課題番号：
研究課題名 ：
主任研究者名 ：分担研究者名 ：２４指１０３
新しい病原因子を標的とした結核ワクチンの開発
切替 照雄
切替 照雄、濱端 崇、秋山 徹、岡村 匡史、舩渡川 圭次

キーワード ： 結核菌、ワクチン、ゲノム、病原因子
研究成果 ：【目的】最近の調査で、BCG が成人結核に対して効果的でないことが明らかになった。 このように、新しいワクチンの開発は、結核の根絶のために必要である。 新しいワクチンの開発のために最も高いプライオリティーは、新しい標的分子の選抜とそのメカニズムの理解である。 この研究において、我々は、結核菌と宿主の相互作用に重要な因子を同定する。
【方法】NCGM2242 プロジェクト： 強毒結核菌 Erdman 株とその変異体株（NCGM2242）の全ゲノムを分析する。 PE_PGRS62 プロジェクト：結核菌遺伝子破壊株の相補株を作製した。結核菌
PE_PGRS62 タンパク質に結合する宿主タンパク質を同定する。Peroxiredoxin 1 プロジェクト：
BALB/c バックグラウンドの Peroxiredoxin 1 欠損マウスを作製する。
【結果】NCGM2242 プロジェクト： 16s rRNA 点突然変異 U1406A（結核菌では U1399A）について記述する。この点変異は結核菌の病原性を非常に低下させるが、抗生物質のカナマイシンに抵抗性になった。 また、この弱毒株の変異は臨床分離株で観察されない変異であり、この弱毒株がワク
チン株 BCG にくらべ大きなワクチン効果があることを証明した。この 16S rRNA 突然変異はリボ
ソーム成熟に影響を及ぼすだけでなく、結核抗原 85 複合体や ESAT-6 などの病原因子、結核菌の基礎代謝に関わる酵素を含む約２０％（n = 361）のタンパク質発現を減少させた。さらにミコール酸の生合成酵素が減少したため、ミコール酸の構造が変化していた。
PE_PGRS62 プロジェクト：Δ62Comp の PE_PGRS62 タンパク質の発現回復を観察した。WT、
Δ62/mock と Δ62Comp の間でどのような違いがあるかどうかを調べた。 7H9/ADC 液体培地でそれぞれの菌株の生育速度に違いがなかったが、Δ62/mock 株は J774 細胞で細胞内生存率が減少することを示した。マウスで ΔPE_PGRS62 の弱毒化するかどうかを評価するために、我々はマウスの尾静脈に、WT または ΔPE_PGRS62 を注射した。WT 感染と比較して、ΔPE_PGRS62 株感染で
BALB/c と SCID マウスの生存日数が遅延した。これらの結果は、PE_PGRS62 が結核菌の病原性に必要とされることを証明した。また、新たに結核菌 PE_PGRS62 の結合タンパク質として宿主
Hornerin/S100A18 タンパク質を同定した。
Peroxiredoxin 1 プロジェクト： Peroxiredoxin 1 ノックアウトマウスは、結核菌に感受性でした。
BALB/c バックグラウンドは、C57BL/6 バックグラウンドよりも結核菌への感受性が高いことが示されました。
【考察】NCGM2242 株の rrs 遺伝子は点突然変異し U1406A なために、NCGM2242 株の点突然変異
が、復帰変異することが懸念される。更なる変異が生ワクチン開発のために必要とされる。我々は、
NCGM2242 株で PE_PGRS62 遺伝子を破壊する。特に BALB/c バックグランドで、Preoxiredoxin 1
ノックアウトマウスで結核菌遺伝子破壊株の弱毒化を評価することは容易かもしれない。

Subject No. ：24S103
Title：Attenuated TB vaccine development by gene disruption of TB virulence factors
Researchers ： Teruo Kirikae, Takashi Hamabata, Toru Miyoshi-Akiyama, Tadashi Okamura, Keiji
Funatogawa
Key word ：TB, Vaccine, Genome, virulence factor
Abstract ：【Purpose】
Recent studies have found that BCG is not effective against adult tuberculosis. Thus, the development of a
new vaccine is necessary for the eradication of tuberculosis. The highest priority for the development of a
new vaccine is the identification of a novel target molecule and the understanding of its mechanism. In thisstudy, we will identify M. tuberculosis pathogenic agent, and a host factor that interacts with M. tuberculosis
pathogenic agent.
【Method】
NCGM2242 project: A genome analysis of Erdman strain and its mutant strain (NCGM2242) were performed.
PE_PGRS62 project: A complementary strain of the M. tuberculosis gene knockout strain was constructed.
We identified Hornerin as PE_PGRS62 binding protein. To identify the host protein(s) interacted with the
mycobacterial PE_PGRS62 protein, we perform an immunoprecipitation of protein complexes followed byliquid chromatography– tandem mass spectrometry (LC-MS/MS).
Peroxiredoxin 1 project: Peroxiredoxin 1 deficient mouse with BALB/c background was constructed.
【Result】
NCGM2242 project: We describe a specific 16S rRNA mutation, U1406A (U1399A: M. tuberculosis
number), which confers resistance to kanamycin while highly attenuating M. tuberculosis virulence. We also
demonstrated that this attenuated strain, not observed in clinical isolates, induced greater protection against
virulent M. tuberculosis than the M. bovis strain bacille Calmette-Guérin. This 16S rRNA mutation not only
affected ribosome maturation but decreased the expression of ~20% (n = 361) of mycobacterial proteins,
including central metabolic enzymes and virulence factors, antigen 85 complexes and ESAT-6. Furthermore,
the reduced expression of mycolic acid biosynthesis enzymes resulted in alterations in mycolic acid structure.
PE_PGRS62 project: We observed the recovery of PE_PGRS62 expression in 62Comp. We checked any
difference among WT, 62/mock and 62Comp. Although we confirmed no difference among these strains
grown in 7H9/ADC culture medium, 62/mock mutant showed reduced the survival of intracellular
mycobacteria in J774 cells. To evaluate the virulence attenuation of PE_PGRS62 in mice, we injected WT
or PE_PGRS62 into tail vein of mice. There was a significant delayed in survival with the PE_PGRS62
mutant relative to WT in BALB/c and SCID mice. These results demonstrate that PE_PGRS62 is required forM. tuberculosis virulence. We identified a host Hornerin/S100A18 protein as the mycobacterial PE_PGRS62
protein binding protein.
Peroxiredoxin 1 project: Peroxiredoxin 1 deficient mouse was susceptible to M. tuberculosis infection.
BALB/c background exhibited greater susceptibility to M. tuberculosis infection than C57BL/6.
【Discussion】As U1406A is mutation in rrs gene of NCGM2242 strain, we are concerned for revertant
mutation in this strain. Further mutation is required for live vaccine development. We will disrupt
PE_PGRS62 gene in NCGM2242 strain. It might be easy to evaluate the attenuation of M. tuberculosis
mutant in Peroxiredoxin 1 deficient mouse, especially with BALB/c background.

Researchers には、分担研究者を記載する。

２４指１０３
研究課題名 ： 新しい病原因子を標的とした結核ワクチンの開発
主任研究者名 ： 切替 照雄
分担研究者名 ： 濱端 崇、秋山 徹、岡村 匡史、舩渡川 圭次

新規の結核ワクチンを開発するためには
新たな病原因子を同定することが必須である

概要

目 的
新たな結核菌病原因子及びこれらと相互作用する
宿主防御因子の同定
対 象‐遺伝子破壊により同定した新規病原性タンパク質‐当研究部で分離した薬剤耐性結核菌NCGM2242株
方 法-遺伝子改変技術、マウス結核菌感染実験や結核菌ゲノム解読を実施することで対象の生物学的性状を解明する-これらの知見をもとに結核ワクチンの候補を作製する

◇：2242生菌
接種群◆：2242 死菌
接種群
■：対象群

0

30

60

90

120

細胞内生存菌数

100
生 80
存 60
率 40
（％） 200

結核菌（野性型
PE_PGRS62破壊株
株）
PE_PGRS6相補株

研究成果

150

感染後日数

ワクチン効果 弱毒株INCGM２242

PE_PGRS62は病原因子

弱毒結核菌NCGM2242株の変異の同定
結核菌弱毒株NCGM2242の全ゲノムを解読し、リボソーム点突然変異による基礎
代謝酵素や病原因子のタンパク質発現が減少していることを明らかにした。
ワクチン標的分子の同定
結核菌細胞壁タンパク質のひとつであると考えられているPE_PGRS62の遺伝子を破壊し、マウス生存日数の遅延の確認により病原因子として新規に同定した。
マウス結核モデルの構築
ペルオキシレドキシン１欠損マウスは結核感受性が高かった。戻し交配によりさらに感受性を高めたBALB/cペルオキシレドキシン１欠損マウスを作製した。
結核ワクチン標的分子を選抜
NCGM2242株のリボゾームrrsの点突然変異を決定し、PE_PGRS62を病原因子として同定した。復帰変異を防ぐためにNCGM2242株のPE_PGRS62を破壊する。

