Recombinant cytokines and antibodies.
Mouse IL-6, IL-12, IL-21, IL-23, and TGFβ-1 were purchased from R&D Systems. Mouse IL-2, IL-4, IL-7, and IL-15 were purchased from PeproTech. Antibodies to CD4 (GK1.5), CD3 (145-2C11), CD28 (37.51), CD11c (HL3), B220 (RA3-6B2), γc (4G3), IL-7Rα (SB199), Thy1.2 (30-H12), CD44 (IM7), CD62L (MEL-14), CCR5 (C34-3448), CXCR5 (2G8), and IL-17 (TC11-18H10; for IL-17A) were purchased from BD. Antibodies to CD30L (RM153), OX40L (RM134L), and CCR7 (4B12) were purchased from eBioscience. Anti-CCR6 antibody (clone 140706) was obtained from R&D Systems. Both anti-CXCR3 and -CCR4 antibodies were purchased from Abcam. α-GalCer was obtained from AXXORA, LLC.
C57BL/6J WT mice (The Jackson Laboratory), Rag2−/−
mice, and Rag2−/−
mice (Taconic) were purchased as indicated. Stat3fl/fl
mice were bred with mice expressing Cre under the control of the MMTV-LTR (MMTV-Cre
) to produce Stat3fl/fl; MMTV-Cre
mice (provided by L. Hennighhausen [National Institute of Diabetes and Digestive and Kidney Diseases] and D. Levy [New York University, New York, NY]) (30
). All animal experiments were performed according to the National Institutes of Health (NIH) guidelines for laboratory animals and were approved by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) Animal Care and Use Committee.
Isolation of cells and cell culture.
Single-cell suspensions were prepared from spleens of healthy 8–10-wk-old mice. All cell cultures were performed in RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine, 5 mM Hepes, 100 U/ml Pen-Strep, and 2.5 μM 2-ME at 37°C for 4, 24, or 48 h. Cells stained with the appropriate antibodies were isolated by flow cytometric cell sorting using a Mo-Flo cell sorter (Dako). Whole splenocytes or isolated cells were cultured in the presence of 20 ng/ml IL-23, 20 ng/ml IL-4, 10 ng/ml IL-6, 20 ng/ml IL-7, 20 ng/ml IL-15, 20 ng/ml IL-2, 20 ng/ml IL-12, 100 ng/ml IL-21, or 5 ng/ml TGFβ-1.
Flow cytometric analysis and intracellular cytokine staining.
Cells were stimulated for 2 h with 50 ng/ml PMA and 1 μg/ml Iono, followed by incubation with BFA (GolgiPlug; BD) for an additional 2 h. Cells were fixed in 4% formyl saline and permeabilized with 0.1% saponin permeabilization buffer after surface staining. PE-conjugated anti–IL-17 antibody was used to detect intracellular cytokine levels (BD). Stained cells with the appropriate antibodies were analyzed on a flow cytometer (FACSCalibur; BD). Events were collected and analyzed with FlowJo software (Tree Star, Inc.). To evaluate production of IL-17A in vivo, Rag2−/−
mice were injected i.p. with 5 or 12.5 mg zymosan (Sigma-Aldrich). Control animals received PBS. To assess in vivo intracellular cytokine levels, 0.25 mg BFA (Sigma-Aldrich) was simultaneously injected i.v., as previously described (21
RNA isolation and measurement of cytokines.
Total RNA was isolated using TRIzol reagent (Invitrogen) from freshly isolated CD4+CD3+CD62L−CD44+ memory T cells; CD4+CD3+CD62LhighCD44low naive T cells; CD19+ B cells, Th17 cells polarized with IL-6, TGFβ-1, and anti-CD3/-CD28 for 3 d; and CD4+CD3−CD11c−B220− LTi-like cells. cDNA was synthesized with the TaqMan Reverse Transcription kit (Applied Biosystems). TaqMan primers and probes for mouse IL-17A, IL-17F, IL-22, IL-23R, IL-12Rβ1, IL-12Rβ2, RORc (for RORγt), IL-17RA, IL-6Rα, CCR6, AHR, GATA3, and 18SrRNA (as endogenous control) were purchased from Applied Biosystems. Samples were analyzed by using a sequence detection system (ABI PRISM 7700; Applied Biosystems).
The amounts of cytokines in the culture supernatant were measured using mouse IL-17 Quantikine assay kits (for IL-17A; R&D Systems) and the mouse IL-22 ELISA construction kit (Antigenix America Inc.) according to the manufacturers' instructions. IL-23–mediated cell stimulations were performed at cell concentrations of 4 × 106 cells/ml. Samples were measured in duplicate against the standard curve of the assay.
Statistical significance was determined by the Student's t test. P < 0.05 was considered to indicate a significant difference.
Online supplemental material.
Fig. S1 shows IL-17A production by WT or Rag2−/−
splenocytes in the presence of IL-23 with γc-dependent cytokines, anti-CD3/-CD28, or α-GalCer. Fig. S2 shows that IL-17A–producing CD3−
cells do not express myeloid markers. Fig. S3 shows three populations in CD4+
cells from Rag2−/−
spleens. Fig. S4 shows that LTi-negative subsets produce minimal IL-17A. Fig. S5 shows IL-17A production by LTi-like cells in the gut. Fig. S6 shows the surface expression of various markers on LTi-like cells. Fig. S7 shows mRNA expression of various factors in LTi-like cells after IL-23 stimulation with IL-7. Fig. S8 shows IL-22 and IL-17A production by isolated NK cells. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20072713/DC1