Immune response to dermatomyositis-specific autoantigen, transcriptional intermediary factor 1γ can result in experimental myositis
Naoko Okiyama ,1 Yuki Ichimura,1 Miwako Shobo,1 Ryota Tanaka,1 Noriko Kubota,1 Akimasa Saito,1 Yosuke Ishitsuka,1,2 Rei Watanabe,1,2 Yasuhiro Fujisawa,1 Yoshiyuki Nakamura,1 Akihiro Murakami,3 Hisako Kayama,4 Kiyoshi Takeda,5 Manabu Fujimoto1,6

<この項は書きかけです。順次追記します。>

表題（仮訳）：皮膚筋炎特異的自己抗原である転写中間因子 1γ に対する免疫応答は、実験的筋炎を引き起こす可能性。

ABSTRACT

Objectives

To investigate whether autoimmunity to transcriptional intermediary factor 1 (TIF1)γ, a ubiquitous nuclear autoantigen for myositis-specific autoantibodies detected in patients with dermatomyositis (DM) is pathogenetic for inflammatory myopathy.

Methods

Wild-type, β2-microglobulin-null, perforin-null, Igμ‐null and interferon α/β receptor (IFNAR)-null mice were immunised with recombinant human TIF1γ whole protein. A thymidine incorporation assay was　performed using lymph node T cells from TIF1γ-immunised mice.
Plasma was analysed using immunoprecipitation followed by western blot analysis and enzyme-linked immunosorbent assays. Femoral muscles were　histologically and immunohistochemically evaluated.
CD8+ or CD4+ T cells isolated from lymph node T cells or IgG purified from plasma were adoptively transferred to naïve mice. TIF1γ-immunised mice were treated with anti-CD8 depleting antibody and a Janus kinase inhibitor, tofacitinib.

Results

Immunisation with TIF1γ-induced experimental myositis presenting with necrosis/atrophy of muscle fibres accompanied by CD8+ T cell infiltration successfully in wild-type mice, in which TIF1γ-specific T cells and antihuman and murine TIF1γ IgG antibodies were　detected. The incidence and severity of myositis were significantly lower in β₂-microglobulin-null, perforin-null, CD8-depleted or IFNAR-null mice, while Igμ‐null mice developed myositis normally. Adoptive transfer of CD8+ T cells induced myositis in recipients, while transfer of CD4+ T cells or IgG did not. Treatment with tofacitinib inhibited TIF1γ-induced myositis.

Conclusions

Here we show that TIF1γ is immunogenic enough to cause experimental myositis, in which CD8+ T cells and type I interferons, but not CD4+ T cells, B cells or antibodies, are required. This murine model would be a tool for understanding the pathologies of DM.

概要（仮訳）

目的

転写中間因子 1 (TIF1) γ、皮膚筋炎 (DM) 患者で検出された筋炎特異的自己抗体のユビキタス核自己抗原に対する自己免疫が炎症性ミオパシーの病原性であるかどうかを調査します。

方法

野生型、β2-ミクログロブリン欠損、パーフォリン欠損、Igμ欠損、およびインターフェロンα/β受容体(IFNAR)欠損マウスを組換えヒトTIF1γ全タンパク質で免疫化した。 TIF1γ免疫マウス由来のリンパ節T細胞を用いて、チミジン取り込みアッセイを行った。
血漿は、免疫沈降法、続いてウエスタンブロット分析および酵素結合免疫吸着アッセイを使用して分析されました。大腿筋を組織学的および免疫組織化学的に評価した。
リンパ節 T 細胞から分離した CD8+ または CD4+ T 細胞、または血漿から精製した IgG をナイーブマウスに養子移入しました。 TIF1γ免疫マウスは、抗CD8枯渇抗体およびヤヌスキナーゼ阻害剤であるトファシチニブで処理しました。

結果

TIF1γ 特異的 T 細胞と抗ヒトおよびマウス TIF1γ IgG 抗体が存在する野生型マウスで、CD8+ T 細胞浸潤を伴う筋線維の壊死/萎縮を呈する TIF1γ 誘発実験的筋炎の免疫化が成功しました。筋炎の発生率と重症度は、β2-ミクログロブリン欠損マウス、パーフォリン欠損マウス、CD8枯渇マウス、またはIFNAR欠損マウスで有意に低かったが、Igμ欠損マウスは正常に筋炎を発症した。 CD8+ T 細胞の養子移入はレシピエントに筋炎を誘発したが、CD4+ T 細胞または IgG の移入は誘発しなかった。トファシチニブによる治療は、TIF1γ誘発性筋炎を抑制しました。

結論

ここで、TIF1γ が実験的筋炎を引き起こすのに十分な免疫原性を示すことを示します。この実験では、CD8+ T 細胞と I 型インターフェロンが必要ですが、CD4+ T 細胞、B 細胞、または抗体は必要ありません。このマウスモデルは、DM の病態を理解するための道具になります。

INTRODUCTION
Idiopathic inflammatory myopathies (IIMs) include dermatomyositis (DM), polymyositis, immune-mediated necrotising myopathy (IMNM), inclusion body myositis and antisynthetase syndrome (ASS), characterised by inflammation of muscles and other　organs.1 2 Autoimmunity mediates these diseases, as　a number of myositis specific autoantibodies have been identified3–6 and are associated with distinct clinical features.7 In DM, five autoantibodies have been identified: anti-Mi-2, antimelanoma differentiation-associated gene 5, antitranscriptional intermediary factor 1 (TIF1),8 9 antinuclear matrix protein 2 and antismall ubiquitin-like modifier activating enzyme. While autoantibodies against various nuclear/cytoplasmic components serve as diagnostic tools for systemic autoimmune diseases, a direct causative role for most of them has been questioned. TIF1γ, a major antigen of anti-TIF1 antibodies, is a 155 kDa nuclear protein belonging to the tripartite motif superfamily.8 9 Anti-TIF1γ antibody is present in a quarter of adult/juvenile patients with DM10 11 and is associated with an increased risk of malignancies in elderly patients.12–14 TIF1γ was found to be overexpressed not only in tumours15 but also in muscle tissues, especially in regenerating atrophic perifascicular myofibers and in skin.16 17 Our observations revealed that pregnancy might trigger the development of anti-TIF1γ antibody-positive DM,18 which would be related to overexpression of TIF1γ antigen in the embryo and mammary epithelial cells during pregnancy.19 20 While this evidence suggests the aetiologic roles of TIF1γ, it remains unknown whether autoimmunity to TIF1γ is directly involved in disease pathogenesis. Here we show that experimental myositis can develop following immunisation with recombinant TIF1γ protein.

序章　（仮訳）

特発性炎症性ミオパシー（IIM）には、皮膚筋炎（DM）、多発性筋炎、免疫介在性壊死性ミオパシー（IMNM）、封入体筋炎、抗シンテターゼ症候群（ASS）を含み、筋肉や他の臓器の炎症を特徴とします.1 2 自己免疫がこれらの疾患を媒介します。多くの筋炎特異的自己抗体が同定されており 3-6 、明確な臨床的特徴と関連しています.7 DMでは、抗Mi-2、抗メラノーマ分化関連遺伝子5、抗転写中間因子1（TIF1）、 8 9 抗核マトリックスタンパク質 2 およびアンチスモールユビキチン様修飾因子活性化酵素。さまざまな核/細胞質成分に対する自己抗体は、全身性自己免疫疾患の診断ツールとして機能しますが、それらのほとんどに対する直接の原因となる役割は疑問視されています。抗 TIF1 抗体の主要な抗原である TIF1γ は、三者モチーフスーパーファミリーに属する 155 kDa の核タンパク質です。 12–14 TIF1γ は、腫瘍 15 だけでなく筋肉組織、特に萎縮性線維束周囲筋線維の再生中および皮膚でも過剰発現していることが判明しました. この証拠は、TIF1γの病因学的役割を示唆しているが、TIF1γに対する自己免疫が疾患の病因に直接関与しているかどうかは不明のままである. . ここでは、実験的筋炎が組換えTIF1γタンパク質による免疫後に発症する可能性があることを示しています。