強毒結核菌Kurono株の全ゲノム配列解析を完了した

結核菌病原因子PE_PGRS62タンパク質の機能を解析した PE_PGRS62を病原因子として同定した
結核菌
PE_PGRS62はHrnrと特異的に結合した
cell lysate

結核菌（WT）

ノックアウト

PE_PGRS62ノックアウト
Hyg
pMV306

PE_PGRS62コンプリ
Hyg

BALB/cの生存率

100%
80%

Putative
TransMembrane
Domains 504

1

GFP-IP
+

+
+
+ +

+
+
+ + +

PE_PGRS62

60%

コンプリ

40%

350

WT

20%

62FL
62TM

351
Myc

0%
50

pMV30662FL-Myc +62TM-Myc
62TM-Myc
GFP-Hrnr1-691 +

100

150

Hrnr binding domain

200

感染後の日数

62TM
Ca2+ Ca2+

691

Hrnr/S100a18

GFP S100

TM

1st IP: HA-ユビキチン
2nd IP: GFP-PE_PGRS62

全長

PE_PGRS62に結合する宿主タンパク質の検索

Hrnr

Hrnr依存的な結核菌の細胞外へのイジェクション
J774 MøGFP

M. tuberculosis

merge

？
62

膜貫通領域を有している Ub
PE_PGRS62に結合する宿
主の細胞質タンパク質

HRNR

humanHRNR
別名S100A18？
TM（同定したタンパク質）

J774 MøGFP-Hrnr

M. tuberculosis

merge

課題番号
：24 指 103
研究課題名 ：新しい病原因子を標的とした結核ワクチンの開発
主任研究者名 ：切替 照雄
分担研究者名 ：切替 照雄
キーワード ：結核菌、遺伝子破壊株、マクロファージ
研究成果 ：【目的】
BCG に代わる次世代ワクチンの開発を目指して、複数変異を導入する親株として弱毒結核菌でワクチン効果を示す NCGM2242 株を得た。全ゲノムシークエンスの結果から NCGM2242 株は rrs 遺伝子の点変異体であることが明らかになったが、この変異がプラスミドにより rrs 発現を相補することで、種々のフェノタイプ（菌の増殖性、薬剤耐性の獲得、リボソーム RNA の成熟、ワクチン効果など）が回復するかどうかを調べる必要がでてきた。相補実験の結果、NCGM2242 株が rrs の点変異が原因
でワクチン効果があることが確認された場合は、復帰変異が起こらないように既に同定している病原因子 PE_PGRS62 の破壊を行いワクチン候補株（NCGM2242 バックグラウンド・PE_PGRS62 ノックアウト）の作製を目指す。
病原因子として同定した結核菌 PE_PGRS62 や PE_PGRS62 結合タンパク質である Prdx1 の機能解析を通じて、結核菌感染に重要な役割を果たす新たな宿主因子を発見する。
【方法】
rrs を相補するために rrs を含むオペロン領域（〜8kbp）をエピソーマルなプラスミド（ゼオシン耐性）に挿入し、NCGM2242 株にエレクトロポレーションで導入、rrs コンプリ株を作製する。
PE_PGRS62 の結合タンパク質であった Hornerin を安定発現する J774 マクロファージを作製し、結核菌感染における Hornerin の役割を共焦点レーザー顕微鏡により解析する。
【結果】
ゼオシン耐性マーカー遺伝子を有するプラスミドを作製した（pHI191）。rrs オペロン８kbp を
pHI191 に挿入し、NCGM2242 株に導入した（rrs コンプリ株）。rrs コンプリ株が、病原性復帰傾向であることを調べるために、マウスへの感染実験を実施中である。
Hornerin の発現依存的に結核菌がマクロファージ細胞外にイジェクトすることが観察された。
【考察・結論】
NCGM2242 の全ゲノムシーケンスにより rrs 変異が明らかとなりリボソーム変異により低代謝なことがわかった。しかしながら、rrs 以外の変異の可能性がないかどうかを示すために、rrs コンプリ株の作製および病原性復帰や表現型解析が必要になった。rrs コンプリ株が完成したので、NCGM2242 株の弱毒化が rrs の変異だけであることが証明できそうである（実験中）。
同定した宿主因子である Hornerin が結核菌感染に重要な役割を果たす可能性が示唆された。したがって、より信憑性が高い実験結果を得るために、導入済みの CRISPR/Cas9 システムを用いた迅速なHornerin ノックアウトマウスの作製を行い、結核菌感受性を調べる必要がでてきた。現在、Hornerin
ノックアウト BALB/c マウスを作製中である。

課題番号：24 指 103
研究課題名 ：新しい病原因子を標的とした結核ワクチンの開発
主任研究者名 ：切替 照雄
分担研究者名 ：濱端 崇
キーワード ：結核菌、PE_PGRS62、Hornerin
研究成果 ：【目的】
結核は国際的な再興感染症であり、予防には BCG が用いられている。BCG は乳幼児期の重傷化す
る結核を予防できるが、成人の結核を予防する次世代ワクチンが開発されていない。そこでワクチンの開発を目指し、これまでに作製した弱毒株である遺伝子ノックアウト結核菌の病原性低下のメカニ
ズムを明らかにするために、PE_PGRS62 タンパク質の宿主細胞内における機能を明らかにする。
【方法】
結核菌 PE_PGRS62 タンパク質に結合する宿主タンパク質の同定：同定した結核菌病原因子タンパク質 PE_PGRS62 の機能を明らかにするために、宿主細胞内で PE_PGRS62 を発現させ観察を行った。結合
タンパク質を同定するために２段階の免疫沈降、具体的には GFP-PE_PGRS62 と HA-ユビキチンを共発現させた 293T 細胞の抽出液を用いて HA 抗体による１回目の免疫沈降後に GFP 抗体による２回目の免疫沈降を行い、得られた特異的な銀染色バンドを LC-MS/MS 解析し、PE_PGRS62 に結合する宿主タンパ
ク質を同定する。
結核菌 PE_PGRS62 タンパク質を発現する肺胞 II 型上皮細胞 A549 の作製：A549 細胞に安定的に GFPPE_PGRS62 を発現させるために、レトロウィルスベクター（pMX-puro、pVSV-G プラスミドと Plat-GP細胞によるシステム、Cell biolabs 社）を使用し GFP-PE_PGRS62 遺伝子を含む DNA 断片を A549 細胞に導入する。ピューロマイシン耐性細胞株を限界希釈により分離し、生育後に細胞抽出液を作製しAnti-GFP 抗体でウェスタンブロッティング解析を行うことで GFP-PE_PGRS62 が安定発現した A549
細胞をクローニングする。
【結果・考察】PE_PGRS62 タンパク質を 293T 細胞に過剰発現させるとベシクルに局在することがわかった。このベシクルに関与している細胞質タンパク質を同定するために、細胞質に豊富に存在しているユビキチン
タンパク質を過剰発現・免疫沈降してから GFP-PE_PGRS62 の免疫沈降を行ったところ、結合タンパク質として Hornerin を同定した。Hornerin と PE_PGRS62 の結合が C 末端領域の推定膜貫通領域であることも確認できた。
Hornerin はケラチノサイトで発現量が多いことがわかっていた。結核菌感染に関係しそうな A549
細胞（肺胞 II 型上皮細胞株）と Jurkat 細胞（T 細胞株）で Hornerin が比較的多く発現していることがわかってきた。マクロファージの他に肺胞 II 型上皮細胞は結核菌が直接感染することが知られており、この細胞内で PE_PGRS62 タンパク質が機能する可能性が考えられたため、GFP-PE_PGRS62 を安定発現する A549 細胞を作製した（クローン No.11 細胞）。
A549 細胞内で GFP_PGRS62 はベシクルに局在することが確認され、このベシクルがラメラ封入体であるかどうかを確認するために、クローン No.11 細胞に安定的に ABCA3-Myc を発現する細胞株を作製中である。
A549 細胞はサーファクファクタントタンパク質の分泌等が不完全であることが知られている。これまでに我々の研究グループが同定した PE_PGRS62 結合タンパク質である宿主 Prdx1 は細胞質型のペルオキシレドキシンとして知られているが、A549 細胞において TGF-b1 刺激依存的に細胞外に分泌することが報告されている。Prdx は細胞内では抗酸化作用を示すと考えられているが、Prdx がマクロフ
ァージや樹状細胞を TLR2/TLR4 依存的に IL-23、IL-1b、TFN-a などの炎症性サイトカインを強く産生させることが知られている。現在、A549 細胞を用いた Prdx1 の分泌を調べる実験系を構築中である。
Prdx1 の分泌を PE_PGRS62 と Hornerin がどのように制御するか明らかにすることで結核菌の新たな感染戦略を発見できる可能性が十分ある。

