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. 2008 May 12;205(5):1019-27.
doi: 10.1084/jem.20071133. Epub 2008 Apr 21.

IL-6-dependent spontaneous proliferation is required for the induction of colitogenic IL-17-producing CD8+ T cells

Masaki Tajima  1 , Daiko Wakita, Daisuke Noguchi, Kenji Chamoto, Zhang Yue, Kazunori Fugo, Harumichi Ishigame, Yoichiro Iwakura, Hidemitsu Kitamura, Takashi Nishimura
Affiliations

IL-6-dependent spontaneous proliferation is required for the induction of colitogenic IL-17-producing CD8+ T cells

Masaki Tajima et al. J Exp Med. .

Abstract

We propose a novel role for interleukin (IL) 6 in inducing rapid spontaneous proliferation (SP) of naive CD8(+) T cells, which is a crucial step in the differentiation of colitogenic CD8(+) T cells. Homeostasis of T cells is regulated by two distinct modes of cell proliferation: major histocompatibility complex/antigen-driven rapid SP and IL-7/IL-15-dependent slow homeostatic proliferation. Using our novel model of CD8(+) T cell-dependent colitis, we found that SP of naive CD8(+) T cells is essential for inducing pathogenic cytokine-producing effector T cells. The rapid SP was predominantly induced in mesenteric lymph nodes (LNs) but not in peripheral LNs under the influence of intestinal flora and IL-6. Indeed, this SP was markedly inhibited by treatment with anti-IL-6 receptor monoclonal antibody (IL-6R mAb) or antibiotic-induced flora depletion, but not by anti-IL-7R mAb and/or in IL-15-deficient conditions. Concomitantly with the inhibition of SP, anti-IL-6R mAb significantly inhibited the induction of CD8(+) T cell-dependent autoimmune colitis. Notably, the transfer of naive CD8(+) T cells derived from IL-17(-/-) mice did not induce autoimmune colitis. Thus, we conclude that IL-6 signaling is crucial for SP under lymphopenic conditions, which subsequently caused severe IL-17-producing CD8(+) T cell-mediated autoimmune colitis. We suggest that anti-IL-6R mAb may become a promising strategy for the therapy of colitis.

