IL-23 receptor and IL-17A expression is up-regulated in T cells from lupus-prone mice and correlated with disease progress. Cells were extracted from the lymph nodes of 2-mo-old B6 mice, predisease 2-mo-old lupus-prone B6/lpr, established disease 5- mo-old B6/lpr, and severely diseased 8-mo-old B6/lpr mice (four mice used in each group). The cells were incubated in DMEM with plate-bound anti-CD3 Ab in the presence (■) or absence of IL-23 (□) for 6 days. Total mRNA was extracted, reverse transcribed, and analyzed by TaqMan PCR with specific primers for the IL-23 receptor (IL-23R) and IL-17A. The expression of IL-23R as a ratio of IL-23R mRNA:β-actin mRNA is shown in A. The expression of IL-17A as a ratio of IL-17A mRNA:β-actin mRNA is shown in B. The data represent one of three independent experiments.

IL-23 stimulation expands the CD4⁻CD8⁻ T cells derived from both control MRL/MPJ and lupus-prone MRL/lpr mice. A, Cells were extracted from the lymph nodes of 4-mo-old MRL/lpr and MRL/MPJ mice. The cells were incubated in DMEM with plate-bound anti-CD3 Ab in the presence or absence of IL-23 for 6 days. Cells were stained with anti-CD3, anti-CD4, anti-CD8 fluorescent Abs. We show FACS analysis of the CD3⁺ cells derived from MRL/lpr and MRL/MPJ mice and treated with or without IL-23. One of three independent experiments is shown. B, Cells were extracted from the lymph nodes of 4-mo-old MRL/lpr and MRL/MPJ mice and sorted into CD3⁺CD4⁺ and CD3⁺CD4⁻CD8⁻ (DNT) cells. The cells were incubated (at a concentration of 2 × 10⁵ cells/well) in DMEM with plate-bound anti-CD3 and anti-CD28 Abs in the presence or absence of IL-23 for 6 days. At the end of the incubation, the cells in each well were counted. The total number of cells from each group is shown in the graph (mean ± SD of three independent measurements, one of three independent experiments). *, p < 0.001).

IL-23 treatment leads to an increase in the number of IL- 17A⁺ lymphocytes and does not affect the percentage of B cells. Lymphocytes from 5- mo-old control B6 and lupus-prone B6/lpr mice were extracted (three mice in each group) and stimulated with anti-CD3 (2 µg/ml) with or without IL-23 (20 ng/ml) for 48 h. These cells were thereafter injected i.p. in Rag-1^−/− mice (5 million in each mouse). A, The number of IL-17A-expressing T cells that were injected into each Rag1^−/− mouse was determined by intracellular cytokine staining, followed by FACS analysis. We show the number of IL17A⁺ T cells that were transferred from all four different groups into the Rag-1^−/− mice. B, The percentage of CD19⁺ cells in the lymph node extracts treated with or without IL-23 were determined using FACS analysis (four animals in each group).

IL-17A⁺CD3⁺ cells are found in kidneys of Rag-1^−/− mice that were injected with IL-23-treated B6/lpr-derived lymphocytes. Rag-1^−/− mice that were injected with lymphocytes derived from control B6 and B6/lprmice were sacrificed 4 wk after transfer. Kidney sections were prepared according to data described in Materials and Methods. The sections were stained with CD3-FITC Ab and anti-IL-17A Ab according to the data in Materials and Methodsand imaged using a confocal microscope. A, Staining of kidney sections of Rag-1^−/− mice that were injected with B6-derived lymphocytes (first row), B6-derived lymphocytes treated with IL-23 (second row), B6/lpr-derived lymphocytes (third row), and B6/lpr-derived lymphocytes treated with IL-23 (fourth row). One representative experiment of 5 (for the B6 groups) and 1of 12 (for the B6/lprgroups) are shown. Bar, 50 µm. B, IL-17A-expressing cells infiltrate the kidneys of Rag-1^−/− mice that are injected with lymphocytes derived from lupus-prone mice. Rag-1^−/− mice that were injected with lymphocytes derived from control B6 and B6/lpr mice were sacrificed 4 wk after transfer. The kidneys were treated with collagenase for 30 min before cell extraction. IL-17A-expressing cells were determined by intracellular cytokine staining, followed by FACS analysis (gate on lymphoid population). A representative FACS analysis is shown. C, Cumulative results from recipient mice that were injected with lymphocytes from control B6 and B6/lpr lymphocytes treated with or without IL-23 (*, p < 0.001). White arrows point to individual cells. LN, Lymph node.

Rag-1^−/− mice injected with IL-23-treated B6/lpr-derived lymphocytes have enlarged glomeruli in their kidneys. Rag-1^−/− mice that were injected with lymphocytes derived from control B6 and B6/lpr mice were sacrificed 4 wk after transfer. Kidney sections were prepared according to the data in Materials and Methods and stained with H&E and PAS according to methods described in Materials and Methods. A, We show the H&E (first column) and PAS (second column) staining of kidney sections of Rag-1^−/− mice that were injected with B6-derived lymphocytes (first row), B6-derived lymphocytes treated with IL-23 (second row), B6/lprderived lymphocytes (third row), and B6/lpr-derived lymphocytes treated with IL-23 (fourth row); original magnification, ×400 for both H&E and PAS); one representative experiment of 5 (for the B6 groups) and one of 12 (for the B6/lpr groups) are shown. B, The glomerular area from the abovementioned sections was measured according to methods described in Materials and Methods. The mean ± SD of the area of 100 glomeruli (derived from 5 mice in the B6 groups and 12 mice in the B6/lpr groups) is plotted (*, p < 0.0001). LN, Lymph node.