課題番号：24 指 103
研究課題名 ：新しい病原因子を標的とした結核ワクチンの開発
主任研究者名 ：切替 照雄
分担研究者名 ：秋山 徹

研究成果

：

【目的】新規の結核ワクチンを開発するためには、新たな病原因子を同定することが必須である。
本研究では、新たな結核菌病原因子およびこれらと相互作用する宿主防御因子を同定するための基盤として、結核菌の全ゲノム情報を詳細に解析するために、結核菌実験株として動物実験などに汎用される Kurono 株の全ゲノム配列解析を実施した。
【方法】Kurono 株は全ゲノム配列を次世代シーケンサにより取得した。Annotation は glimmer 3.0
による CDS 抽出後、代表的な結核菌株である H37Rv 株の配列を参照しながら、in sillico molecularcloning（インシリコバイオロジー社）で自動的に予備 annotation を行った後、マニュアルで補正した。
【結果】Kurono 株の全ゲノム配列の解析を完了した。総コンティグ塩基数は 4,455,125,637bp であり、既知の結核菌のゲノムサイズのほぼ 100 倍のデータ量を得ることができた。約 50 倍 coverageの Illumina1 ショートリードによる SNP レベルのエラーの有無について確認したところ、エラー
はまったく検出されなかった。Kurono 株の全ゲノム塩基数は 4,415,078bp で他の結核菌と同様 rrs
オペロンは 1 個のみ存在した（図１）。蛋白質コード領域は 4340 個と推定された。tRNA 遺伝子
数は 53 個、tmRNA 数は 1 個、IS6110 配列は 14 個存在した。既知の薬剤耐性に寄与する変異は認められなかった。Large sequence polymorphisms 法による系統解析では、Kurono 株はEuro-American 系統に分類された。
現在までに全ゲノム配列が完全決定された結核菌は Kurono 株を含めて 16 株である。全ゲノム配列を用いた系統解析では、Kurono 株は H37Rv と最も近縁であり、SNP レベルの変異はわずか 818個だった。これらの SNP の約 40%は、結核菌ゲノムの中で特に変動が大きいとされている、PE-PPE
ファミリ蛋白質の中に存在していた。
。
【考察・結論】これまでに他の菌種で実施したゲノム解析の経験を元に、結核菌実験株として著名である Kurono 株の全ゲノム配列解析を完了した。

課題番号：24指103

研究課題名：新しい病原因子を標的とした結核ワクチンの開発

主任研究者名 ：切替 照雄
分担研究者名 ：岡村 匡史
キーワード：マウス、結核、感受性遺伝子、連鎖解析

研究成果

：

分担研究課題：結核ワクチン評価モデルの開発およびマウス結核感受性遺伝子の同定[目的]

BCG ワクチンは小児の結核性髄膜炎や粟粒結核を有効に予防するものの、成人の肺結核には無効であることが大規模な疫学検査から明らかとなっている。従って、成人の肺結核に対して本当に有効なワクチンは未だ存在しない。その原因の一つとして、有用な結核感染モデ
ル動物の開発が遅れていることが上げられる。我々は、結核の病原因子として、PE_PGRS62を同定し、PE_PGRS62 は宿主細胞のペルオキシレドキシン(Prdx1)と特異的に結合し、Prdx1
遺伝子欠損マウスでは、結核菌に対する感受性が著しく高い事を見出した。さらに、Balb/c
マウスは、C57BL/6 比べ、遺伝的に結核菌に高感受性を示す形質を有している。本研究では、以上の知見およびツールを用いて、新たな結核菌評価モデルを開発するとともに、結核菌病
原因子と相互作用する宿主防御因子を同定することを目的とする。
[結果および考察]
１．結核菌高感受性マウスを用いたワクチン候補株の検証
Prdx1 遺伝子を欠損することで、マウスは結核菌に対する感受性が著しく高まる。さらに、結核菌に感受性が高い系統である BALB/c に Prdx1 欠損を導入した結核菌高感受性マウス（Prdx1-/--BALB/c）は、ワクチン候補株やリコンビナント病原因子のワクチン効果をより効率的に評価できるモデルとして期待される。ワクチン候補株である弱毒株 NCGM2242 を結核高感受性 Prdx1-/--BALB/c に接種し、そのワクチン効果を検証した。その結果、NCGM2242 を接種したことで、逆に発症が早まってしまった。NCGM2242 にワクチン効果はなかったが、Prdx1-/--BALB/c は、BALB/c よりも結核菌に高感受性であるため、ワクチンの安全性およびその効果の検証に有用であると考えられた。
２．Prdx1 欠損マウス結核感染モデルによる新規宿主防御遺伝子の同定
C57BL/6 と BALB/c の系統間では、結核菌に対する著しい感受性の違いがある。結核菌に抵抗性である Prdx1-/--B6 と感受性である Prdx1-/-- BALB/c を交配した F1 は、結核菌に抵抗性であることがわかった。
F1 と Prdx1-/--BALB/c の交雑群を作成し、結核菌の感染実験を行った。
体重減少および致死を指標に、交雑群の連鎖解析を行った結果、マウス第 1 染色体に有意（ P=0.0094 ） な 連 鎖 が 見 ら れ た 。 第 1 染 色 体 に 存 在 す る 結 核 菌 抵 抗 性 遺 伝 子 座 を 、Prdx1-/--BALB/c に導入したコンジェニック系統を作製しており、現在 N6 世代（100%Prdx1-/--BALB/c に置換）を作製した。今後、樹立したコンジェニック系統の結核菌感染後の体重変化、生存日数等を指標に第 1 染色体に存在する結核菌抵抗性遺伝子を同定する。

課題番号：24 指 103
研究課題名 ：マウス結核感染実験による結核ワクチン評価系構築
主任研究者名 ：切替 照雄
分担研究者名 ：舩渡川 圭次
キーワード ：結核感染実験、ワクチン、マウス
研究成果 ：【目的】新規の結核ワクチンを開発するためには、新たな病原因子を同定することが必須である。
本研究では、結核菌強毒株 Erdman 由来 NCGM2242 株のマウス感染実験を実施し、その病原性を評価する。また、NCGM2242 株のワクチン効果を検証する。宿主防御因子候補分子 Peroxiredoxin1（Prdx1）のノックアウトマウスに結核菌を感染させ、結核菌に対する感受性を評価する。結核菌
感染で発現が増加する宿主 microRNA155 のノックアウトマウスが結核感受性かどうか調べる。
【方法】感染実験に供する結核菌は、標準株 Erdman 株、NCGM2242 株を用いた。マウスは、
C57BL/6 野生マウス、BALB/c 野生マウスおよび BALB/c をバックグラウンドに持つ Prdx1 ノック
アウトマウス（Prdx1-/-, BALB/c マウス）の雄、８週齢を準備した。対数増殖期まで培養した結核菌を遠心して回収し、PBS で洗い、1×107 CFU/ml になるよう調製した。マウスの尾静脈より結核菌（2×106 CFU）を感染させた。感染させたマウスは、P3A 施設内のケージで飼育した。ワクチン効
果を検証する場合は、接種菌量を 2×105, 4CFU にし、接種１ヶ月後、Erdman 株（2×105, 4 CFU）を感染させた。
【結果】NCGM 2242 株の LD50 試験：NCGM 2242 株の LD50 を決定するため、2×107 生菌数を
BALB/c マウスの腹腔に接種したが、300 日経過しても全て生存した。これに対して、Erdman 株を感染させた群では、平均 114 日（2×105 生菌数接種群）、136 日（2×104 生菌数接種群）、236 日（2×103 生菌数接種群）生存した。NCGM2242 株のワクチン効果試験（１）：NCGM2242 株のワクチン効果を検証するため、NCGM2242 株の生菌及び死菌を BALB/c マウスに接種し、４週後
Erdman 株を感染させた。何も接種しなかったコントロール群、および NCGM 2242 死菌を接種した群は、平均 44 日生存したのに対し、NCGM2242 生菌を接種した群は、平均 102 日生存した。
NCGM2242 株のワクチン効果試験（２）：NCGM2242 株のワクチン効果を BCG と比較するため、NCGM 2242 株及び BCG を BALB/c マウスに接種し、４週後 Erdman 株を感染させた。何も接種しないコントロール群は、平均 129 日で死亡した。BCG を接種した群は、平均 161 日（2×103 生菌数
接種群）もしくは 190 日（2×104 生菌数接種群）生存した。それに対して、NCGM 2242 株を接種した群は、平均 245 日（2×103 生菌数接種群）もしくは 258 日（2×104 生菌数接種群）生存した。
全ゲノムシークエンスにより、NCGM2242 株は rrs（別名 MTB000019 遺伝子）が変異していることがわかった（rrs の相補実験を計画中）。また、この変異により NCGM2242 株はカナマイシン
耐性およびハイグロマイシン耐性になった。
Prdx1 ノックアウトマウスおよび miR-155 ノックアウトマウスは結核菌に感受性が高まっていた。
また、BALB/c バックグラウンドの Prdx1 ノックアウトマウスも結核感受性であった。
【考察・結論】NCGM2242 株のワクチン効果：NCGM2242 株の生菌でワクチン効果が見られた。
それに対し、熱で処理した死菌ではワクチン効果は、ほとんど見られなかった。死菌は、マウスの体内ですぐ処理されてしまい免疫が成立しない。
NCGM2242 株のリボソーム変異を相補（コンプリメンント）する追加実験が必要になった。
NCGM2242 株はリボソーム変異により複数の抗生物質耐性になったため、ゼオシンの耐性マーカ
ーを有するプラスミドを構築した。このプラスミドへ rrs 発現に必要なオペロン領域８kbp を挿入
しコンプリメント用プラスミドを得た。このプラスミドを NCGM2242 株に導入し、NCGM2242 の
rrs 変異が回復することでワクチン効果などに影響があるかどうか調べる必要がある。
miR-155 は結核菌感染で発現が増加していた。miR-155 ノックアウトマウスは Th2 優位であり
Th1 応答が正常ではなかったため結核菌への抵抗性が著しく低下していた。

研究発表及び特許取得報告について

課題番号：24指103

研究課題名：

新しい病原因子を標的とした結核ワクチンの開発

主任研究者名：

切替

照雄

論文発表
論文タイトルA silent mutation of mabG1 confers
isoniazid resistance on Mycobacterium
tuberculosis.

