (YFP+) of all CD4+ T cells peaked at day 10. To analyze the cytokine production of Th17 cells, we compared YFP+ Th17 with non-Th17 cells. The main finding of this study is that Th17 cells maintained IL-17A expression in experimental glomerulonephritis, and only a small subset of these cells expressed IFN-γ. In contrast, non-Th17 cells expressed high amounts of IFN-γ, and a minor IFN-γ–negative subset also expressed IL-13. Results obtained with the IL-17A fate reporter in the chronic model of pristane-induced lupus nephritis revealed almost identical results, demonstrating that Th17 cells have a stable phenotype in autoimmune kidney diseases.

In two recent landmark studies, a shift of Th17 to Th1 cells in the course of experimental autoimmune encephalomyelitis and experimental colitis was shown (18, 21), whereas acute cutaneous infection with Candida albicans did not result in the deviation of Th17 cells to the production of alternative cytokines (18). This suggests that the specific inflammatory environment in the target organ can directly influence the plasticity of Th17 cells, but more studies are needed for a better understanding of the underlying mechanisms. The role of IL-23 stimulation of Th17 cells in different organs, which seems to be required for simultaneous expression of IL-17A and IFN-γ, the induction of T-bet in Th17 cells, and the subsequent shift from Th17 to Th1 cells (18), might be of great interest in this context.

After having shown that Th17 cells have a rather stable phenotype in two models of renal autoimmune diseases, we sought to therapeutically interfere with the stability of these cells. To induce transdifferentiation of renal Th17 cells into anti-inflammatory IL-10–producing T cells, we induced NTN in IL-10 reporter mice (IL-10GFP) (22) and treated them with an anti-CD3 Ab, a treatment strategy that we previously used to induce tolerogenic Th17 cells mainly in the intestine (25). The mechanisms of CD3-specific Ab-induced tolerance have been extensively studied before, but the exact mechanisms are still incompletely understood and a matter of ongoing debate. Potential mechanisms include expansion of Foxp3+ Treg (37) and type 1 regulatory (Tr1) cells (25), induction of a regulatory phenotype in Th17 cells (23), and transdifferentiation of Th17 cells into Tr1 cells (24). Our results demonstrate that IL-10 can be induced in renal IL-17A+ T cells by anti-CD3 treatment in a model of crescentic glomerulonephritis. Importantly, Th17 cells maintained IL-17A expression and there was no induction of IFN-γ in Th17 fate-mapped cells. These data show that Th17 cells in mice treated with anti-CD3 Abs may acquire a regulatory phenotype. The potential therapeutic relevance of this intervention was further substantiated by the observation of reduced renal injury in nephritic mice after anti-CD3 treatment. However, we do not yet know whether these IL-10–producing Th17 cells represent a stable Treg cell subset. In addition, it is unclear whether a further transdifferentiation into Tr1 T cells can be induced.

In conclusion, using adoptive transfer experiments and studies in fate reporter mice, we demonstrated that renal Th17 cells have very limited spontaneous plasticity in acute and chronic models of glomerulonephritis. However, using CD3-specific Ab treatment, we were able to promote an IL-10–producing regulatory phenotype of Th17 cells in the kidney. A better understanding of renal Th17 cell flexibility might provide the basis for interventions that modulate effector Th17 cells and that push immune responses from proinflammatory to beneficial anti-inflammatory responses.

This work was supported by Deutsche Forschungsgemeinschaft Grants SFB1192/A5 (to C.F.K. and S.H.), SFB1192/A1 (to U.P.), and KFO 228: PA 754/7-2 (to U.P. and C.F.K.); Emmy-Noether-Programme Grants TU316/2-1 (to J.-E.T.) and HU1714/3-1 (to S.H.); and grants from the Deutsche Gesellschaft für Nephrologie (to C.F.K. and J.-E.T.). T.K. received a stipend from the Werner Otto Foundation.

The online version of this article contains supplemental material.

Abbreviations used in this article:

EAE

experimental autoimmune encephalomyelitis

GN

glomerulonephritis

NTN

nephrotoxic nephritis

PAS

periodic acid–Schiff

Tr1

type 1 regulatory

Treg

T regulatory.

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The authors have no financial conflicts of interest.

Supplementary data