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Figures

Figure 1.
Figure 1.
Adoptive transfer of naive CD8+ T cells into RAG2−/− mice causes severe autoimmune colitis. 5 × 105 CD44lowCD62L+ naive CD8+ T cells from C57BL/6 mice were intravenously injected into RAG2−/− mice. (A) Body weights of untreated (n = 5), CD8+ T cell–transferred (n = 5), or CD4+ T cell–transferred (n = 5) mice were monitored for 6 wk. Percentages of the resultant body weights against preinjection body weights were calculated every week. The means and SDs are indicated. (B) Colon tissues were obtained from control or CD8+ T cell–transferred mice after 9 wk. Morphology of the representative colon tissues is shown. Bar, 1 cm. (C) HE staining was performed on sections of the colon tissues. Histological pictures of the representative tissues are shown at two different magnifications. Means and SDs of the colitis score are indicated in the bar graph. Bars: (left) 1 mm; (right) 200 μm. (D) Serum KC and SAA levels of untreated or CD8+ T cell–transferred mice were measured by ELISA. The means and SDs are indicated. (E) Cytokine (IFN-γ and IL-17) production by CD8+ T cells from pLNs and mLNs of the adoptively transferred mice 1, 4, and 6 wk after cell transfer. IFN-γ–producing cells in mLNs or pLNs, and IL-17–producing cells in mLNs or pLNs are indicated. Means and SDs of the percentages of cytokine producing cells are shown. (F) Colon sections from untreated or CD8+ T cell–transferred mice were stained with DAPI, anti-CD8 mAb, and anti-CD11b mAb. Bars, 200 μm.
Figure 2.
Figure 2.
Kinetics of SP essential for inducing pathogenic effector memory T cells in mLNs. 5 × 105 cells naive CD8+ T cells from C57BL/6 mice were labeled with CFSE and intravenously injected into RAG2−/− mice. (A) Proliferation of CD8+ T cells was monitored by flow cytometry 3, 5, and 7 d after the injection. The representative FACS profiles are indicated in the figure. (B) CD44 expression and cytokine production levels of CD8+ T cells from pLNs and mLNs in the adoptively transferred mice were examined by staining with mAbs against CD44, IFN-γ, or IL-17 at day 5. The representative FACS profiles are shown. Percentages are indicated. (C) CFSE-labeled CD8+ T cells from OT-1–TCR transgenic mice were intravenously injected into RAG2−/− mice. Proliferation of the CD8+ T cells in mLNs was analyzed at day 7. The representative FACS profiles are indicated. (D) CFSE-labeled CD8+ T cells from C57BL/6 mice were intravenously injected into untreated and antibiotic-treated RAG2−/− mice. Proliferation of the CD8+ T cells in the mLNs of the untreated and antibiotic-treated mice was analyzed by FACS. (E) CFSE-labeled CD8+ T cells from C57BL/6-background Ly5.1 mice were intravenously injected into RAG2−/− or OT-1/RAG2−/− mice. Proliferation of the CD8+ T cells in the pLNs or mLNs of OT-1/RAG2−/− mice was analyzed by FACS.
Figure 3.
Figure 3.
Critical role of IL-6 for SP while inducing pathogenic effector T cells. 5 × 105 naive CD8+ T cells from C57BL/6 mice were labeled with CFSE and intravenously injected into RAG2−/− mice. (A) IL-7, IL-15, IL-6, and TGF-β mRNA expression levels in pLNs or mLNs of untreated RAG2−/− mice were evaluated by real-time PCR. The means and SDs (n = 4 per group) are indicated. (B) SP in mLNs of RAG2−/− mice treated with anti–IL-7R mAb and RAG2/IL-15 double-knockout mice were observed 7 d after the injection. The representative FACS profiles are indicated. (C) Proliferation of CD8+ T cells in the pLNs and mLNs of RAG2−/− mice treated with anti–IL-6R mAb was observed, and the representative FACS profiles are indicated. (D) Cytokine (IFN-γ and IL-17) production by CD8+ T cells in the pLNs and mLNs of anti–IL-6R mAb–treated RAG2−/− mice. The representative FACS profiles are shown. Percentages are indicated.
Figure 4.
Figure 4.
Anti–IL-6R mAb inhibits CD8+ T cell–dependent autoimmune colitis. 5 × 105 naive CD8+ T cells from C57BL/6 mice were intravenously injected into RAG2−/− mice. (A) Body weight of untreated (n = 4), naive CD8+ T cell–transferred (n = 3), or naive CD8+ T cell–transferred/anti–IL-6R mAb–treated (n = 3) mice were monitored for 9 wk. Body weight percentages against the respective preinjection values were calculated every week. The means and SDs are indicated. (B) Colon tissues were obtained from untreated, control, or anti–IL-6R mAb–treated mice 9 wk after the cell transfer. Morphology of the representative colon tissues is shown. Bar, 1 cm. (C) HE staining was performed on the colon tissues from the untreated, control, or anti–IL-6R mAb–treated mice. The representative histological micrographs are shown at two different magnifications. Means and SDs of the colitis score are indicated in the bar graph. Bars: (left) 1 mm; (right) 200 μm. (D) TNF-α, IL-1β, IL-17A, and IFN-γ mRNA expression levels in the colon tissues from the untreated, control, and anti–IL-6R mAb–treated mice were determined by real-time PCR. The means and SDs are indicated.
Figure 5.
Figure 5.
Requirement of Tc17 cells in autoimmune colitis. 5 × 105 naive CD8+ T cells from C57BL/6 WT and IL-17A−/− mice were transferred into RAG2−/− mice. (A) Body weights of untreated (n = 4), WT naive CD8+ T cell–transferred (n = 4), or IL-17A−/− naive CD8+ T cell–transferred (n = 4) mice were monitored for 9 wk after the injection. Body weight percentages against the respective preinjection values were calculated every week. The means and SDs are indicated. (B) Colon tissues were obtained from the untreated, WT naive CD8+ T cell–transferred, or IL-17A−/− naive CD8+ T cell–transferred mice. Morphology of the representative colon tissues is shown. Bar, 1 cm. (C) Colon sections from the untreated, WT naive CD8+ T cell–transferred, or IL-17A−/− naive CD8+ T cell–transferred mice were stained with HE. The representative histological micrographs are shown at two different magnifications. Bars: (left) 1 mm; (right) 200 μm. (D) CD8+ T cells from the pLNs or mLNs of the adoptively transferred mice were intracellularly stained with anti–IFN-γ mAb and anti–IL-17 mAb. The representative FACS profiles are indicated. Percentages are shown.
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