Figure 1

Development of experimental myositis dependent on the immune response to transcriptional intermediary factor 1 (TIF1)γ. (A) Sodium dodecyl sulphate-polyacrylamide　gel electrophoresis (second lane) followed by a western blotting assay using anti-TIF1γ antibodies (third lane)　revealed that the purified protein has a molecular weight (MW) of about 150 kDa, which is consistent with TIF1γ whole protein. MW markers are　shown in first lane. (B) The upper panel shows our immunisation protocols. Mice received intraperitoneal injection (i.p.) of pertussis toxin (PT), and intradermal injection of an emulsion of complete Freund’s adjuvant with/without TIF1γ protein at the back and foot pads. The graph shows histological scores for experimental　myositis in the hamstrings and quadriceps of mice immunised once (n=8)　and four times (n=10) with TIF1γ whole protein, and mice immunised with　adjuvant only four times (n=6). Dots and bars represent individuals and medians with interquartile ranges (IRs), respectively. *p<0.05 by Kruskal-Wallis　test with Dunn’s multiple comparisons test. (C–E) Representative myositis (yellow arrows) with muscle fibre atrophy (C) or necrosis (D), and perifascicular atrophy (E) observed in H&E-stained　sections of muscle tissues from TIF1γ-immunised mice. Bars
represent 50 μm. (F) 3H thymidine incorporation (cpm) in T cells from regional lymph nodes of TIF1γ-immunised mice (n=3) co-cultured
with bone marrow-derived dendritic cells (BMDCs) presenting TIF1γ. T cells from mice immunised BMDCs pulsed with adjuvant only (n=3) or without antigen were used as control T cells and control BMDCs, respectively. Bars represent means with SE of the mean (SEM). *p<0.05, and **p<0.01 by two-way　ANOVA with Tukey’s multiple comparisons test. (G) IgGs binding TIF1γ antigens from K562 and EL-4 cell lysates, the MWs of which were between　150 and 250 kDa, were detected in the plasma of TIF1γ-immunised mice (n=4), but not in plasma from mice immunised with adjuvant only (n=3). (H)Enzyme-linked　immunosorbent assay　conducted with plasma from TIF1γ-immunised mice (n=10) and mice　immunised with adjuvant only (n=7). The titre index calculation method　is detailed in online supplemental materials and methods. Dots and bars represent individuals and means with SEM, respectively. ***p<0.001 by Student’s t-test. Data are representative of two independentexperiments.

図 1 転写中間因子（仮訳）

1 (TIF1)γ に対する免疫応答に依存する実験的筋炎の発症。 (A) >ドデシル硫酸ナトリウム - ポリアクリルアミドゲル電気泳動 (2 番目のレーン) に続いて、抗 TIF1γ 抗体を使用したウェスタンブロッティングアッセイ (3 番目のレーン) により、精製されたタンパク質の分子量 (MW) が約 150 kDa であることが明らかになりました。 TIF1γ全タンパク質を使用。 MWマーカーは最初のレーンに表示されます。 (B) 上のパネルは、私たちの予防接種プロトコルを示しています。百日咳毒素（PT）の腹腔内注射（i.p.）と、TIF1γタンパク質を含む/含まない完全フロイントアジュバントのエマルジョンを、マウスの背部と足蹠に皮内注射した。グラフは、TIF1γ 全タンパク質で 1 回 (n=8) および 4 回 (n=10) 免疫したマウス、およびアジュバントのみで 4 回免疫したマウス (n=6) のハムストリングスおよび大腿四頭筋における実験的筋炎の組織学的スコアを示しています。ドットとバーは、それぞれ四分位範囲 (IR) を持つ個人と中央値を表します。 *Dunn の多重比較検定を使用した Kruskal-Wallis 検定による p<0.05。 (C–E) TIF1γ免疫マウスの筋組織のH&E染色切片で観察された、筋線維萎縮(C)または壊死(D)、および線維束周囲萎縮(E)を伴う代表的な筋炎(黄色の矢印)。バーは 50 μm を表します。 (F) TIF1γを提示する骨髄由来樹状細胞 (BMDC) と共培養した TIF1γ 免疫マウス (n=3) の局所リンパ節由来の T 細胞における 3 H チミジン取り込み (cpm)。アジュバントのみ（ｎ＝３）または抗原なしでパルスしたマウス免疫化ＢＭＤＣからのＴ細胞を、それぞれ対照Ｔ細胞および対照ＢＭＤＣとして使用した。バーは、平均の SE (SEM) で平均を表します。 * p < 0.05、および ** p < 0.01 は、Tukey の多重比較検定を使用した双方向 ANOVA による。 (G) K562 および EL 4 細胞溶解物からの TIF1γ 抗原に結合する IgG は、その分子量が 150 ～ 250 kDa であり、TIF1γ 免疫マウス (n = 4) の血漿で検出されましたが、免疫マウスの血漿では検出されませんでした。アジュバントのみ (n=3)。 (H) TIF1γ免疫マウス(n=10)およびアジュバントのみで免疫したマウス(n=7)からの血漿を用いて実施した酵素免疫測定法。力価指数の計算方法は、オンラインの補足資料および方法で詳しく説明されています。ドットとバーは、それぞれSEMでの個人と平均を表します。 ***スチューデントの t 検定による p < 0.001。データは、2 つの独立した実験の代表です。

Figure 2

Infiltration of inflammatory cells and upregulation of major histocompatibility complex (MHC) class I molecules in muscle tissues. (A–D) Immunohistochemical (IHC) analyses of CD8 (A), CD4 (B), CD11b (C) and B220 (D) on the mononuclear cells infiltrating into the endomysium areas of the muscle tissues of TIF1γ-immunised mice. (E–G) IHC analyses of MHC class I molecules on the cell membranes of the muscle fibres in control adjuvant-treated mice (E) and in TIF1γ- immunised mice (F), compared with the isotype-control antibody-stained samples from TIF1γ immunised mice (G) Data are representative of three independent experiments.

図 2(仮訳)

筋肉組織における炎症細胞の浸潤と主要組織適合遺伝子複合体 (MHC) クラス I 分子のアップレギュレーション。 (A–D) TIF1γ 免疫マウスの筋肉組織の筋内膜領域に浸潤する単核細胞に対する CD8 (A)、CD4 (B)、CD11b (C)、および B220 (D) の免疫組織化学 (IHC) 分析。 (E–G) コントロールアジュバント処理マウス (E) および TIF1γ 免疫マウス (F) の筋線維の細胞膜上の MHC クラス I 分子の IHC 分析と、アイソタイプコントロール抗体染色サンプルとの比較。 TIF1γ 免疫マウス (G) データは 3 つの独立した実験の代表です。

METHODS

Mice

Female C57BL/6 (B6) mice 8–10 weeks of age were purchased from Charles River. Beta2-microglobulin (β2MG)-null, perforin-null and Igμ-null (μMT) B6 mice and interferon α/β receptor (IFNAR)-null B6 mice21 were purchased from The Jackson Laboratory and B&K Universal. All experiments were carried out under specific pathogen-free conditions in accordance with the University of Tsukuba’s ethics and safety guidelines for animal experiments.

Recombinant human TIF1γ protein

A full-length human TIF1γ gene (GenBank accession number: AF119043) was His-tagged at its 3’ end and inserted into pFastBac1 vector for baculovirus expression (invitrogen).The detailed protocol for the expression and purification of recombinant TIF1γ protein is described in online supplemental materials and methods. Human TIF1γ whole protein is 93.3% homologous with the murine protein.

Induction of experimental myositis

Mice were immunised intradermally with 200 μg of TIF1γ protein
emulsified in complete Freund’s adjuvant (CFA) containing 100 μg of heat‐killed Mycobacterium butyricum (Difco) once in the back and in foot pads along with an intraperitoneal injection of 250 ng of pertussis toxin (PT, Wako Junyaku). Other mice were immunised intradermally with the CFA emulsion containing TIF1γ protein four times weekly at multiple sites of the back and foot pads. The same time as the last (fourth) intradermal injection of the emulsion, 250 ng of PT was once injected intraperitoneally. Mice treated with CFA (weekly, 4 times) and PT were used as controls. These immunisation protocols are presented in figure 1B. Following the evaluation method for C protein-induced myositis (CIM),22 three H&E-stained sections from the hamstring and quadricep each were blinded to the intervention and examined histologically for necrosis/atrophy of muscle fibres accompanied by mononuclear cell infiltration. The scoring system is detailed in online supplemental materials and methods.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting assay and immunoprecipitation followed by western blot assay

Fifty microliters of mouse plasma were combined with 50 μL of Protein G Sepharose 4 Fast Flow (GE Healthcare) for 2 hours at room temperature. Antibody-bound
sepharose beads were washed with immunoprecipitation (IPP) buffer (10 mM tris-HCl, pH 8.0; 50 mM NaCl and 0.1% 4-nonylphenyl- polyethylene glycol (BioVision)) and incubated with extracts from 1×107 K562 human cells and EL-4 murine cells (American Type Culture Collection (ATCC)), respectively, for 2 hours at 4℃. Purified recombinant TIF1γ protein, K562-precipitated protein and EL-4-precipitated protein were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% polyacrylamide gels, applied to Mini-PROTEANTGX precast gels (4%–15%, Bio-Rad Laboratories). The gel on which recombinant TIF1γ protein was applied was stained with Bio-Safe Coomassie Stain (Bio-Rad Laboratories). For western blotting (WB) assay, proteins were transferred onto nitrocellulose membranes using a wet transfer apparatus (Mini Trans-Blot Cell, Bio-Rad Laboratories) from the gels. The membranes were blocked with 5% skim milk and incubated with rabbit antihuman/murine TIF1γ polyclonal antibody (NB100-57498, Novus Biologicals), antihuman TIF1γ polyclonal antibodies (LS-C408048, LifeSpan Biosciences) and antimurine TIF1γ polyclonal antibodies (ab47062; Abcam), respectively, overnight at 4℃. They were washed with tris-buffered saline with Tween 20, incubated with peroxidase-labelled goat antirabbit IgG polyclonal antibodies (sc-2004, Santa Cruz Biotechnology) and then visualised using SuperSignal West Pico (Thermo Fisher Scientific).