CD3⁺CD4⁻CD8⁻ T cells from lupus-prone MRL/lpr mice express IL-17A at a higher level than CD3⁺CD4⁺ T cells from both lupus-prone and control mice. Lymph nodes (LN) and spleens were harvested from lupus-prone MRL/lpr and control MRL/MPJ mice. The cells were isolated from the tissue as described in Materials and Methods and stained with anti-CD3, anti-CD4, and anti-CD8 Abs and with anti-IL-17A Ab (by intracellular staining) and analyzed by FACS. A, FACS analysis showing IL-17A expression in CD3⁺ lymph node-derived cells from both control (MPJ) and lupus-prone (MRL) mice. One representative experiment of four experiments is shown. B, Cumulative results showing IL-17A expression in lymph node-derived cells (n = 8 MPJ, n = 9 MRL mice, 4 mo old). C, FACS analysis showing IL-17A expression in CD3⁺ splenocytes from both control (MPJ) and lupus-prone (MRL) mice. One representative experiment of four experiments is shown. D, Cumulative results showing IL-17A expression in splenocytes (n = 8 MPJ, n = 9 MRL mice, 4 mo old). E, The total number of CD3⁺CD4⁺ (CD4⁺) and CD3⁺CD4⁻CD8⁻ (DNT) cells isolated from the lymph nodes (first two bars) and the spleens (last two bars) were measured. The total number of CD4⁺ and DNT was multiplied by the percentage of IL-17A-expressing cells for each group as measured by FACS. We show a bar graph comparing the total number of cells (CD4⁺ and DNT) that express IL-17A in both control and lupus-prone mice. *, p < 0.0001. SSC, Side scatter.

IL-23 induces IL-17A production in CD4⁺ and particularly in CD4⁻CD8⁻ T cells. Cells were extracted from the lymph nodes of 5-mo-old B6, B6/lpr, 4-mo-old MRL/MPJ, and MRL/lpr mice. The cells were incubated in DMEM with plate-bound anti-CD3 Ab in the presence or absence of IL-23 for 6 days. IL-17A⁺ T cells were measured using intracellular staining according to methods described in Materials and Methods. A, Histograms of IL-17A expression in IL-23-treated (blue line) vs control-treated (red line) T cells are depicted. Specifically, histograms for B6 (first column), B6/lpr (second column), MRL/MPJ (third column), MRL/lpr (fourth column) CD4⁺ (first row), and CD4⁻CD8⁻ (second row) are depicted. B, Cumulative results from IL-23-treated T cells from eight mice for the B6 and B6/lpr groups. C, Cumulative results from IL-23-treated T cells from seven mice for the MRL/MPJ and MRL/lpr groups are shown. *, p < 0.0001.

IL-17A- expressing T cells infiltrate the kidneys of lupus-prone mice. Frozen kidney sections from both 5-mo-old MRL/MPJ and MRL/lpr mice were stained with anti-CD3-FITC and anti-IL-17 Abs, followed by goat anti-rat IgG Texas Red fluorescent secondary Ab. The slides were imaged using a confocal microscope (one of four independent experiments). White arrows point to individual cells.

IL-17A⁺CD3⁺ cells are found in the spleen of Rag-1^−/− mice that were injected with IL-23-treated B6/lpr-derived lymphocytes. Rag-1^−/− mice that were injected with lymphocytes derived from control B6 and B6/lpr mice were sacrificed 4 wk after transfer. Spleen sections were prepared according to the data in Materials and Methods. The sections were stained with CD3-FITC Ab and anti-IL-17A Ab according to the methods described in Materials and Methods and imaged using a confocal microscope. Staining of splenic sections of Rag-1^−/− mice that were injected with B6-derived lymphocytes (first row), B6-derived lymphocytes treated with IL-23 (second row), B6/lpr-derived lymphocytes (third row), and B6/lpr-derived lymphocytes treated with IL-23 (fourth row). Bar, 50 µm. White arrows point to individual cells. LN, Lymph node.

Rag-1^−/− mice injected with IL-23-treated B6/lpr-derived lymphocytes show significant IgG, IgM, and C3 deposition in the kidneys. Rag-1^−/− mice that were injected with lymphocytes derived from control B6 and B6/lpr mice were sacrificed 4 wk after transfer. Frozen kidney sections were prepared according to the data in Materials and Methods and used for immunofluorescent studies. The sections were stained with IgG-Alexa Fluor 488, C3d-Alexa Fluor 488, IgM-Alexa Fluor 488, and DAPI. We show staining of kidney sections of Rag-1^−/− mice that were injected with B6-derived lymphocytes (first row), B6-derived lymphocytes treated with IL-23 (second row), B6/lpr-derived lymphocytes (third row), and B6/lpr-derived lymphocytes treated with IL-23 (fourth row). The merge columns depict merging of the respective Ab staining with DAPI staining (one representative experiment of 5 (for the B6 groups) and 1 of 12 (for the B6/lpr groups) are shown). Bar, 50 µm. LN, Lymph node.