著者
Ando H,
MiyoshiAkiyama T,
Watanabe S,
Kirikae T

掲載誌

Mol Microbiol

Okada T, Nitta
T, Kaji K,
Takashima A,
Oda H,
Differential function of Themis CABITPLoS One
Tamehiro N,
domains during T cell development.
Goto M,
Okamura T,
Patrick MS and
Suzuki H.
*Ogawa, M.,*Okamura, T.,
Ishikura, S.,
Doi, K.,
Matsuzaki, H.,
Tanaka, Y.,
Zfat-Deficiency Results in a Loss of CD3 Ota, T.,ζ Phosphorylation with Dysregulation of Hayakawa, K.,
PLoS OneERK and Egr Activities Leading toSuzuki, H.,
Impaired Positive Selection.
Tsunoda, T.,
Sasazuki, T.
and Shirasawa,
S.*The authors
equally
contributed tothis work.

掲載号

年

91(3)

2014

9: e89115

2014

8, e76254

2013

学会発表
タイトル
A silent mutation in mabA confers
isoniazid resistance on Mycobacterium
tuberculosis .

発表者

学会名

安藤弘樹, 秋山
第87回日本細菌学
徹, 渡邊真弥,
東京
会総会
切替照雄

舩渡川圭次, 渡邊真弥, 祝弘樹,
蓜島由二, 奥村16S リボソーム RNA の解読領域の変異は結香世, 加藤雅子,
核菌を弱毒化する
橋本雅仁, 切替
富美子, 秋山徹,
切替照雄

場所

年月
2014年3月

第87回日本細菌学
東京会総会

2014年3月

Peroxiredoxin 1 contributes to host defense
against Mycobacterium tuberculosis.

松村和典, 祝弘樹, 加藤雅子,
第87回日本細菌学
舩渡川圭次, 切
東京会総会
替富美子, 秋山徹, 切替照雄

2014年3月

Peroxiredoxin 1 contributes to hostdefense against Mycobacterium
tuberculosis.

松村和典, 祝弘樹, 加藤雅子,
第87回日本細菌学
舩渡川圭次, 切
東京会総会
替富美子, 秋山徹, 切替照雄

2014年3月

研究発表及び特許取得報告について

その他発表(雑誌、テレビ、ラジオ等)
タイトル

発表者

発表先

場所

年月日

該当なし

特許取得状況について※出願申請中のものは(

発明名称)記載のこと。
登録番号

特許権者(申請者）
登録日(申請日)（共願は全記載）

結核菌の菌株の同定方法及び遺伝子変異の検 PCT/JP2014/051 秋山 徹、切替照
2014年1月22日
出方法
208
雄、奥村香世※該当がない項目の欄には「該当なし」と記載のこと。
※主任研究者が班全員分の内容を記載のこ

出願国

国際出願

Zfat-Deficiency Results in a Loss of CD3f
Phosphorylation with Dysregulation of ERK and EgrActivities Leading to Impaired Positive Selection
Masahiro Ogawa1,2., Tadashi Okamura3., Shuhei Ishikura1,2, Keiko Doi1,2, Hiroshi Matsuzaki1,
Yoko Tanaka1, Takeharu Ota1,2, Kunihiro Hayakawa4, Harumi Suzuki4, Toshiyuki Tsunoda1,2,
Takehiko Sasazuki5, Senji Shirasawa1,2*
1 Department of Cell Biology, Faculty of Medicine, Fukuoka University, Jonan-ku, Fukuoka, Japan, 2 Central Research Institute for Advanced Molecular Medicine, Fukuoka
University, Jonan-ku, Fukuoka, Japan, 3 Division of Animal Models, Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine,Tokyo, Japan, 4 Department of Immunology and Pathology, National Institute for Global Health and Medicine, Chiba, Japan, 5 Institute for Advanced Study, Kyushu
University, Fukuoka, Japan

Abstract
The human ZFAT gene was originally identified as a susceptibility gene for autoimmune thyroid disease. Mouse Zfat is a
critical transcriptional regulator for primitive hematopoiesis and required for peripheral T cell homeostasis. However, its
physiological roles in T cell development remain poorly understood. Here, we generated Zfatf/f-LckCre mice anddemonstrated that T cell-specific Zfat-deletion in Zfatf/f-LckCre mice resulted in a reduction in the number of
CD4+CD8+double-positive (DP) cells, CD4+single positive cells and CD8+single positive cells. Indeed, in Zfatf/f-LckCre DP
cells, positive selection was severely impaired. Defects of positive selection in Zfat-deficient thymocytes were not restored in
the presence of the exogenous TCR by using TCR-transgenic mice. Furthermore, Zfat-deficient DP cells showed a loss of
CD3f phosphorylation in response to T cell antigen receptor (TCR)-stimulation concomitant with dysregulation of
extracellular signal-related kinase (ERK) and early growth response protein (Egr) activities. These results demonstrate that
Zfat is required for proper regulation of the TCR-proximal signalings, and is a crucial molecule for positive selection throughERK and Egr activities, thus suggesting that a full understanding of the precise molecular mechanisms of Zfat will provide
deeper insight into T cell development and immune regulation.
Citation: Ogawa M, Okamura T, Ishikura S, Doi K, Matsuzaki H, et al. (2013) Zfat-Deficiency Results in a Loss of CD3f Phosphorylation with Dysregulation of ERK
and Egr Activities Leading to Impaired Positive Selection. PLoS ONE 8(10): e76254. doi:10.1371/journal.pone.0076254
Editor: Troy A Baldwin, University of Alberta, Canada
Received May 10, 2013; Accepted August 22, 2013; Published October 3, 2013
Copyright: ! 2013 Ogawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of the Science and a Grant-in-Aid
for the FCAM from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: sshirasa@fukuoka-u.ac.jp. These authors contributed equally to this work.

Introduction

Japanese and Korean population [7,8] and several common
diseases including hypertension and cancer [9,10]. Of greatinterest is that a genetic variant of ZFAT is reported to be strongly
associated with interferon-b responsiveness in multiple sclerosis[11] and the severity of Hashimoto’s disease [12]. However, theexact functions of ZFAT during T cell development remain
unknown.
T cells must be reactive to foreign pathogens, but tolerant toself-antigens. These features are formed during T cell developmentin the thymus [13]. CD4+CD8+double-positive (DP) cells expressing complete abTCR complexes undergo positive selection, fordifferentiation into mature CD4+single positive (CD4SP) cells or
CD8+single positive (CD8SP) cells [14–16]. DP cells that recognize
self-peptide and major histocompatibility complex (pMHC)
molecules with low affinities receive survival signals and differentiate into mature single positive cells; this process is known as
positive selection. Accumulating evidence suggests that mitogenactivated protein kinase (MAPK) signaling pathways and themolecules in this pathway play critical roles in the regulation of the

ZFAT was originally identified as a candidate susceptibility gene
for autoimmune thyroid disease [1]. ZFAT contains 18 zinc-finger
domains and one AT-hook and is an evolutionally conserved genefrom fish to humans, and the Zfat protein is highly expressed in thethymus and the spleen in adult mice [2]. We previously reported
that Zfat-deficient (Zfat2/2) mice are embryonic lethal byembryonic day 8.5, and Zfat is a critical transcriptional regulator
for primitive hematopoiesis [3], and that ZFAT is functionallyinvolved in the regulation of apoptosis of mouse embryonic
fibroblasts and MOLT24 cells [4,5].
Just recently, we generated Zfatf/f-Cd4Cre mice [6], and showedthat Zfat-deficient mice exhibited a reduction in the number of
peripheral T cells with decreased surface expression of IL-7Ra andT cell antigen receptor (TCR)-stimulation-induced expression of
CD25 and IL-2, indicating that Zfat is required for peripheral T
cell homeostasis [6]. On the other hand, genetic variants of ZFAT
have also been reported to be associated with adult height in

PLOS ONE | www.plosone.org

1

October 2013 | Volume 8 | Issue 10 | e76254

Role of Zfat in Positive Selection

However, a difference in the expression levels of Bcl-2, a prosurvival factor induced by IL-7-mediated signaling [27,28], was
not observed between the genotypes (Figure S1B), suggesting thatthe reduced expression of IL-7Ra did not play a functionallysignificant role in the reduction of T cells in the thymus of Zfatf/fLckCre mice.

cellular fate during T cell development [17]. Extracellular signalrelated kinase (ERK) is activated by phosphorylation throughsequential activation of Ras, Raf and MEK transduced by TCRstimulation, and proper ERK activation is essential for positive
selection [18–20].
Egr1, Egr2 and Egr3 are zinc-finger transcription factors of theearly growth response protein (Egr) family, and they are rapidly
induced in response to TCR-stimulation [21–23]. Egr1- or Egr2deficiency was reported to result in defects of positive selection andsurvival of T cells [24,25]. Egr3-deficiency was reported to cause
the impaired proliferation of thymocytes during transition from
DN to DP cells [26]. Thus, TCR-stimulation-induced Egrexpression is thought to be essential for the proper progressionof T cell development in the thymus.
In this study, we generated Zfatf/f-LckCre mice and showed thatthey exhibited a loss of CD3f phosphorylation with dysregulationof ERK and Egr activities leading to impaired positive selection.
This is the first report demonstrating that Zfat is required forproper regulation of the TCR-proximal signalings, and is a crucial
molecule for positive selection in the thymus.