方法(仮訳)

マウス

8 ～ 10 週齢のメスの C57BL/6 (B6) マウスを Charles River から購入しました。 Beta2-microglobulin (β2MG)-null、perforin-null、および Igμ-null (μMT) B6 マウスおよびインターフェロン α/β 受容体 (IFNAR)-null B6 マウスは、Jackson Laboratory および B&K Universal から購入しました。すべての実験は、筑波大学の動物実験に関する倫理および安全ガイドラインに従って、特定の無菌条件下で実施されました。

組換えヒト TIF1γ タンパク質

完全長のヒト TIF1γ 遺伝子 (GenBank アクセッション番号: AF119043) の 3' 末端に His タグが付けられ、バキュロウイルス発現用の pFastBac1 ベクター (invitrogen) に挿入されました。組換え TIF1γ タンパク質の発現と精製に関する詳細なプロトコルは、オンラインの補足資料と方法。ヒト TIF1γ 全タンパク質は、マウスタンパク質と 93.3% の相同性があります。

実験的筋炎の誘導

マウスは、200μgのTIF1γタンパク質で皮内免疫されました
百日咳毒素（PT、和光純薬）250 ngの腹腔内注射とともに、100μgの加熱殺菌されたMycobacterium butyricum（Difco）を含む完全フロイントアジュバント（CFA）で乳化させた。他のマウスは、ＴＩＦ１γタンパク質を含有するＣＦＡエマルジョンを週４回、背中および足蹠の複数の部位で皮内に免疫した。最後（４回目）のエマルションの皮内注射と同時に、２５０ｎｇのＰＴを腹腔内に１回注射した。 CFA（毎週、4回）およびPTで処理されたマウスをコントロールとして使用しました。これらの予防接種プロトコルは、図 1B に示されています。 C タンパク質誘発性筋炎 (CIM) の評価方法 22 に従って、ハムストリングと大腿四頭筋のそれぞれの 3 つの H&E 染色切片を介入に対して盲検化し、単核細胞浸潤を伴う筋線維の壊死/萎縮について組織学的に調べた。採点システムについては、オンラインの補足資料と方法で詳しく説明されています。

ドデシル硫酸ナトリウム - ポリアクリルアミドゲル電気泳動およびウエスタンブロットアッセイおよび免疫沈降、その後のウエスタンブロットアッセイ

50マイクロリットルのマウス血漿を50μLのProtein G Sepharose 4 Fast Flow (GE Healthcare)と室温で2時間混合しました。抗体結合セファロースビーズを免疫沈降 (IPP) バッファー (10 mM トリス-HCl、pH 8.0; 50 mM NaCl および 0.1% 4-ノニルフェニル-ポリエチレングリコール (BioVision)) で洗浄し、1 × 107 K562 ヒト細胞からの抽出物および EL- マウス細胞 (American Type Culture Collection (ATCC)) 4 個、4℃で 2 時間。精製組換え TIF1γ タンパク質、K562 沈殿タンパク質、および EL-4 沈殿タンパク質を、10% ポリアクリルアミドゲルを使用したドデシル硫酸ナトリウム - ポリアクリルアミドゲル電気泳動 (SDS-PAGE) によって分画し、Mini-PROTEANTGX プレキャストゲル (4% ～ 15%) に適用しました。バイオ・ラッド・ラボラトリーズ）。組換えTIF1γタンパク質を適用したゲルをBio-Safe Coomassie Stain (Bio-Rad Laboratories)で染色した。ウェスタンブロッティング（WB）アッセイでは、タンパク質をゲルから湿式転写装置（Mini Trans-Blot Cell、Bio-Rad Laboratories）を使用してニトロセルロース膜に転写しました。メンブレンを 5% スキムミルクでブロックし、ウサギ抗ヒト/マウス TIF1γ ポリクローナル抗体 (NB100-57498、Novus Biologicals)、抗ヒト TIF1γ ポリクローナル抗体 (LS-C408048、LifeSpan Biosciences)、および抗マウス TIF1γ ポリクローナル抗体 (ab47062; Abcam) とインキュベートしました。、それぞれ、4℃で一晩。それらをTween 20を含むトリス緩衝生理食塩水で洗浄し、ペルオキシダーゼ標識ヤギ抗ウサギIgGポリクローナル抗体（sc-2004、Santa Cruz Biotechnology）とともにインキュベートし、SuperSignal West Pico（Thermo Fisher Scientific）を使用して視覚化しました。

Figure 3

TIF1γ-specific B cell linages and antibodies are not required for the development of TIF1γ-induced myositis. (A) Histologic scores for experimental myositis in hamstrings and quadriceps of μMT mice immunised with TIF1γ whole protein (n=9) were equal to TIF1γ- immunised wild-type (WT) mice (n=12). Dots and bars represent individuals and medians with IRs, respectively. (B) Histologic scores for experimental myositis in the hamstrings and quadriceps of recipient mice (n=12) with adoptive intravenous transfer of IgGs purified from pooled plasma of TIF1γ-immunised mice or control recipients (n=10) of IgGs from pooled plasma of mice immunised with adjuvant only. Dots and bars represent individuals and medians with IRs, respectively. Data are representative of two independent experiments. IRs, interquartile ranges.

図 3(仮訳) TIF1γ 特異的 B 細胞系統および抗体は、TIF1γ 誘発性筋炎の発症には必要ありません。 (A) TIF1γ全タンパク質で免疫化されたμMTマウス(n=9)のハムストリングスおよび大腿四頭筋における実験的筋炎の組織学的スコアは、TIF1γで免疫化された野生型(WT)マウス(n=12)と等しかった。ドットとバーは、それぞれ IR を持つ個人と中央値を表します。 (B) レシピエントマウス (n = 12) のハムストリングスおよび大腿四頭筋における実験的筋炎の組織学的スコア。アジュバントのみで免疫したマウス。ドットとバーは、それぞれ IR を持つ個人と中央値を表します。データは、2 つの独立した実験の代表です。 IR、四分位範囲。

Figure 4

TIF1γ-specific CD8+ T cells are critical for the development of TIF1γ-induced myositis. (A) Histological scores for experimental myositis in the hamstrings and quadriceps of WT mice (n=11), β2-microgloblin (β2MG)-null mice (n=8), and perforin-null
mice (n=10) 2 weeks after fourth immunisation with TIF1γ whole protein. Dots and bars represent individuals and medians with IRs, respectively. *p<0.05, and **p<0.01 by Kruskal-Wallis test with Dunn’s multiple comparisons test. (B) Histological scores for experimental myositis of TIF1γ-immunised mice treated with anti-CD8 depleting antibody (n=7) compared with those treated with control antibody (n=7). Dots and bars represent individuals and medians with IRs, respectively. **p<0.01 by Mann-Whitney U test. (C) Histological scores for experimental myositis in the hamstrings and quadriceps of recipient mice (n=10) following adoptive intravenous transfer of TIF1γ-activated T cells originally purified from pooled lymph node cells of TIF1γ-immunised mice compared with TIF1γ-activated T cells originally from pooled lymph node cells of mice immunised with adjuvant only (n=10). Dots and bars represent individuals and medians with IRs, respectively. *p<0.05 by Mann-Whitney U test. (D) Representative myositis (yellow arrows) in HE-stained sections of muscle tissues from TIF1γ-specific T cell-recipients. Bars represent 50 μm. (E) Histological scores for experimental myositis in hamstrings and quadriceps of TIF1γ-CD8 recipient mice (n=10) following adoptive intravenous transfer of CD8+ T cells purified from TIF1γ-specific T cells compared with transfer of TIF1γ-CD4 recipient mice (n=8) with adoptive intravenous transfer of CD4+ T cells purified from TIF1γ-specific T cells and control CD8 recipients (n=5) following adoptive intravenous transfer of CD8+ T cells purified from T cells of mice immunised with adjuvant only. Lack of myositis in control CD4 recipients (n=2) following adoptive intravenous transfer of CD4+ T cells purified from T cells of mice immunised with adjuvant only. Dots and bars represent individuals and medians with IRs, respectively. **p<0.01 by Kruskal-Wallis test with Dunn’s multiple comparisons test. Data are representative of two independent experiments. IRs, interquartile ranges.

