IL-17A secretion by CD8+ T cells supports Th17-mediated autoimmune encephalomyelitis - PubMed (original) (raw)

. 2013 Jan;123(1):247-60.

doi: 10.1172/JCI63681. Epub 2012 Dec 10.

Sylvia Heink, Axel Pagenstecher, Katharina Reinhard, Josephine Ritter, Alexander Visekruna, Anna Guralnik, Nadine Bollig, Katharina Jeltsch, Christina Heinemann, Eva Wittmann, Thorsten Buch, Olivia Prazeres da Costa, Anne Brüstle, Dirk Brenner, Tak W Mak, Hans-Willi Mittrücker, Björn Tackenberg, Thomas Kamradt, Michael Lohoff

Affiliations

IL-17A secretion by CD8+ T cells supports Th17-mediated autoimmune encephalomyelitis

Magdalena Huber et al. J Clin Invest. 2013 Jan.

Abstract

IL-17-producing CD8+ T (Tc17) cells are detectible in multiple sclerosis (MS) lesions; however, their contribution to the disease is unknown. To identify functions of Tc17 cells, we induced EAE, a murine model of MS, in mice lacking IFN regulatory factor 4 (IRF4). IRF4-deficient mice failed to generate Tc17 and Th17 cells and were resistant to EAE. After adoptive transfer of WT CD8+ T cells and subsequent immunization for EAE induction in these mice, the CD8+ T cells developed a Tc17 phenotype in the periphery but could not infiltrate the CNS. Similarly, transfer of small numbers of WT CD4+ T cells alone did not evoke EAE, but when transferred together with CD8+ T cells, IL-17-producing CD4+ (Th17) T cells accumulated in the CNS and mice developed severe disease. Th17 accumulation and development of EAE required IL-17A production by CD8+ T cells, suggesting that Tc17 cells are required to promote CD4+ T cell-mediated induction of EAE. Accordingly, patients with early-stage MS harbored a greater number of Tc17 cells in the cerebrospinal fluid than in peripheral blood. Our results reveal that Tc17 cells contribute to the initiation of CNS autoimmunity in mice and humans by supporting Th17 cell pathogenicity.

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Figures

Figure 1

Figure 1. Tc17 differentiation depends on IRF4.

(A and F) Purified WT and _Irf4–/–_CD8+ T cells activated via CD3/28 plus cytokines as indicated were stained for IFN-γ, IL-17, or Foxp3. (B and D) mRNA expression of the indicated genes analyzed by quantitative real-time RT-PCR in Irf4–/– (black) or WT (white) CD8+ T cells. Data (± SD) represent PCR duplicates. (C and G) WT or _Irf4–/–_CD8+ T cells transduced with retroviruses expressing RORγt-GFP (RORγt), Eomes-VP16-GFP (Eomes), Foxp3-Thy1.1 (Foxp3), or the control viruses MSCV-GFP (MIG) or MSCV-Thy1.1 (MIT) activated as indicated and stained for IL-17 or IFN-γ. Analyses were performed on a (C) GFP+ or (G) Thy1.1+GFP+ gate. (E) Western blot for Eomes and β-actin in WT and _Irf4–/–_CD8+ T cells after 72 hours of activation. (A, C, F, and G) Numbers represent percentages of positive cells. (A–G) Data are representative of 3 independent experiments.

Figure 2

Figure 2. Tc17 cells do not migrate into the CNS in an IRF4-deficient environment.

(A and B) Mean clinical scores (± SEM) of MOG37–50-induced EAE in WT mice (n = 4) and Irf4–/– mice (n = 4) (A) without or (B) with transfer of congenic 107 CD45.2–CD8+ WT T cells. (C) Absolute T cell number per CNS (endogenous or transferred, averages of pooled cells of 4 mice). (D) CD8+ gate of cells from LNs and spleens of Irf4–/– mice substituted with 107 CD45.2–CD8+ WT T cells stained for CD8, CD45.2, IL-17, or IFN-γ at day 19 after immunization. Numbers represent percentages of cells in the respective quadrant. (A–D) Data are representative of 6 independent experiments.

Figure 3

Figure 3. CD8+ T cells mutually interact with CD4+ T cells to induce EAE.

(A) Mean EAE scores (± SEM) combining 2 independent experiments of MOG37–50-immunized Irf4–/– mice (n = 6) that received sorted congenic 2.5 × 106 CD45.1+CD44loCD8+ and/or 104 CD62LhiCD45.1+ 2D2 T cells. P values were calculated comparing the scores of Irf4–/– mice transferred with 2D2 cells alone or in combination with CD8+ T cells. (B and D) Absolute numbers in the CNSs of Irf4–/– mice of (B) T cells (mean ± SEM, n = 4) or (D) CD8+ T cells after transfer of 2D2 or CD8+ T cells alone or in combination. (C) Absolute numbers of CD8+ compared with CD4+ T cell numbers after cotransfer of 2D2 and CD8+ T cells. (C and D) Averages of pooled cells of 4 mice at day 15 after immunization. (E) Histology of spinal cords at day 15 after immunization. Immunochemically stained cells were detected as red-brown foci. Scale bar: 100 μm. KB, Klüver-Barrera. (F) Flow cytometry of gated CD4+ or CD8+ CNS cells after PMA/ionomycin restimulation. Numbers represent percentages of cells in the respective quadrant. (A–F) The experiments were repeated 4 times with consistent results. *P < 0.05; ***P < 0.001.

Figure 4

Figure 4. CD4+ T cells require CCR6 to cooperate with CD8+ T cells for infiltration into the CNS.

