Highly purified Th17 cells from BDC2.5NOD mice convert into Th1-like cells in NOD/SCID recipient mice

Abstract

Th17 cells are involved in the pathogenesis of many autoimmune diseases, but it is not clear whether they play a pathogenic role in type 1 diabetes. Here we investigated whether mouse Th17 cells with specificity for an islet antigen can induce diabetes upon transfer into NOD/SCID recipient mice. Induction of diabetes in NOD/SCID mice via adoptive transfer of Th1 cells from BDC2.5 transgenic mice was prevented by treatment of the recipient mice with a neutralizing IFN-γ-specific antibody. This result suggested a major role of Th1 cells in the induction of disease in this model of type 1 diabetes. Nevertheless, transfer of highly purified Th17 cells from BDC2.5 transgenic mice caused diabetes in NOD/SCID recipients with similar rates of onset as in transfer of Th1 cells. However, treatment with neutralizing IL-17-specific antibodies did not prevent disease. Instead, the transferred Th17 cells, completely devoid of IFN-γ at the time of transfer, rapidly converted to secrete IFN-γ in the NOD/SCID recipients. Purified Th17 cells also upregulated Tbet and secreted IFN-γ upon exposure to IL-12 in vitro and in vivo in NOD/SCID recipients. These results indicate substantial plasticity of Th17 commitment toward a Th1-like profile.

Figure 1. Th17 cells cause diabetes in NOD/SCID mice.

(A) Intracellular staining for IL-17 and IFN-γ of polarized Th17 or Th1 BDC2.5 cells was performed on day 4 of culture. Cells were transferred into NOD/SCID recipients and incidence of diabetes for Th1 (n = 5) (filled symbols) and Th17 (n = 5) (open symbols) transfer is shown. (B) Intracellular staining of CD4⁺Vβ4⁺ T cells in the PLNs on day 8 after transfer is shown. (C) Intracellular staining was performed as in A, followed by transfer into antibody-treated NOD/SCID recipients. Th17 were transferred into isotype control–treated (n = 4) (open diamonds) or anti–IFN-γ–treated (n = 5) (filled squares) hosts. Th1 were transferred into isotype-treated (n = 4) (open triangles) or anti–IFN-γ–treated (n = 5) (filled diamonds) hosts. (D) Representative FACS plots of CD4⁺Vβ4⁺ T cells in PLNs and pancreata of isotype–treated (left panels) or anti–IFN-γ–treated (right panels) hosts 10 days after transfer. Numbers indicate the percentage of cells in each quadrant.

Figure 2. Anti–IFN-γ treatment inhibits activation of CD11b⁺ cells but not the effect on diabetes by anti–IL-17A administration.

(A) Histogram of MHC class II expression (left panel) on CD11b-expressing cells isolated from the pancreata of isotype- (thick line) or anti–IFN-γ–treated (thin line) NOD/SCID recipients, and MFI of MHC class II on individual samples (right panel). Horizontal bars represent mean MHC class II MFI, and individual points represent MHC class II MFI of CD11b-infiltrating cells from individual mice. (B) mRNA expression of iNOS and programmed death ligand 1 (PD-L1) is shown. Mean ± SEM. (C) Th17 and Th1 BDC2.5 T cells were adoptively transferred into antibody-treated NOD/SCID recipients. Th1 was transferred into isotype control–treated (open triangles) or anti–IL-17A–treated (filled diamonds) hosts. Th17 was transferred into isotype-treated (open diamonds) or anti–IL-17A–treated (filled squares) hosts. Mice were injected with 2 mg of isotype (OX-1) or anti–IL-17A (MM17-F3) on days 0, 2, 4, and 6. n = 5.

Figure 3. Spiked Th1 cells do not outgrow Th17.

(A) Day 4 intracellular staining of Th17 (upper-left panel) or GFP⁺ Th1 (upper-right panel) cells. Cells were transferred as Th17 (n = 5) (filled diamonds), Th17 plus 1% GFP⁺ Th1 (n = 5) (open squares), or 1% GFP⁺ Th1 alone (n = 5) (× symbols), and diabetes development is shown (bottom panels). (B) Percentage of GFP⁺ Vβ4⁺ T cells in tissues on day 9 after transfer. Top panels show representative FACS plots of CD4 versus GFP gated on all Vβ4 T cells recovered from pancreas (left panel) and PLNs (right panel) from a Th17 plus 1% GFP Th1 recipient mouse. Horizontal bars represent mean percentage of GFP⁺ of total Vβ4⁺ T cells from 5 samples, and individual points represent percentage of GFP⁺ of total Vβ4⁺ T cells of individual mice. Numbers indicate the percentage of cells in each quadrant.

Figure 4. Purified Th17 T cells convert in vivo.

(A) FACS plots of tetramer staining for IL-17 versus IFN-γ before (left dot plot) or after (middle dot plot) purification. IL-17 versus Foxp3 profile of purified cells (right dot plot). Diabetes onset in NOD/SCID recipients of highly purified Th17 cells (filled diamonds) (right panel). Numbers indicate the percentage of cells in each quadrant. (B) Representative FACS plots gated on CD4⁺Vβ4⁺ T cells from PLNs, pancreata, and mesenteric lymph nodes (MLNs) 8 days after transfer of highly purified Th17 cells, showing IL-17 versus IFN-γ intracellular staining. Numbers indicate the percentage of cells in each quadrant (A and B). (C) Summary graphs showing the percentage of cells producing IL-17, IL-17 plus IFN-γ, or IFN-γ alone. Horizontal bars represent mean percentage of CD4⁺ cells positive for stated cytokines from 6 samples, and individual symbols represent percentage of CD4⁺ cells positive for stated cytokines for individual mice. **P < 0.01, Mann-Whitney U test (n = 6). These data are representative of 3 independent experiments.

Figure 5. In vitro conversion of highly purified Th17 cells.

(A) mRNA expression relative to Hprt for stated transcription factors, cytokines, and receptors, comparing highly purified Th17 cells and Th1 cells. (B) Representative FACS plot showing intracellular staining of highly purified Th17 cells, cultured in the presence of IL-23 and the cytokines indicated, on day 3 following purification. Numbers indicate the percentage of cells in each quadrant. (C) mRNA expression relative to Hprt, comparing highly purified Th17 cells cultured with IL-23, with (+) or without (–) IL-12, for stated transcription factors, cytokines, and receptors. Mean ± SEM.

Figure 6. Kinetic analysis of transcriptional markers for Th1 program, following adoptive transfer of purified Th17 cells.

Purified Th17 cells were transferred into NOD/SCID recipients, and 5 mice were sacrificed on both day 3 and 6 after transfer. PLNs and pancreata were harvested, and CD4⁺Vβ4⁺ T cells were pooled and then isolated by MoFlo cell sorting followed by RNA extraction. Whole transcriptome amplification (QIAGEN) was then performed to create a cDNA template for real-time PCR. Depicted are mRNA expression levels relative to Hprt for stated transcription factors, cytokines, and receptors, comparing starting Th17 population (Start Th17) and (upper row) PLNs residing T cells and (bottom row) pancreata infiltrating T cells on day 3 and 6 after transfer. ND, none detected.