図 4(仮訳) TIF1γ 特異的 CD8+ T 細胞は、TIF1γ 誘発性筋炎の発症に重要です。 (A) WT マウス (n=11)、β2-ミクログロブリン (β2MG)-null マウス (n=8)、およびパーフォリン-null のハムストリングスおよび大腿四頭筋における実験的筋炎の組織学的スコア

マウス (n = 10) TIF1γ 全タンパク質による 4 回目の免疫の 2 週間後。ドットとバーは、それぞれ IR を持つ個人と中央値を表します。 *p < 0.05、および ** p < 0.01 は、Dunn の多重比較検定を使用した Kruskal-Wallis 検定による。 (B) コントロール抗体 (n = 7) で処理したマウスと比較した、抗 CD8 枯渇抗体 (n = 7) で処理した TIF1γ 免疫マウスの実験的筋炎の組織学的スコア。ドットとバーは、それぞれ IR を持つ個人と中央値を表します。 **p<0.01 マン・ホイットニー U 検定による。 (C) TIF1γ活性化T細胞と比較した、TIF1γ免疫マウスのプールされたリンパ節細胞から元々精製されたTIF1γ活性化T細胞の養子静脈内移入後のレシピエントマウス(n=10)のハムストリングスおよび大腿四頭筋における実験的筋炎の組織学的スコアアジュバントのみで免疫したマウスのプールされたリンパ節細胞に由来します (n=10)。ドットとバーは、それぞれ IR を持つ個人と中央値を表します。 *マン・ホイットニーのU検定によるp<0.05。 (D) TIF1γ特異的T細胞レシピエントからの筋肉組織のHE染色切片における代表的な筋炎(黄色の矢印)。バーは 50 μm を表します。 (E) TIF1γ-CD4 レシピエントマウスの移植と比較した、TIF1γ-特異的 T 細胞から精製した CD8+ T 細胞の養子静脈内移植後の TIF1γ-CD8 レシピエントマウス (n = 10) のハムストリングスおよび大腿四頭筋における実験的筋炎の組織学的スコア (n = 10) 8) アジュバントのみで免疫したマウスの T 細胞から精製した CD8+ T 細胞を養子静脈内移入した後、TIF1γ 特異的 T 細胞およびコントロール CD8 レシピエント (n=5) から精製した CD4+ T 細胞を養子静脈内移入した。アジュバントのみで免疫したマウスの T 細胞から精製した CD4+ T 細胞の養子静脈内移入後のコントロール CD4 レシピエント (n=2) における筋炎の欠如。ドットとバーは、それぞれ IR を持つ個人と中央値を表します。 ** p < 0.01、ダンの多重比較検定を使用したクルスカル・ウォリス検定による。データは、2 つの独立した実験の代表です。 IR、四分位範囲。

Enzyme-linked immunosorbent assay
The plasma samples collected from immunised mice 2 weeks
after the last immunisation were evaluated in our established
enzyme-linked
immunosorbent assay (ELISA) system (online
supplemental materials and methods).
Immunohistochemical analyses
CD8, CD4, CD11b and B220-positive
cells and upregulation of
H‐2Kb molecules were detected on sections from muscle tissue
samples (online supplemental materials and methods).
In vivo depletion of CD8+ T cells
To deplete CD8+T cells, mice were intraperitoneally injected
with purified rat anti-CD8α
depleting monoclonal antibody
(53.67.2) in the protocol as shown previously22 and as described
in online supplemental materials and methods.
Adoptive transfer of T cells or IgG
T cells were purified from the inguinal and popliteal lymph
node (LN) cells of immunised mice 2 weeks after the fourth
immunisation using CD3 T cell enrichment columns (R&D
systems). Three million T cells were cultured with 1.5×105
TIF1γ-pulsed mature bone marrow-derived
dendritic cells
(BMDCs, generated in the protocol shown in online supplemental
materials) and 100 U/mL recombinant murine IL-2
(PeproTec) in 2 mL RPMI1640 with 10% fetal bovine serum
(FBS) for 72 hours using 24-well
culture plates. CD8‐positive
or CD4‐positive T cells were sorted with MACS magnetic
beads (Miltenyi Biotech). Flow cytometric analyses showed
that the purities of the sorted CD8+ and CD4+ T cells were

95%, and that CD11c-positive
DCs were absent. IgG was
purified from the plasma of immunised mice 2 weeks after
the fourth immunisation using protein G columns (Ab-Rapid
SPiN Ex, ProteNova). One million whole T cells, 4×105
CD8-positive
or CD4-positive
T cells or 500 μg of IgG were
intravenously injected into recipient mice that had been
pretreated with CFA.23 The muscles of the hind legs were
evaluated histologically 2 weeks after transfer.

Figure 5 Type I interferon (IFN) signalling in the pathogenesis of TIF1γ-induced myositis. (A) Fold changes in mRNA levels of type I IFN-related

genes, Mx1, Isg15, Osa1 and Osa3, which were normalised against β-actin mRNA levels, in muscle tissues from naïve, adjuvant-treated,
and TIF1γ-
immunised mice 2 weeks after fourth immunisation. n=2–6 in each group. Bars represent the means with SEMs. *p<0.05, and **p<0.01 by ordinary
one-way
analysis of variance. (B) Histological scores for experimental myositis in the hamstrings and quadriceps of wild-type
(n=5) and IFN α/β
receptor-null
mice (n=6) 2 weeks after the fourth TIF1γ immunisation. Dots and bars represent individuals and the medians with IRs, respectively.
*p<0.05 by Mann-Whitney
U test.

Real-time quantitative polymerase chain reaction analyses

As shown in online supplemental materials and methods and
online supplemental table 1), real-time
quantitative polymerase
chain reaction (RT-qPCR)
analyses were performed
on total RNA extracted from the muscle tissue samples.

Treatment with the Janus kinase inhibitor tofacitinib

In accordance with a previous report,24 tofacitinib (MedChemExpress)
in 0.5% methylcellulose/0.025% Tween 20 was
orally administrated to mice at 12.5 or 50 mg/kg Twice daily
from the day of fourth immunisation of TIF1γ.

Statistical analysis

Data were analysed with Prism 8 (GraphPad Software). P
values less than 0.05 were considered significant.