Zfat-deficiency did not Affect the Transition of thymic DN
to DP Cells
As the proportion and number of DN cells in Zfatf/f-LckCre miceseemed to be slightly increased compared with those of Zfatf/f mice
(Figure 1C, 1D), we next analyzed whether LckCre-mediated Zfatdeficiency would affect the T cell development at the DN stage.
The proportions and total numbers of the DN subsets, i.e., DN1,
DN2, DN3 and DN4, in Zfatf/f-LckCre mice did not show
significant differences with those of Zfatf/f mice, although the total
numbers of DN3 and DN4 subsets seemed to be slightly elevatedin Zfatf/f-LckCre mice (Figure 2A, 2B). At the DN3 stage,
thymocytes undergo b-selection through the pre-TCR signaling,leading to the transition from DN3a (CD25+CD442CD27loFSClo)to DN3b (CD25+CD442CD27hiFSChi) cells [29,30]. The proportion of DN3b cells was comparable between the genotypes(Figure 2C), and the expression levels of intracellular TCRb
(icTCRb) in Zfatf/f-LckCre mice were comparable to those of Zfatf/f
mice during the transition from the DN3 (CD25+icTCRb+) to
DN4 (CD252icTCRb+) stage (Figure 2D), which together
indicated that the thymocytes in Zfatf/f-LckCre mice normallypassed through b-selection and transition from DN3 to DN4 cells.
We then addressed the transition from the DN4 to DP stage by
analyzing an ex vivo culture system using Tst-4/DLL1 cells, which
can support T cell development to the DP stage [31]. DN4 cells
were sorted and then cultured on a monolayer of Tst-4/DLL1
cells. After 3 days of the culture, we confirmed a decline in Zfat
expression in the thymocytes sorted from Zfatf/f-LckCre thymus(Figure 2E), and observed the comparable proportions of the
production of DP cells between Zfatf/f and Zfatf/f-LckCre thymocytes (Figure 2F), indicating no blockade of the transition of DN4
to DP cells in Zfatf/f-LckCre mice. Taken together, Zfat-deficiency
did not affect the transition of thymic DN to DP cells including bselection and transitions from DN3 to DN4 cells, and from DN4 to
DP cells. In agreement with a decrease in DP cells in vivo
(Figure 1D), however, the total number of the produced DP cells of
Zfatf/f-LckCre mice in ex vivo culture system was significantly
reduced compared with that of Zfatf/f mice (Figure 2G), suggestingthat there may be a defect in the proliferation or survival during
the transition from DN to DP stage in the Zfat-deficient
thymocytes.

Results
Reduction in the Number of Thymic DP, CD4SP and
CD8SP cells in Zfatf/f-LckCre MiceTo clarify the physiological roles of Zfat in T cell developmentin the thymus, we crossed Zfatf/f mice [6] with LckCre transgenic
mice. The deletion of Zfat in Zfatf/f-LckCre thymocytes was
confirmed by an immunoblot analysis. While Zfat was detected
specifically in the nuclear fraction of Zfatf/f thymocytes, the protein
was hardly observed in Zfatf/f-LckCre thymocytes (Figure 1A),
indicating the efficient deletion of Zfat in the thymocytes of Zfatf/fLckCre mice. During the transition of DN stages in the Zfatf/f mice,
the Zfat expression levels in the DN1 (CD252CD44+) and DN2(CD25+CD44+) subsets was low, whereas Zfat expression in the
DN3 (CD25+CD442), DN4 (CD252CD442) and DP subsets was
detected at a higher level (Figure 1B). In contrast, in Zfatf/f-LckCre
mice, Zfat expression in the DN3 subset was slightly decreased
compared with that of Zfatf/f mice, whereas Zfat expression in
DN4 and DP subsets was apparently decreased compared withthat of Zfatf/f mice (Figure 1B). These results indicated that theZfat expression in Zfatf/f-LckCre mice was abolished at the DN4stage.
In Zfatf/f-LckCre mice, the proportions of CD4SP and CD8SP
cells, but not DP cells, were remarkably reduced and the total
number of thymocytes, DP cells, CD4SP cells and CD8SP cells
was significantly decreased compared with that of Zfatf/f mice
(Figure 1C, 1D). On the other hand, the proportion and total
number of DN cells in Zfatf/f-LckCre mice seemed to be slightly
increased compared with those of Zfatf/f mice, however, thedifference of the total number of DN cells was not statisticallysignificant (Figure 1C, 1D). Consistent with the decreased
proportions and total number of CD4SP and CD8SP cells in
the Zfatf/f-LckCre thymus, a reduction in the proportion of
TCRb+T cells in both the spleen and lymph nodes (LNs) was
observed in Zfatf/f-LckCre mice (Figure 1E). The proportion and thetotal number of CD4+or CD8+T cells in the spleen and LNs weresignificantly reduced in Zfatf/f-LckCre mice in comparison to thoseof Zfatf/f mice (Figure 1E, 1F). These results demonstrated that
Zfat-deficiency results in impaired T cell development in thethymus.
In the thymus, a slight reduction in the expression levels of IL7Ra on CD4SP and CD8SP cells in Zfatf/f-LckCre mice wasobserved compared with those from Zfatf/f mice (Figure S1A).
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Impaired Positive Selection in Thymocytes of Zfatf/fLckCre Mice
Loss of Zfat did not affect the DN to DP transition, in spite ofsignificant reduction in the number of DP cells. We next analyzed
an involvement of Zfat in positive selection of the DP cells. During
the positive selection, TCRbintCD692 DP cells initially show a
TCRbintCD69+transitional phenotype (P-I; Figure 3A) after the
TCR/pMHC interaction. After the positive selection, P-I cells
become TCRbhiCD69+cells (P-II; Figure 3A) and then differentiate into CD4SP or CD8SP cells (TCRbhiCD692) (P-III;
Figure 3A) [20]. A considerable reduction in the proportion of
P-I cells in thymocytes (1.4% versus 3.87%, respectively,
Figure 3A) and DP cells (1.65% versus 4.35%, respectively,
Figure 3B) from Zfatf/f-LckCre mice was observed compared withthat of Zfatf/f mice, indicating the existence of impaired positive
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Role of Zfat in Positive Selection

Figure 1. Reduction in the number of thymocytes and peripheral T cells in Zfatf/f-LckCre mice. (A) An immunoblot of Zfat in nuclear (N) or
cytoplasmic (C) fractions of thymocytes from Zfatf/f and Zfatf/f-LckCre mice. CREB1 and PLCc1 were used as controls of nuclear and cytoplasmic
fractions, respectively. (B) An immunoblot of Zfat in DN1 and DN2 (DN1,2: CD42CD82CD44+), DN3 (CD42CD82CD25+CD442), DN4(CD42CD82CD252CD442) or DP thymocytes from Zfatf/f and Zfatf/f-LckCre mice. Actin was used as a loading control. (C) Flow cytometry analysisof the surface expression of CD4 and CD8 on thymocytes from Zfatf/f (top) and Zfatf/f-LckCre (bottom) mice at 6 to 7 weeks of age. Data arerepresentative of three independent experiments. (D) Total numbers of thymocytes, thymic DP, DN, CD4SP or CD8SP cells from Zfatf/f (black bars) andZfatf/f-LckCre (white bars) mice at 6 to 7 weeks of age. The data are the mean 6 standard deviation (s.d.); n = 6; * P,0.05; ** P,0.01; n.s., notsignificant. (E) Flow cytometry analysis of TCRb+T cells and B220+B cells (left) or CD4+and CD8+T cells (right) in the spleen and LNs of Zfatf/f (top) andZfatf/f-LckCre (bottom) mice. Data are representative of three independent experiments. (F) Total numbers of CD4+T, CD8+T or B220+B cells in the
spleen from Zfatf/f (black bars) and Zfatf/f-LckCre (white bars) mice. The data are the mean 6 s.d.; n = 6; ** P,0.01; n.s., not significant.
doi:10.1371/journal.pone.0076254.g001

selection in the Zfat-deficient DP thymocytes. However, no
obvious alterations in the rearrangements of TCRachains (FigureS2), the surface expression of TCRb (Figure 3C) and CD45

(Figure 3D) [32] on DP thymocytes were observed between the
genotypes. In addition, the expression levels of CD5, which is
known to be correlated with the avidity of TCR/pMHC