(A) Mean clinical scores (± SEM) of MOG37–50-immunized Irf4–/– mice (n = 4) substituted with 107 WT CD45.2–CD8+ T plus 2.5 × 106 WT CD45.2+CD4+ T cells or _Ccr6–/–_CD45.2+CD4+ T cells. P values were calculated comparing the scores of Irf4–/– mice transferred with WT CD8+ T cells plus either WT CD4+ or _Ccr6–/–_CD4+ T cells. (B) qRT-PCR for Ccr6 mRNA in Irf4–/– (black) or WT (white) CD4+ T cells stimulated for 2 days as described. Data (± SD) of PCR duplicates. (C) Histology of spinal cords at day 26 after immunization: H&E staining (scale bar: 100 μm); immunochemically stained cells were detected as brown foci (scale bar: 50 μm). (A–C) The experiments were repeated twice with consistent results. **P < 0.005; ***P < 0.001.

Figure 5

Figure 5. IL-17A competence of CD4+ T cells is not required for their pathogenicity.

(A) Mean clinical scores (± SEM) of MOG37–50-immunized Irf4–/– mice (n = 4) substituted with 2.5 × 106 WT CD4+ T cells or _Il17a–/–_CD4+ T cells with or without 107 WT CD8+ T cells. P values were calculated comparing the scores of Irf4–/– mice transferred with WT CD4+ T cells alone or WT CD8+ T cells plus _Il17a–/–_CD4+ cells. (B) Absolute numbers of T cells (mean ± SEM, n = 4) in the CNSs of Irf4–/– mice after transfer of WT CD4+ T cells alone or of WT CD8+ T cells in combination with either WT CD4+ or _Il17a–/–_CD4+ T cells. (C) Flow cytometry of gated CD4+ CNS cells stained for IL-17A, IL-17F, or IFN-γ. Numbers represent percentages of cells in the respective quadrant. (A–C) The experiments were repeated twice with consistent results. *P < 0.05; **P < 0.005; ***P < 0.001.

Figure 6

Figure 6. CD8+ T cells fail to accelerate pathogenicity of committed Th17 cells.

(A) 2D2 cells were polarized under Th17 conditions in vitro for 3 days. Thereafter, Irf4–/– mice (n = 4) were substituted with these cells either alone or in combination with WT 2.5 × 106 CD44loCD8+ T cells and subsequently immunized with MOG37–50. Mean clinical scores (± SEM) are shown. (B) Absolute numbers of T cells (mean ± SEM, n = 4) in the CNSs of Irf4–/– mice after transfer of Th17 cells alone or in combination with CD44loCD8+ T cells. (C) Flow cytometry of transferred in vitro–differentiated 2D2 cells polarized under Th17 conditions and of gated CD4+ CNS cells stained for IL-17A or IFN-γ. Numbers represent percentages of cells in the respective quadrant. (A–C) The experiments were repeated twice with consistent results.

Figure 7

Figure 7. IL-17A competence of CD8+ T cells accelerates CD4+ T cell encephalitogenicity.

(A and B) Mean EAE scores (± SEM) combining 2 independent experiments of MOG37–50-immunized Irf4–/– (n = 6) mice that received sorted 2.5 × 106 CD44loCD8+ cells from WT, Ccr6–/–, or Il17a–/– mice and/or 104 CD62Lhi 2D2 T cells. P values were calculated comparing (A) the scores of Irf4–/– mice transferred with 2D2 alone or 2D2 in combination with _Ccr6–/–_CD8+ T cells and (B) the scores of mice transferred with 2D2 T cells in combination with WT CD8+ T cells or in combination with _Il17a–/–_CD8+ T cells. (C) Histology of spinal cords at day 13 after immunization: Klüver-Barrera staining (scale bar: 100 μm); immunochemically stained cells were detected as brown foci (scale bar: 50 μm). (A–C) The experiments were repeated twice with consistent results. *P < 0.05; **P < 0.005; ***P < 0.001.

Figure 8

Figure 8. Tc17 cells promote Th17 differentiation via direct cell contact in vitro.

(A) Purified CD4+ T cells were mixed 1:1 with in vitro–differentiated WT or Il17a–/– Tc17 cells or their SNs with or without exogenous rmIL-17A and stimulated via CD3/CD28. After 72 hours, CD4+ T cells were sorted, and mRNA expression of the indicated genes was analyzed by qRT-PCR. Data (± SD) of PCR duplicates. (B) ELISA for IL-17A and IFN-γ produced by sorted CD4+ T cells after restimulation for 24 hours with anti-CD3. (C) ELISA for IL-17A in SNs of 72 hours cocultures as described for A. (B and C) Data (± SD) of ELISA duplicates. (D) WT or Il17a–/– Tc17 cells differentiated in vitro for 96 hours were restimulated with PMA/ionomycin in the presence or absence of brefeldin A and stained for IL-17A, IL-17F, and IFN-γ either on their surface (left) or intracellularly (ICS) after permeabilization (right). Numbers represent percentages of cells in the respective quadrant. (A–D) Data are representative of two independent experiments.

Figure 9

Figure 9. Frequency of Tc17 cells in peripheral blood and CSF of control patients and patients with early-stage MS.

(A and B) IL-17+ cells among gated CD8+ T cells from CSF and peripheral blood (PBMCs) obtained from patients with early-stage MS (CIS/eMS, n = 17) as well as from patients with noninfectious headache (control, n = 17). PBMC and CSF lymphocytes were restimulated with CytoStim human. Thereafter, the cells were stained for surface CD8 and then fixed, permeabilized, and stained for intracellular IL-17. The box plots depict the minimum and maximum values (whiskers) and the upper and lower quartiles (top and bottom edges of the box). The median is identified by a line inside the box. The length of the box represents the interquartile range. P values were calculated with Mann-Whitney U test.

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