RESULTS

TIF1γ-immunised mice develop experimental myositis

SDS-PAGE
and WB revealed that the purified recombinant
human whole TIF1γ protein contained few contaminants
(figure 1A). Wild-type
(WT) B6 mice immunised with the
TIF1γ protein four times weekly developed myositis 2
weeks after the fourth immunisations in their hamstrings
and quadriceps as assessed histologically. The incidence
rate and the median ((IQRs) of histologic scores were
70% and 0.5 (0–0.5), while none of the mice treated only
with adjuvant was affected (p=0.0143; figure 1B). The
mice immunised once with TIF1γ rarely did (12.5% and
0 [0–0]; figure 1B) as well as the mice treated only with
adjuvant (p>0.9999). No mice exhibited significant weight
loss during the observation period. Histologic analysis of
muscles from the mice immunised with TIF1γ four times
showed atrophy and necrosis of muscle fibres accompanied
by infiltrating mononuclear cells in the perifascicular
and endomysial sites of the muscle tissue (figure 1C,D),
sometimes (in one per five samples) typical perifascicular
atrophy (figure 1E). Myositis was still observed in 60% of
the immunised mice 3 weeks after the fourth immunisation
(incidence rate, 60%; median (IQRs) of histologic score,
0.500 (0–1.250); n=5), and some mice also presented
myositis 1 week later (60%, 0.500 (0–0.625); n=5). No
inflammation was observed in other organs, including skin,
cardiac muscles and lungs.
Thymidine incorporation assay was performed as shown
in online supplemental materials and methods. T cells
from mice with TIF1γ-induced myositis (TIM) proliferated
significantly more than T cells from control mice treated
only with adjuvants when cocultured with TIF1γ-presenting
BMDCs (the means±SEMs of 3H thymidine incorporation
were 35899±7411 and 3940±2086 (cpm), respectively;
p=0.0022). T cells from TIM or control mice did not proliferate
when cocultured with BMDCs presenting no specific
antigen (11055±3156 and 3600±1782 (cpm), respectively,
p=0.6218; figure 1F). IPP-WB
analysis demonstrated the
existence of not only antihuman TIF1γ antibodies reacting
to the human cell (K562) lysate but also antimurine TIF1γ
autoantibodies reacting to the murine cell (EL-4) lysate in
the plasma from TIM mice, but not in that from CFA-treated
control mice (figure 1G). Our ELISA system demonstrated
higher titers of anti-TIF1γ
antibodies in TIM mice (the
mean±SEM of titre index was 82.8±2.2) compared with
control mice (0.1±0.5, p=0.0005; figure 1H).
CD8+ T cells predominantly adhere to muscle fibres, which
upregulate major histocompatibility complex class I
molecules, in the muscle tissues of TIM mice
Immunohistochemical analyses of the muscle tissues of
TIM mice revealed that CD8+ cells predominantly infiltrated
into the endomysium areas and adhered to the muscle
fibres (figure 2A). On the other hand, only a few CD4+ cells
(figure 2B), CD11b+ macrophages (figure 2C), and B220+
B cells (figure 2D) infiltrated into the endomysium areas.
Moreover, major histocompatibility complex (MHC) class
I molecules were upregulated on the cell membranes of the
muscle fibres in TIM mice (figure 2F) compared with those
in control adjuvant-treated
mice (figure 2E) and the isotype-control
antibody-stained
TIM samples (figure 2G).

Figure 6 Inhibitory effect of tofacitinib on TIF1γ-induced myositis. (A) Histological scores for experimental myositis in the hamstrings and

quadriceps of TIF1γ-immunised mice treated with low dose (12.5 mg/kg, two times per day; n=7) or high dose (50 mg/kg, two times per day; n=7)
tofacitinib from the fourth day of TIF1γ immunisation compared with control vehicle-treated
TIF1γ-immunised mice (n=6) and control vehicle-treated
mice immunised with adjuvant alone. Dots and bars represent individuals and medians with IRs, respectively. *p<0.05, and **p<0.01 by Kruskal-Wallis
test with Dunn’s multiple comparisons test. (B–E) Representative H&E-stained
sections of muscle tissues from TIF1γ-immunised mice treated
with control vehicle (B) low-dose
tofacitinib, (C) high-dose
tofacitinib and (D) control vehicle-treated
mice immunised without any antigens (E).
Yellow arrows show myositis and bars represent 50 μm. (F) Total T cells in the regional lymph nodes per mouse. TIF1γ-immunised treated vehicle
control (n=5), low-dose
tofacitinib (n=5) and high-dose
tofacitinib (n=5), and vehicle control-treated
mice immunised without any antigens (n=3),
were counted after T cell purification. Dots/squares/triangles and bars represent medians and interquartile ranges, respectively. (G) Proliferation of
purified T cells from TIF1γ-immunised mice treated with vehicle, low-dose
tofacitinib, and high-dose
tofacitinib compared with those from control
vehicle-treated
mice immunised without any antigens when co-cultured
with bone marrow-derived
dendritic cells presenting TIF1γ (DC-TIF1γ)
or with
dendritic cells lacking antigen. Bars represent means with SEMs. ***p<0.001, and ****p<0.0001 by two-way
analysis of variance (ANOVA) with
Tukey’s multiple comparisons test. (H) ELISA of plasma from TIF1γ-immunised mice treated with control vehicle, low-dose
tofacitinib, or high-dose
tofacitinib (n=6–7, each) compared with mice immunised with adjuvant alone (n=3). Dots and bars represent individuals and means with SEMs,
respectively. **p<0.05, **p<0.01, and ***p<0.0001 by ordinary one-way
ANOVA.

TIF1γ-specific B cell linages and antibodies are not required

for the initiation of TIM
μMT mice, which completely lack B cell lineages, developed
myositis (the incidence and the median±IQR of histologic
scores were 67% and 1.000 (0–1.250)) at a similar incidence and
severity as observed in WT mice (83% and 0.625 (0.500–1.000),
p=0.9014) when immunised with TIF1γ emulsion (figure 3A).
Moreover, intravenous adoptive transfer of the IgG purified
from pooled plasma of TIM mice did not induce myositis in
recipient mice (figure 3B).