Figure 2. Zfat-deficiency did not affect the transition of thymic DN3 to DP cells. (A) Flow cytometry analysis of the surface expression of
CD44 and CD25 on CD42CD82 DN thymocytes from Zfatf/f and Zfatf/f-LckCre mice at 6 to 7 weeks of age. The numbers in the quadrants indicate thepercent DN1 (upper left), DN2 (upper right), DN3 (lower right) or DN4 (lower left) subsets in DN thymocytes from the indicated genotypes. Data arerepresentative of three independent experiments. (B) Total numbers of DN1, DN2, DN3 or DN4 cells from Zfatf/f (black bars, n = 3) and Zfatf/f-LckCre(white bars, n = 3) mice at 6 to 7 weeks of age. The data are the mean 6 standard deviation (s.d.); n.s., not significant. (C) Flow cytometry analysis of
the surface expression of CD27 and forward scatter (FSC) of DN3 thymocytes. The gated area indicates DN3b (CD27hiFSChi) subsets. Data arerepresentative of three independent experiments. (D) Flow cytometry analysis of TCRb-negative DN (CD42CD82TCRb2) cells. The intracellular
expression of TCRb (icTCRb) and surface expression of CD25 are shown. Data are representative of three independent experiments. (E-G) Sorted DN4cells were co-cultured with Tst-4/DLL1 cells for 3 days and analyzed by an immunoblot of Zfat (E) and flow cytometry for the surface expression of
CD4 and CD8 (F). The proportions (F) or total numbers (G) of CD4+CD8+DP thymocytes from Zfatf/f (top, black bar, n = 3) or Zfatf/f-LckCre (bottom,
white bar, n = 3) mice are shown. The data are the mean 6 standard deviation (s.d.); ** P,0.01. * P,0.05.
doi:10.1371/journal.pone.0076254.g002

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Role of Zfat in Positive Selection

Figure 3. Impaired positive selection in Zfatf/f-LckCre thymus. (A, B) A scheme of the intermediates of positive selection defined by changes in
expression of TCRb and CD69: TCRbintCD69+(P-I), TCRbhiCD69+(P-II) and TCRbhiCD692 (P-III) are generated sequentially as shown by arrows (A, left).
Flow cytometry analysis of the surface expression of CD69 and TCRb on total thymocytes (A) or DP cells (B) from the indicated genotypes at 6 to 7
weeks of age. The numbers indicate the proportion of the gated area. Data are representative of three independent experiments. (C-E) Surface
expression of TCRb on DP cells (C), CD45 on DP cells (D) or CD5 on DP, CD4+CD82TCRb+or CD42CD8+TCRb+cells (E) from Zfatf/f (gray-filled) and Zfatf/fLckCre (black line) mice at 6 to 7 weeks of age. Data are representative of three independent experiments. (F, G) Flow cytometry analysis for thesurface expression of CD4 and CD8 on the thymocytes and total numbers of CD4+CD8int, CD8SP or CD4SP cells from OT-I Zfatf/f (black bar) and OT-I
Zfatf/f-LckCre mice (white bar) (F) or from OT-II Zfatf/f (black bar) and OT-II Zfatf/f-LckCre mice (white bar) (G). The numbers indicate the proportion of
the gated area. The data are the mean 6 standard deviation (s.d.); n = 3; ** P,0.01. Data are representative of three independent experiments.
doi:10.1371/journal.pone.0076254.g003

Zfatf/f mice (Figure 3G). Together, these results confirmed that
positive selection is impaired in the Zfat-deficient thymocytes.

interaction [33], on DP, CD4SP and CD8SP cells werecomparable between the genotypes (Figure 3E). These results
strongly indicated that the abTCR recombination and TCR/
pMHC avidity are normal during T cell development in Zfatf/fLckCre mice.
To further confirm the impaired positive selection in Zfatf/fLckCre mice, we crossed ovalbumin (OVA)-specific MHC class Irestricted TCR transgenic (OT-I) [34] or MHC class II-restricted
TCR transgenic mice (OT-II) [35] with Zfatf/f-LckCre mice, andexamined the developmental fate of thymocytes in OT-I Zfatf/fLckCre and OT-II Zfatf/f-LckCre mice. It is known that DP cells passthrough the CD4+CD8int transitional stage before complete
differentiation into either CD4SP or CD8SP cells [14]. As
expected, not only the proportions but also the total numbers of
CD8SP and CD4+CD8int cells were significantly decreased in OTI Zfatf/f-LckCre mice compared with those of OT-I Zfatf/f mice
(Figure 3F), suggesting that MHC class I-restricted positive
selection in OT-I Zfatf/f-LckCre mice was impaired. In addtion,
MHC class II-restricted positive selection in OT-II Zfatf/f-LckCre
mice was also impaired, as both the proportions and the total
numbers of CD4SP and CD4+CD8int cells were remarkably
reduced in OT-II Zfatf/f-LckCre compared with those of OT-II
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Zfat-deficiency Impaired CD3f Phosphorylation with
Defects in ERK1/2 Activation
To elucidate mechanisms of the impaired positive selection
observed in Zfatf/f-LckCre DP cells, we examined phosphorylationof molecules working at the signaling transduced by TCRstimulation. Phosphorylations of ERK1/2 induced by TCRstimulation, which is known to be critical in the positive selection,
were markedly decreased in Zfatf/f-LckCre thymocytes comparedwith those of Zfatf/f thymocytes (Figure 4). In agreement with thedefects in ERK1/2 activation, the phosphorylations of both
MEK1/2 and c-Raf, which are located upstream of the ERK
signaling pathway, were also reduced in the Zfatf/f-LckCre
thymocytes compared with those of Zfatf/f thymocytes (Figure 4).
Furthermore, the phosphorylations of Zap70 and PLCc1 were
diminished in Zfatf/f-LckCre thymocytes (Figure 4). Finally, TCR
stimulation-induced phosphorylation of CD3f was virtually
ablated in the Zfatf/f-LckCre thymocytes, and the phosphorylatedCD3f at non-stimulated status was also apparently diminished dueto the Zfat-deficiency (Figure 4). However, phosphorylated status
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Role of Zfat in Positive Selection

with those of OT-II Zfatf/f mice (Figure S3B), and together
these findings suggested that the impaired phosphorylation of
ERK and Egr expression induced by TCR-stimulation wascaused by molecules other than TCR itself.
To address whether MEK activation is essential for ERK
mediated Egr inductions under the TCR-stimulated condition, we
stimulated DP cells in the presence of U0126, an inhibitor of
MEK1/2 [37]. We found that the inductions of Egr1 and Egr2
were considerably reduced by the treatment with U0126 both inZfatf/f and Zfatf/f-LckCre DP cells (Figure 5B), indicating that theenhancements of Egr1 and Egr2 expression by TCR-stimulation
were essentially regulated by the MEK-ERK axis. On the other
hand, the Egr3 induction was just partly reduced by the treatment
with U0126 in Zfatf/f DP cells, whereas the Egr3 expression in theZfatf/f-LckCre DP cells seemed to be slightly increased or unalteredby TCR-stimulation in the presence of U0126 (Figure 5B). These
results suggested the possibility that Egr3 expression may beregulated by other signaling pathways in addition to the MEKERK pathway.
While the levels of Egr mRNA expression in the unstimulated
DP cells were comparable between the Zfatf/f and Zfatf/f-LckCre
genotypes (Figure 5C), each of the Egr1, Egr2 and Egr3 mRNA
inductions by TCR-stimulation were robustly suppressed in Zfatf/fLckCre DP cells compared with those of Zfatf/f DP cells (Figure 5D),
suggesting that Zfat is required for proper Egr gene expression
induced by TCR-stimulation. However, considering the fact thatthe expressions of Egr proteins were slightly decreased in theunstimulated Zfatf/f-LckCre DP cells (Figure 5A), Zfat-deficiency
might affect the turnover or degradation of Egr proteins as well as
the expression of Egr mRNAs. Taken together, these results
indicated that impairment of the TCR-stimulation-induced Egrinductions in the Zfat-deficient thymocytes leads to the defects ofpositive selection.

at Tyr505 of Lck, which phosphorylates tyrosine residues in
ITAMs of CD3f in response to TCR-stimulation [36], wascomparable between the genotypes. Taken together, these results
indicate TCR signaling cascade is strikingly attenuated in Zfatf/fLckCre DP cells at CD3f phosphorylation concurrent with ERK1/
2 phosphorylation. Moreover, the impaired positive selection in
Zfatf/f-LckCre DP cells is thought to be attributed to defects in theactivation of ERK1/2 induced by TCR-stimulation.