TIF1γ-specific CD8+ T cells are involved in the initiation of TIM

β2MG-null
mice lacking MHC class I expression and perforin-null
mice rarely developed TIM (figure 4A). While the incidence
rate and the median histologic score with IQRs for TIM were
82% and 1.000 (0.500–1.500) in WT mice, they were 29% and
0 (0–0.375) in β2MG-null
and 40% and 0 (0–0.500) in perforin-null
mice (p=0.0054 and p=0.0123 vs WT mice, respectively).
Mice treated with anti-CD8
depleting antibody rarely presented
TIM (the incidence rate and the median histologic score with
IQRs, 71.4% and 0.500 (0–0.500)) compared with control mice
(100% and 1.250 (1.250–1.500), p=0.0006; figure 4B).
Moreover, adoptive transfer of enriched TIF1γ-specific T
cells derived from TIM mice-induced
TIM-like
myositis with an
incidence of 50% in naïve recipient mice (the median (IQRs) of
histologic scores was 0.250 (0–0.500)), while transfer of CFA-treated
mouse-derived
T cells stimulated by TIF1γ-presenting
BMDCs did not (p=0.0325, figure 4C,D). Adoptive transfer
of CD8+ T cells from TIM mice-induced
myositis with a high
incidence (90%) as well as muscle damage (the median (IRs) of
histologic scores was 0.750 (0.500–1.063)); however, transfer of
CD4+ T cells from TIM mice did not (p=0.0010) nor did CD8+
T cells from CFA-treated
control mice (p=0.0067, figure 4E).
Type I interferon partially mediates the pathogenesis of TIM
Our RT-qPCR
analyses revealed that the mRNA expression
of type I interferon (IFN)-related
genes, Mx1 and Osa3, was
significantly upregulated in the muscle tissues of the TIM mice
(the mean±SEM, 11.3±4.49 and 3.66±0.11) compared with
those of naïve mice (0.86±0.14 and 0.95±0.11; p=0.0205 and
0.0063, respectively); however, Osa3 mRNA expression was
also upregulated in adjuvant-treated
control mice (5.96±1.00,
p=0.0019 vs naïve mice, figure 5A). Upregulation of mRNA
expression for other type I IFN-related
genes, Isg15 and Oas1,
was not observed in the muscle tissues of TIM mice or control
mice (figure 5A). IFNAR-null
mice developed milder myositis
(the medians (IQRs) of the histological scores, 1.250 (0.750–
1.500)) than WT mice after TIF1γ immunisations (2.000
(2.250–1.500), p=0.0433, figure 5B).
Treatment with a JAK inhibitor, tofacitinib, inhibits the
development of TIM
TIF1γ-immunised mice treated with high dose (50 mg/kg, two
times per day) or low dose (12.5 mg/kg, two times per day) of
tofacitinib starting on the fourth day of TIF1γ immunisation
developed milder myositis (the medians (IQRs) of histologic
scores were 0.500 (0–0.500) and 0.500 (0–0.7500), respectively)
at a lower incidence rate (57% and 57%, respectively)
than vehicle control-treated
mice (1.500 (0.938–1.563);
p=0.0075 and 0.0873, respectively; 100% incidence; figure).
T cell counts in regional LNs of mice treated with low/high-dose
tofacitinib did not differ from those of vehicle-treated
mice (the medians (IRs) of T cell counts were 72 [45 – 110]
and 43 [36 – 52] vs 68 [59 – 75] [×105], p>0.9999 and
p=0.0909, figure 6F). Moreover, there was no inhibition of
ex vivo proliferation of T cells purified from regional LNs
of tofacitinib-treated
TIF1γ-immunised mice when cocultured
with TIF1γ-presenting BMDCs (figure 6G). This effect
was significant when compared with that of mice immunised
with adjuvant alone (p=0.0002, 0.0007 and 0.0009 for
TIF1γ-immunised mice treated with vehicle, low-dose
and
high-dose
tofacitinib, respectively). Index values of TIF1γ-specific
antibodies in low-dose
and high-dose
tofacitinib-treated
TIF1γ-immunised mice (the means±SEMs were 84.7±15.3 and
131.0±17.1, respectively) were also equal to those in vehicle-treated
TIF1γ-immunised mice (110.5±7.6; p=0.3713 and
0.3713, respectively; figure 6H).
DISCUSSION
Our findings indicate that immunity to TIF1γ can contribute
to the development of myositis in mice. This is the first study
to demonstrate the immune response to a DM-specific
autoantigen
that can induce myositis. Therefore, this new experimental
murine model, which we termed TIM, closely mimics human
pathogenesis, especially in the initiation phase.
A number of animal models for IIMs have been established,
including infectious, genetic and antigen-induced
models.25 26 While experimental autoimmune myositis27 28 and
CIM22 29 completely depend on immune responses specific to
muscular antigens, myosin and C protein, TIM is induced via
autoimmunity generated in response to a ubiquitous intracellular
molecule, which has been identified as an autoantigen in
humans suffering DM. In addition to a previous report showing
that muscle and lung inflammation could be induced by immunisation
with purified epitopic peptides derived from conspecific
histidyl-tRNA
synthetase (Jo-1) as a murine model for ASS,30
our results indicate that experimental myositis can be induced
by immunisation with the DM-specific
autoantigen. In our TIM
model, mice immunised by xenogeneic (human) TIF1γ protein
developed autoimmunity to conspecific TIF1γ resulting in
experimental myositis, which might be due to epitope spreading
to a counterpart conspecific molecule as shown in experimental
autoimmune encephalomyelitis.31
TIM is initiated by cytotoxic CD8+ T cells, which evokes
infiltration of CD8+ T cells and MHC class I upregulation in
muscle fibres of patients with IIM.32–36 While it has also been
proven that genetically modified mice with overexpression of
MHC class I in muscle tissues naturally develop myositis via
endoplasmic reticulum (ER) stress,34 37 our experiments showed
that adoptive transfer of TIF1γ-specific CD8+ T cells, but not of
TIF1γ-specific CD4+ T cells, caused myositis in recipient mice.
This suggests that autoaggressive CD8+ T cells are indicative of
the development of myositis.
In contrast, B cells and autoantibodies themselves are not
required for the development of TIM. Previous clinical reports
indicated that the titres of anti-TIF1γ
antibody were related to
the conditions of DM38 and/or the presence of internal malignancies.
39 40 Our findings indicate that while the immune
response against TIF1γ is likely to mediate the induction of
myositis, the development of anti-TIF1γ
autoantibodies may be
an epiphenomenon lacking direct pathogenic roles. In contrast,
transfer of human IgGs from patients with IMNM, which
contained antisignal recognition particle or anti-3-hydroxy-
3-methylglutaryl-
CoA
reductase antibodies, corroborated the idea
that complement can provoke muscle deficiency in recipient
mice.41 The difference between our experiments and this study
may clarify the differences in pathogenesis of DM and IMNM.
Immunohistochemistry and gene-expression
analyses of
muscle and skin biopsy samples revealed that type I IFN expression
correlates with DM pathogenesis.42 43 Janus kinase (JAK)1
mediates downstream effects of type I IFN, and it has been
reported that ruxolitinib, a JAK1/2 inhibitor, is effective for the
treatment of DM case, some of which were positive for anti-TIF1γ
antibody.44–46 In addition, a report presented that treatment
with tofacitinib, a JAK1/3 inhibitor, also improved myositis
in a case of anti-TIF1γ
antibody-positive
DM.47 Our experiments
revealed that deficiency of IFNAR partially inhibits the development
of TIM. Treatment of myositis with tofacitinib after the
initiation of immunity to TIF1γ was effective; however, it did
not result in significant inhibitory effects on the TIF1γ-specific
T cells and antibodies. The mechanism underlying these results
could be that the activation of type I IFN pathway induces
myotube atrophy and impairs endothelial cells angiogenesis.45
Collectively, the limitations of this murine model include
the lack of several DM-like
phenomenons (specific rash, define
muscle weakness with persistent myositis and upregulation of
some type I INF-related
genes in the muscle) and predominant
infiltration of CD8 T cells to muscle fibres, which is not usually
observed in patients with DM. Nevertheless, our new model
based on autoimmunity against the ubiqutous interacellular
antigen, TIF1γ, provides a useful tool to investigate the pathologic
mechanisms of anti-TIF1γ
antibody-positive
DM.
Author affiliations
1Department of Dermatology, Faculty of Medicine, University of Tsukuba, Tsukuba,
Ibaraki, Japan
2Department of Integrative Medicine for Allergic and Immunological Disease,
Graduate School of Medicine, Osaka University, Suita, Osaka, Japan
3Medical and Biological Laboratories Co Ltd, Nagoya, Aichi, Japan
4Institute for Advanced Co-Creation
Studies, Osaka University, Suita, Osaka,
Japan
5Department of Microbiology and Immunology, Graduate School of Medicine, Osaka
University, Suita, Osaka, Japan
6Department of Dermatology, Graduate School of Medicine, Osaka University, Suita,
Osaka, Japan
Acknowledgements Parts of this work were presented at the 3 rd Global
Conference on Myositis, 2019.
Contributors Study concept and design: NO and MF. Acquisition and analysis and
interpretation of data: all authors. Drafting of the manuscript: NO and MF. Critical
revision of the manuscript for important intellectual content: NO and YI. Obtained
funding: NO and MF.
Funding Supported by KAKENHI from the Japan Society for the Promotion of
Science (JSPS, 18K08263 for Naoko Okiyama).
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval All animal studies were approved by the Institutional Animal
Care and Use Committee of University of Tsukuba. This study did not involve human
participants.
Provenance and peer review Not commissioned; externally peer reviewed
Data availability statement All data relevant to the study are included in the
article or uploaded as supplementary information.
Supplemental material This content has been supplied by the author(s). It
has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have
been peer-reviewed.
Any opinions or recommendations discussed are solely those
of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and
responsibility arising from any reliance placed on the content. Where the content
includes any translated material, BMJ does not warrant the accuracy and reliability
of the translations (including but not limited to local regulations, clinical guidelines,
terminology, drug names and drug dosages), and is not responsible for any error
and/or omissions arising from translation and adaptation or otherwise.
ORCID iD
Naoko Okiyama http:// orcid. org/ 0000- 0002- 5398- 0773

REFERENCES

1 Schmidt J. Current classification and management of inflammatory myopathies. J
Neuromuscul Dis 2018;5:109–29.
2 Selva-O’Callaghan
A, Pinal-Fernandez
I, Trallero-Araguás
E, et al. Classification and
management of adult inflammatory myopathies. Lancet Neurol 2018;17:816–28.
3 Lundberg IE, de Visser M, Werth VP. Classification of myositis. Nat Rev Rheumatol
2018;14:269–78.
4 Benveniste O, Goebel H-H,
Stenzel W. Biomarkers in inflammatory Myopathies-An
expanded definition. Front Neurol 2019;10:554.
5 McHugh NJ, Tansley SL. Autoantibodies in myositis. Nat Rev Rheumatol
2018;14:290–302.
6 Mammen AL, Casciola-Rosen
L, Christopher-Stine
L, et al. Myositis-Specific
autoantibodies are specific for myositis compared to genetic muscle disease. Neurol
Neuroimmunol Neuroinflamm 2015;2:e172.
7 Okiyama N, Fujimoto M. Cutaneous manifestations of dermatomyositis
characterized by myositis-specific
autoantibodies. F1000Res 2019;8. doi:10.12688/
f1000research.20646.1. [Epub ahead of print: 21 Nov 2019].
8 Targoff IN, Mamyrova G, Trieu EP, et al. A novel autoantibody to a 155-kd
protein is
associated with dermatomyositis. Arthritis Rheum 2006;54:3682–9.
9 Kaji K, Fujimoto M, Hasegawa M, et al. Identification of a novel autoantibody
reactive with 155 and 140 kDa nuclear proteins in patients with dermatomyositis: an
association with malignancy. Rheumatology 2007;46:25–8.
10 Betteridge Z, Tansley S, Shaddick G, et al. Frequency, mutual exclusivity and clinical
associations of myositis autoantibodies in a combined European cohort of idiopathic
inflammatory myopathy patients. J Autoimmun 2019;101:48–55.
11 Hamaguchi Y, Kuwana M, Hoshino K, et al. Clinical correlations with dermatomyositis-specific
autoantibodies in adult Japanese patients with dermatomyositis: a multicenter
cross-sectional
study. Arch Dermatol 2011;147:391–8.
12 Fujimoto M, Hamaguchi Y, Kaji K, et al. Myositis-Specific
anti-155/140 autoantibodies
target transcription intermediary factor 1 family proteins. Arthritis Rheum
2012;64:513–22.
13 Fiorentino DF, Chung LS, Christopher-Stine
L, et al. Most patients with cancer-associated
dermatomyositis have antibodies to nuclear matrix protein NXP-2 or
transcription intermediary factor 1γ. Arthritis Rheum 2013;65:2954–62.
14 Venalis P, Selickaja S, Lundberg K, et al. Association of Anti-Transcription
intermediary
factor 1γ antibodies with paraneoplastic rheumatic syndromes other than
dermatomyositis. Arthritis Care Res 2018;70:648–51.
15 Kasuya A, Hamaguchi Y, Fujimoto M, et al. TIF1γ-overexpressing, highly progressive
endometrial carcinoma in a patient with dermato-myositis
positive for malignancy-associated
anti-p155/
140 autoantibody. Acta Derm Venereol 2013;93:715–6.
16 Pinal-Fernandez
I, Ferrer-Fabregas
B, Trallero-Araguas
E, et al. Tumour TIF1 mutations
and loss of heterozygosity related to cancer-associated
myositis. Rheumatology
2018;57:388–96.
17 Mohassel P, Rosen P, Casciola-Rosen
L, et al. Expression of the dermatomyositis
autoantigen transcription intermediary factor 1γ in regenerating muscle. Arthritis
Rheumatol 2015;67:266–72.
18 Oya K, Inoue S, Saito A. Pregnancy triggers the onset of anti-transcriptional
intermediary factor 1gamma antibody-positive
dermatomyositis: a case series.
Rheumatology 2019.
19 Yan K-P,
Dollé P, Mark M, et al. Molecular cloning, genomic structure, and expression
analysis of the mouse transcriptional intermediary factor 1 gamma gene. Gene
2004;334:3–13.
20 Hesling C, Lopez J, Fattet L, et al. Tif1γ is essential for the terminal differentiation
of mammary alveolar epithelial cells and for lactation through Smad4 inhibition.
Development 2013;140:167–75.
21 Müller U, Steinhoff U, Reis LF, et al. Functional role of type I and type II interferons in
antiviral defense. Science 1994;264:1918–21.
22 Sugihara T, Sekine C, Nakae T, et al. A new murine model to define the
critical pathologic and therapeutic mediators of polymyositis. Arthritis Rheum
2007;56:1304–14.
23 Okiyama N, Sugihara T, Oida T, et al. T lymphocytes and muscle condition act like
seeds and soil in a murine polymyositis model. Arthritis Rheum 2012;64:3741–9.
24 Okiyama N, Furumoto Y, Villarroel VA, et al. Reversal of CD8 T-cell-
mediated
mucocutaneous graft-versus-
host-
like
disease by the JAK inhibitor tofacitinib. J Invest
Dermatol 2014;134:992–1000.
25 Nagaraju K, Plotz PH. Animal models of myositis. Rheum Dis Clin North Am
2002;28:917–33.
26 Ascherman DP. Animal models of inflammatory myopathy. Curr Rheumatol Rep
2012;14:257–63.
27 Rosenberg NL, Ringel SP, Kotzin BL. Experimental autoimmune myositis in SJL/J mice.
Clin Exp Immunol 1987;68:117–29.
28 Matsubara S, Shima T, Takamori M. Experimental allergic myositis in SJL/J mice
immunized with rabbit myosin B fraction: immunohistochemical analysis and transfer.
Acta Neuropathol 1993;85:138–44.
29 Okiyama N, Sugihara T, Iwakura Y, et al. Therapeutic effects of interleukin-6 blockade
in a murine model of polymyositis that does not require interleukin-17A.
Arthritis
Rheum 2009;60:2505–12.
30 Katsumata Y, Ridgway WM, Oriss T, et al. Species-Specific
immune responses
generated by histidyl-tRNA
synthetase immunization are associated with muscle and
lung inflammation. J Autoimmun 2007;29:174–86.
31 Sgroi D, Cohen RN, Lingenheld EG, et al. T cell lines derived from the spinal cords
of mice with experimental allergic encephalomyelitis are self reactive. J Immunol
1986;137:1850–4.
32 Nagaraju K, Raben N, Villalba ML, et al. Costimulatory markers in muscle of patients
with idiopathic inflammatory myopathies and in cultured muscle cells. Clin Immunol
1999;92:161–9.
33 Bartoccioni E, Gallucci S, Scuderi F, et al. Mhc class I, MHC class II and intercellular
adhesion molecule-1 (ICAM-1) expression in inflammatory myopathies. Clin Exp
Immunol 1994;95:166–72.
34 Nagaraju K, Casciola-Rosen
L, Lundberg I, et al. Activation of the endoplasmic
reticulum stress response in autoimmune myositis: potential role in muscle fiber
damage and dysfunction. Arthritis Rheum 2005;52:1824–35.
35 Arahata K, Engel AG. Monoclonal antibody analysis of mononuclear cells in
myopathies. V: identification and quantitation of T8+ cytotoxic and T8+ suppressor
cells. Ann Neurol 1988;23:493–9.
36 Dalakas MC, Hohlfeld R. Polymyositis and dermatomyositis. Lancet 2003;362:971–82.
37 Nagaraju K, Raben N, Loeffler L, et al. Conditional up-regulation
of MHC class I in
skeletal muscle leads to self-sustaining
autoimmune myositis and myositis-specific
autoantibodies. Proc Natl Acad Sci U S A 2000;97:9209–14.
38 Shimizu K, Kobayashi T, Kano M, et al. Anti-transcriptional
intermediary factor
1-γ antibody as a biomarker in patients with dermatomyositis. J Dermatol
2020;47:64–8.
39 Ceribelli A, Isailovic N, De Santis M, et al. Myositis-Specific
autoantibodies and their
association with malignancy in Italian patients with polymyositis and dermatomyositis.
Clin Rheumatol 2017;36:469–75.
40 Ikeda N, Yamaguchi Y, Kanaoka M, et al. Clinical significance of serum levels of anti-transcriptional
intermediary factor 1-γ antibody in patients with dermatomyositis. J
Dermatol 2020;47:490–6.
41 Bergua C, Chiavelli H, Allenbach Y, et al. In vivo pathogenicity of IgG from patients
with anti-SRP
or anti-HMGCR
autoantibodies in immune-mediated
necrotising
myopathy. Ann Rheum Dis 2019;78:131–9.
42 Uruha A, Nishikawa A, Tsuburaya RS, et al. Sarcoplasmic MxA expression: a valuable
marker of dermatomyositis. Neurology 2017;88:493–500.
43 Okiyama N, Yamaguchi Y, Kodera M, et al. Distinct histopathologic patterns of finger
eruptions in dermatomyositis based on myositis-specific
autoantibody profiles. JAMA
Dermatol 2019. doi:10.1001/jamadermatol.2019.1668. [Epub ahead of print: 10 Jul
2019].
44 Hornung T, Janzen V, Heidgen F-J,
et al. Remission of recalcitrant dermatomyositis
treated with ruxolitinib. N Engl J Med 2014;371:2537–8.
45 Ladislau L, Suárez-Calvet
X, Toquet S, et al. Jak inhibitor improves type I interferon
induced damage: proof of concept in dermatomyositis. Brain 2018;141:1609–21.
46 Aeschlimann FA, Frémond M-L,
Duffy D, et al. A child with severe juvenile
dermatomyositis treated with ruxolitinib. Brain 2018;141:e80.
47 Paik JJ, Christopher-Stine
L. A case of refractory dermatomyositis responsive to
tofacitinib. Semin Arthritis Rheum 2017;46:e19.