TCR-stimulation-induced Egr Expressions were Impairedin the Zfat-deficient Thymocytes
Next, we examined the expression levels of Egr1, Egr2 andEgr3, which are known to be induced by TCR-stimulation.
Both the Egr1 and Egr2 expressions were robustly increased in
the Zfatf/f DP cells after the stimulation with anti-CD3e andanti-CD28 antibodies in vitro (Figure 5A), whereas expression
levels of both Egr1 and Egr2 in the Zfatf/f-LckCre DP cells after
the stimulation were not much induced compared with those of
Zfatf/f DP cells. In addition, it appeared that the Egr1
expression level before the stimulation was also decreased in
the Zfatf/f-LckCre DP cells compared with that of the Zfatf/f DPcells (Figure 5A). On the other hand, Egr3 was constitutively
expressed and slightly increased in the Zfatf/f DP cells after the
stimulation, whereas the expression levels of Egr3 in the Zfatf/fLckCre DP cells were constitutively lower compared with those of
Zfatf/f DP cells and rarely enhanced by TCR-stimulation(Figure 5A). These results indicated that Zfat-deficiency causes
dysregulated expression of Egr protein in the DP cells before
and after TCR-stimulation. Similar experiments were performedon the thymocytes from OT-II Zfatf/f-LckCre mice. Phosphorylation of ERK induced by TCR-stimulation in the thymocytes
from OT-II Zfatf/f-LckCre mice was decreased compared withthat of OT-II Zfatf/f mice (Figure S3A). Furthermore, the levelsof TCR-stimulation-induced Egr1, Egr2 and Egr3 expression in
OT-II Zfatf/f-LckCre thymocytes were all decreased compared

Figure 4. Zfat-deficiency impaired CD3f phosphorylation with defects in ERK1/2 activation. Immunoblots for phosphorylated or total
protein of ERK, MEK1/2, c-Raf, Zap70, PLCc1, CD3f and Lck before or at the indicated time points after the stimulation with cross-linking anti-CD3e
antibody in thymocytes from the indicated genotypes. The values below each image represent the relative ratio of the amount of phosphorylated
protein to total protein. Data are representative of three independent experiments.
doi:10.1371/journal.pone.0076254.g004

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Role of Zfat in Positive Selection

Figure 5. TCR-stimulation induced Egr expressions were impaired in the Zfat-deficient DP thymocytes. (A)Immunoblots for Egr1, Egr2
and Egr3 before or at the indicated time points after the stimulation with plate-bound anti-CD3e and anti-CD28 antibodies in DP cells from theindicated genotypes. Actin was used as a loading control. Data are representative of three independent experiments. (B) Immunoblots for Egr1, Egr2
and Egr3 in DP cells from the indicated genotypes before or at the indicated time points after the stimulation with plate-bound anti-CD3e and antiCD28 antibodies under the condition with U0126 in DMSO or with DMSO alone. Actin was used as a loading control. Data are representative of three
independent experiments. (C, D) Quantitative RT-PCR analysis of Egr1, Egr2 and Egr3 expression before (C) or at the indicated time points after the
stimulation with anti-CD3e and anti-CD28 antibodies (D) in DP cells from Zfatf/f and Zfatf/f-LckCre mice. The relative expression for each gene wasnormalized by the expression of Actb. The results are presented as the value relative to the unstimulated DP cells from Zfatf/f mice. The data are themean 6 s.d.; n.s., not significant.
doi:10.1371/journal.pone.0076254.g005

Discussion

dysregulated signaling downstream of TCR itself. TCR-stimulation triggers activation of the ERK pathway through sequential
activation of Ras, Raf and MEK [17]. Activation of ERK is criticalin the TCR-mediated signaling cascades and an essential
requirement for positive selection in the thymus [19,20].
Both Egr1 and Egr2 expression are critically regulated by
activated ERK transduced by TCR-stimulation and play pivotal
roles in positive selection and survival of DP cells [24,25,38].
Intriguingly, we identified the defects of TCR-stimulated ERK
phosphorylation and Egr inductions in Zfat-deficient DP thymocytes, indicating that impaired Egr induction was at least partially
responsible for the defects of positive selection in Zfatf/f-LckCre
mice. Moreover, Egr3 was also dysregulated in Zfat-deficient
thymocytes. Egr3-deficient mice have been reported to exhibit a
defect in the thymocytes proliferation and a partial block indifferentiation at the DN3 stage [26]. Therefore, a possibility that
Zfat plays an important role in the proliferation of thymocytes
during the DN to DP transition through Egr3 induction is not
excluded.
Decreased phosphorylation of CD3f in Zfatf/f-LckCre thymocytes induced by TCR-stimulation was observed, indicating that
Zfat-deficiency results in impaired activation of TCR signaling at
proximal level. Tyrosines in ITAMs of CD3f are phosphorylated
by Src family kinase Lck, and then the tyrosine-phosphorylated
ITAMs of CD3f serve as docking sites for Zap70 in response to
TCR stimulation [36]. However, Zfat-deficiency did not affectphosphorylation status of Lck in the thymocytes, whereas
phosphorylation of Zap70 was reduced in Zfatf/f-LckCre thymo-

In this study, we demonstrated that Zfat is required for theproper phosphorylation of CD3f, and is a critical regulator for T
cell development in the thymus, especially for positive selection.
The molecular mechanism by which Zfat-deficiency leads toimpaired positive selection would be mainly dependent on the lossof CD3fphosphorylation leading to the impaired ERK activationtransduced by TCR-stimulation. Furthermore, activated ERKmediated expression of Egr1 and Egr2, which are criticalregulators for positive selection, were also decreased in the Zfatf/f-LckCre thymocytes. Taken together, these results suggest that Zfat
is involved in the proper regulation of the TCR-proximal
signalings, which is required for positive selection in the thymus.
Zfatf/f-LckCre mice showed a considerable reduction in thenumber of DP thymocytes. We could not find apparent defects in
T cell development at DN stage or transition of DN to DP cells in
Zfat-deficient thymocytes in vivo and ex vivo experiments. Therefore, the reduction in the number of DP cells in Zfatf/f-LckCre mice
may be due to the Zfat-deficiency at the DP stage. However, Zfat
expression was not completely abolished in the Zfatf/f-LckCre DN3
thymocytes, and thus the possibility that Zfat is necessary forproper development at the DN stage cannot be excluded.
Impaired positive selection in Zfat-deficient thymocytes was notrestored in the presence of OT-I TCR or OT-II TCR transgene,which promote positive selection and differentiation of DP cells
into CD8SP or CD4SP cells. These results strongly indicated that
positive selection is impaired in Zfat-deficient mice, and alsosuggested that the defect of positive selection is caused by the
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Role of Zfat in Positive Selection

ish peroxidase-conjugated secondary antibody (GE Healthcare)and SuperSignal West Pico Chemiluminescent Substrate (Thermo
Scientific) were used for the detection. The quantitative analysis of
the immunoblotting was done using the integration value of eachblot with Image J software (version1.46, US National Institutes of
Health) [42].

cytes. We have not elucidated how exactly Zfat affects the
CD3fphosphorylation in this study. Activation of CD3f isnegatively regulated by SHP21 and SHP22 (SH2 domaincontaining phosphatase-1 and -2) through dephosphorylation[39,40]. Moreover, c-Cbl E3 ubiquitin ligase reduces CD3f levels
at the plasma membrane by stimulating internalization [39,41].
Considering that Zfat is expected to be a transcriptional regulator
in the nucleus [2,3], Zfat might affect the expressions of the genes
involved in the regulation of CD3f phosphorylation, such as SHP1, SHP-2 and c-Cbl. However, elucidation of the precise
molecular mechanisms of Zfat function in regulation of TCR
signaling should await future studies.
Proper activation of TCR signaling is also required for negative
selection in the thymus. Thus, Zfat might be involved in thenegative selection since Zfat-deficiency results in the defect inproximal molecules of TCR complex. However, we have not seen
obvious differences in the T cell developments between H-Y Zfatf/f
and H-Y Zfatf/f-LckCre male mice in preliminarily experiments(data not shown). To establish a role for Zfat in the negative
selection, further studies should be required in the future.
In conclusion, we demonstrated that Zfat-deficiency in DP cells
results in a loss of CD3f phosphorylation with dysregulation of
ERK and Egr activities leading to impaired positive selection in
the thymus, suggesting that Zfat is a pivotal molecule in T cell
development. As the activation of ERK-Egr pathway was notcompletely impaired in the Zfat-deficient thymocytes, the possibility that Zfat plays crucial roles in particular signaling pathways
other than ERK-mediated pathway does exist. Thus, a fullunderstanding of the roles and precise molecular mechanisms ofthe transcriptional regulator Zfat will lead to a better understanding of the orchestrated gene expression programs and provide
deeper insight into T cell development, immune regulation and awide variety of diseases.

Flow Cytometry
Cells from the spleen, lymph nodes and the thymus weredepleted of erythrocytes by hypotonic lysis and stained withfluorophore-conjugated antibodies. A Cytofix/Cytoperm kit (BDBiosciences) was used for analysis of the intracellular detection of
TCRb. Data were collected with a cytometer (FACSAria II; BD
Biosciences) and analyzed with FlowJo software (Tomy Digital
Biology). The fluorophore-conjugated antibodies used for flow
cytometry analysis and their specificities were as follows: CD4(RM4–5), CD8 (53-6.7), B220 (RA3-6B2), CD25 (PC61), CD44
(IM7), CD27 (LG.3A10), TCRb (H57-597), CD69 (H1.2F3),
CD45 (30-F11) and CD5 (53-7.3; all from Biolegend).