Key messages

What is already known about this subject?
►► A number of autoantibodies have
been identified in sera of patients with
dermatomyositis (DM), which are not only
highly disease specific but are associated with
distinct clinical features.
►► One of the autoantigens for the myositis-specific
autoantibodies, transcriptional
intermediary factor 1 (TIF1) γ, is a ubiquitous
intracellular molecule that is often mutated
or overexpressed in tumours and triggers the
development of anti-TIF1γ
antibody-positive
DM.
►► Previously established murine models of
experimental autoimmune myositis are
dependent on immune responses against
muscle tissue-specific
antigens.
What does this study add?
►► Autoimmunity against TIF1γ results in
experimental myositis.
►► The initiation of the experimental myositis is
completely dependent on autoreactive TIF1γ-
specific CD8+ T cells, but not on CD4+ T cells or
IgG.
►► The type I interferon pathway is partially
involved in the pathogenesis of myositis caused
by autoimmunity against TIF1γ.
How might this impact on clinical practice or
future developments?
►► Autoimmunity to TIF1γ is not only a diagnostic
marker for a subset of human DM and may play
a role in the pathogenesis of the DM seen in
patients with these autoantibodies.
►► This new murine model of experimental
myositis might be a useful tool to investigate
pathologic mechanisms of, and to develop
specific treatments for, human anti-TIF1γ
antibody-positive
DM.

Supplemental Materials and Methods

Recombinant human TIF1γ protein.
A full-length human TIF1γ gene (GenBank accession Number: AF119043) was
His-tagged at its 3’ end and inserted into pFastBac1 vector for baculovirus expression
(Invitrogen). Recombinant bacmids produced using the expression vectors were
transfected into SF9 cells using Cellfectin II (Invitrogen). The infected SF9 cells were
incubated for 48 hours at 27 C and then harvested and lysed by sonication. The soluble
cell lysate from 8 - 10  107 cells/15 ml was applied onto a Ni Sepharose 6 Fast Flow
Resin column (GE Healthcare). The purified recombinant protein was collected from 2nd
and 3rd fractions eluted with phosphate buffer containing 200 mM imidazole, and
concentrated to > 1 mg/ml using Centriprep

Centrifugal Filter Devices YM-50
(Millipore).
Histological scoring system for experimental myositis
The histological severity of inflammation in each muscle block of the hamstrings and
quadriceps was graded as follows: grade 1 = involvement of 1-4 muscle fibers; grade
2 = a lesion involving 5–30 muscle fibers; grade 3 = a lesion involving a muscle
fasciculus; and grade 4 = diffuse and extensive lesions. When multiple lesions with the
same grade were found in a single muscle block, 0.5 point was added to the grade. The
histological severity in each mouse was determined as the average of the hamstring and
quadricep blocks.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance
Supplemental material placed on this supplemental material which has been supplied by the author(s) Ann Rheum Dis
Okiyama N, et al. Ann Rheum Dis 2021;0:1–8. doi: 10.1136/annrheumdis-2020-218661
Generation of antigen-pulsed mature bone marrow–derived dendritic cells
(BMDCs).
BMDCs were generated from BM cells of B6 mice cultured in RPMI1640 with 10%
fetal bovine serum (FBS) and 20 ng/ml recombinant murine granulocyte-macrophage
colony stimulating factor (Wako Junyaku) for 8 days. The cells were incubated with 50
μg/ml of the recombinant TIF1γ protein and 1 μg/ml lipopolysaccharide
(Sigma-Aldrich) for an additional 24 hours. More than 70% of the treated cells were
CD11c-positive.
Cell proliferation assay.
T cells were purified from the inguinal and popliteal lymph node (LN) cells of
immunized mice 14 days after the last immunization using CD3 T cell enrichment
columns (R&D systems). Two hundred thousand T cells and 2  104 mature BMDCs
were cultured in 200 μl RPMI1640 with 10% FBS for 3 days using 96-well
round-bottom plates, into which 1 μCi [3H]thymidine was added during the last 18
hours of culture. Proliferation of the T cells was evaluated as radioactivity of harvested
cells counted by a β counter (MicroBetaTM, Perkin Elmer).
Enzyme-linked immunosorbent assay (ELISA).
The plasma samples collected from immunized mice 2 weeks after the last
immunization were diluted at 1:100 or 1:1000 and then incubated in 96-well flat-bottom
microtiter plates, which had been coated with 3 ng/ml of full-length human TIF1γ for 1
hour at 37 ℃. Pooled plasma from three TIF1γ-immunized mice was used as the
positive control and plasma from one CFA-treated mouse was the negative control.
Plates were washed four times then incubated with peroxidase-conjugated goat
anti-murine IgG antibodies (ab205719, Abcam) and visualized by incubation with 3, 3’
5, 5’-tetramethyl-benzidine for 1 hour at room temperature. The reactions were stopped
by 0.5 N sulfuric acid. Optical density (OD) at 450 nm was measured, and each
antibody index was calculated from the formula: ([sample OD - blank OD] / [positive
reference OD - blank OD])  100.
Immunohistochemical analyses (IHC)
Sections (6 μm) from formalin-fixed paraffin-embedded muscle tissue samples
were pretreated with Trilogy (Merck), and stained with rabbit anti‐mouse CD8α
(EPR20305; Abcam), rabbit anti‐mouse CD4 (EPR19514; Abcam), and rat antimouse/
human B220 (RA3‐6B2; BD Biosciences PharMingen) monoclonal antibodies,
and rabbit anti-rat/human/mouse CD11b polyclonal antibodies (product number,
PA5-79533, Invitrogen). Nonspecific staining was blocked with 5-10% bovine serum
albumin (BSA). Bound antibodies were visualized with peroxidase‐labeled antirabbit/
mouse IgG antibody (Envision™+ Dual Link System-HRP; Dako) or goat
anti-rat IgG antibody (Abcam), and associated substrates (Liquid DAB+ Substrate
Chromogen System; Dako). Cryostat‐frozen sections (6 μm) fixed in cold acetone were
preincubated with 5% BSA, stained with mouse anti‐mouse H‐2Kb monoclonal
antibody (AF6‐88.5; BioLegend), and visualized with peroxidase‐labeled goat
anti-mouse/rabbit IgG antibodies and its substrates (Dako). Isotype controls were used
as negative control. The stained sections were evaluated by two independent observers,
who reported results that were comparable.
In vivo depletion of CD8+ T cells
Mice were injected intraperitoneally with 1 mg of purified rat anti-CD8α depleting
monoclonal antibody (53.67.2), produced by hybridoma cells cultured in CELLLine
flasks (WHEATON) with Hybridoma-SFM (Gibco), or purified rat IgG2b (BioXCell)
as a control, for three consecutive days. This treatment started 10 days before the 4th
immunization, and injection of 500 μ of the same antibody was repeated every other day
for 14 days. Complete depletion of CD8+ T cells in the splenocytes were confirmed by
flow cytometric analysis.
Real-time quantitative polymerase chain reaction (RT-qPCR) analyses
Total RNA was extracted from the muscle tissue samples using Trizol Reagent
(Invitrogen). Complementary DNA was synthesized with a High-Capacity cDNA
Reverse Transcription Kit (Thermo Fisher). Levels of expression were detected using
the QuantStudio™ 5 Real-Time PCR Systems (Applied Biosystems) with PrimeTime®
Gene Expression Master Mix and Prime Time qPCR predesigned primers (Integrated
DNA Technologies; Supplemental Table 1). All RT-qPCR analyses were performed in
triplicate. Amplification products were quantified by the comparative CT method. The
mRNA level of each gene was normalized to that of Actb.

Supplemental Table 1. Primers for real-time quantitative polymerase chain

reactions
Target ID
Mx1 Mm.PT.58.12101853.g
Isg15 Mm.PT.58.41476392.g
Osa1b Mm.PT.56α.10289138.g
Osa3 Mm.PT.58.12139602

bottom line and head line

Okiyama N, et al. Ann Rheum Dis 2021;0:1–8. doi: 10.1136/annrheumdis-2020-218661
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance
Supplemental material placed on this supplemental material which hbeen supplied by the author(s) Ann Rheum Dis

first line

Handling editor Josef S
Smolen
►► Additional supplemental
material is published online
only. To view, please visit the
journal online (http:// dx. doi.
org/ 10. 1136/ annrheumdis-
2020- 218661).
For numbered affiliations see
end of article.
Correspondence to
Dr Naoko Okiyama, Department
of Dermatology, Faculty of
Medicine, University of Tsukuba,
Tsukuba 305-8575, Japan;
naoko. okiyama@ md. tsukuba.
ac. jp
Received 21 July 2020
Revised 9 March 2021
Accepted 21 March 2021

参考資料　

医学論文参考文献の参考文献例（１）TIF1γ

＜この記事は個人の過去の経験に基づく個人の感想です。現在所属する組織、業務とは関係がありません。＞

文書履歴(document history)

ver. 0.01 初稿　20230311
ver. 0.02 ありがとう追記 20230312

最後までおよみいただきありがとうございました。

いいね　💚、フォローをお願いします。

Thank you very much for reading to the last sentence.

Please press the like icon 💚　and follow me for your happy life.