In vitro Differentiation of DN3 Cells
Tst-4/DLL1 stromal cells (RCB2118) were provided by the
RIKEN BRC through the National Bio-Resource Project of the
MEXT, Japan. Cells were plated and co-cultured with sorted DN4(CD42CD82CD252CD442) cells. DN4 cells (16105) were
cultured at 37uC with 5% CO2 in RPMI 1640 medium containing
10% FCS, penicillin-streptomycin-glutamine (Life Technologies)
and b-mercaptoethanol (50 nM; Sigma) supplemented withrecombinant mouse IL-7 (0.5 ng/ml; Pepro Tech) and SCF(1 mg/ml; R&D Systems).

In vitro TCR-stimulation
Thymocytes (16107) from mice were stimulated at 37uC with
anti-CD3e antibody (10 mg/ml, 145-2C11; Biolegend) cross-linked
with goat anti-hamster IgG (80 mg/ml; Southern Biotech) in
RPMI 1640 medium containing 10% FCS, penicillin-streptomycin-glutamine (Life Technologies) and b-mercaptoethanol (50 nM;
Sigma). For the stimulation with plate-bound anti-CD3e and antiCD28 antibodies, plates were coated overnight with anti-CD3e(1 mg/ml) and anti-CD28 (3 mg/ml, 37.51; Biolegend) antibodies.
DP cells (56106) were stimulated with plate-bound anti-CD3e andanti-CD28 antibodies under the condition with U0126 (100 mM;
Calbiochem) in DMSO or with DMSO alone.

Materials and Methods
Mice
Zfatf/f mice were originally generated as described previously [6]and backcrossed to a C57BL/6 background over seven generations. Zfatf/f mice were crossed to Lck-Cre mice from Taconic togenerate T-cell-specific Zfat knockout (Zfatf/f-LckCre) mice in the
C57BL/6 background. All the animal experiments were approvedby the Animal Care and Use Committee of the NCGM ResearchInstitute, and the experiments on mice were carried out under theguidelines of the Institutional Animal Care and Use Committee of
Fukuoka University.

Quantitative RT-PCR
Total RNA was extracted by TRIZol reagent with PureLink
RNA Mini Kit (Life Technologies). Superscript VILO cDNA
synthesis kit (Life Technologies) was used for reverse transcription.
Quantitative RT-PCR was performed by using Thunderbird
SYBR qPCR Mix (TOYOBO) with ABI PRISM 7900HT(Applied Biosystems). The PCR primers used for Egr1 59AGGACTTGATTTGCATGGTATTGGA-39 and 59-ATGCAGGGCAGGGTTCTGAG-39,
Egr2
59-GAACCAGGACACCGTGAGATGA-39 and 59-GTAGTGTTGGCAGCTCGGACAG-39, Egr3 59-GACTCGGTAGCCCATTACAATCAGA39 and 59-GAGAGTTCCGGATTGGGCTTC-39 and Actin 59CATCCGTAAAGACCTCTATGCCAAC-39 and 59-ATGGAGCCACCGATCCACA-39.

Immunoblotting
The nuclear and cytoplasmic fractions from thymocytes wereprepared using NE-PER Nuclear and Cytoplasmic Extraction Kit(Pierce). Separated cells were lysed in RIPA buffer [50 mM TrisHCl, pH 7.5, 150 mM NaCl, 1% NP240, 0.5% deoxycholate,
0.1% SDS, protease inhibitor cocktail (Roche)]. Equal amounts of
total lysates were electrophoresed in 7, 8.5 or 4–20% SDSpolyacrylamide gels, and transferred to a nitrocellulose membrane(GE Healthcare). The antibodies used for immunoblotting analysis
and their specificities were as follows: Egr3, phosphorylated ERK,
antibodies for phosphorylated and total Zap70, PLCc1, c-Raf, andLck (all from Cell Signaling Technology); CREB1 (C-21), Erk (K23) and Egr1 (C-19; all from Santa Cruz); total and phosphorylated MEK1/2 (from New England Biolabs); Actin (A2066; from
Sigma); Egr2 (from Proteintech Group); CD3f (from Exbio);
phosphorylated CD3f (A0468; from Assay biotech). Anti-Zfat
antibody was prepared as described previously [2]. The horseradPLOS ONE | www.plosone.org

Statistical Analysis
Data are presented as the means 6 s.d. and statistical analysis
was performed using an unpaired Student’s t-test when comparing

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Role of Zfat in Positive Selection

the means of two groups. Differences of P,0.05 were considered
to be statistically significant (*P,0.05; **P,0.01).

GACCAACGCCAC-39 with Ca Rv primer 59-TTCAGCAGGAGGATTCGGAG-39,
Gapdh
Fw
59-GAACGGATTTGGCCGTATTG-39
and
Gapdh
Fw
59GATGATGACCCTTTTGGCTC-39. Data are representativeof three independent experiments.
(TIF)

Accession Number
GenBank mRNA sequences: Zfat, NT_078782.

Supporting Information

Figure S3 Reduced ERK activation and Egr induction in
OT-II Zfatf/f-LckCre thymocytes. (A) Immunoblots forphosphorylated or total protein of ERK before or the indicatedtime points after the stimulation with cross-linking anti-CD3e
antibody in thymocytes from the indicated genotypes. The values
below each image represent the relative ratio of the amount ofphosphorylated protein to total protein. Data are representative of
three independent experiments. (B) Immunoblots for Egr1, Egr2
and Egr3 before or at the indicated time points after the
stimulation with plate-bound anti-CD3e and anti-CD28 antibodies in DP cells from the indicated genotypes. Actin was used as aloading control. Data are representative of three independent
experiments.
(TIF)

Surface expression of IL-7Ra and intracellular expression of Bcl-2. Flow cytometry analysis of theexpression of surface IL-7Ra (A) and intracellular Bcl-2 (B) in
the DP, CD4SP and CD8SP cells from Zfatf/f (gray-filled) andZfatf/f-LckCre (black line) mice at 6 to 7 weeks of age (left). Data ofthymocytes from Zfatf/f (black bar) and Zfatf/f-LckCre (white bar)
mice were measured as the mean fluorescence intensity (MFI)(right). The fluorophore-conjugated antibodies used for flow
cytometry analysis and their specificities were as follows: IL-7Ra(A7R34) and Bcl-2 (10C4; all from Biolegend). Data arerepresentative of three independent experiments. The data are
the mean 6 s.d.; * P,0.05; ** P,0.01; n.s., not significant.
(TIF)
Figure S1

Figure S2 Rearrangements of TCRa chains in Zfatf/f or

Acknowledgments

Zfatf/f-LckCre thymocytes. Semiquantitative RT-PCR analysis of Va-to-Ca rearrangements in DP thymocytes from theindicated genotypes. Ca-Ca amplification within a Ca region
served as the control. Gapdh, an internal control. Primer sequencesused for amplifications were as follows: Va3 59-CCCAGTGGTTCAAGGAGTGA-39, Va6 59-CTGACTCATGTCAGCCTGAGAG-39, Va8 59-CAACAAGAGGACCGAGCACC-39, Va14 59TGGGAGATACTCAGCAACTCTGG-39,
Va19
59CTGCTTCTGACAGAGCTCCAG-39 and Ca 59-TTCAAAGA-

The authors thank Takahiro Fujimoto, Aya Fujikane, Masato Hamabashiri, Midori Koyanagi and Takami Danno for technical assistance.

Author Contributions
Conceived and designed the experiments: MO SS. Performed theexperiments: MO KD HM YT T. Ota KH HS. Analyzed the data: MO
SI TT. Contributed reagents/materials/analysis tools: T. Okamura TS SS.
Wrote the paper: MO SI SS.

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Differential Function of Themis CABIT Domains during T
Cell Development
Toshiyuki Okada1., Takeshi Nitta1., Kentaro Kaji1, Akiko Takashima1, Hiroyo Oda1, Norimasa Tamehiro1,
Motohito Goto2, Tadashi Okamura2,3, Michael S. Patrick1, Harumi Suzuki1*
1 Department of Immunology and Pathology, Research Institute, National Center for Global Health and Medicine, Ichikawa-shi, Chiba, Japan, 2 Department of Laboratory
Animal Medicine, Research Institute, National Center for Global Health and Medicine, Shinjuku, Tokyo, Japan, 3 Department of Infectious Diseases, Research Institute,
National Center for Global Health and Medicine, Shinjuku, Tokyo, Japan

Abstract
Themis (also named Gasp) is a newly identified Grb2-binding protein that is essential for thymocyte positive selection.
Despite the possible involvement of Themis in TCR-mediated signal transduction, its function remains unresolved andcontroversial. Themis contains two functionally uncharacterized regions called CABIT (cysteine-containing, all-b in Themis)
domains, a nuclear localization signal (NLS), and a proline-rich sequence (PRS). To elucidate the role of these motifs in
Themis’s function in vivo, we established a series of mutant Themis transgenic mice on a Themis2/2 background. Deletionof the highly conserved Core motif of CABIT1 or CABIT2 (Core1 or Core2, respectively), the NLS, or the PRS abolished Grb2association, as well as TCR-dependent tyrosine-phosphorylation and the ability to induce positive selection in the thymus.
The NLS and Core1 motifs were required for the nuclear localization of Themis, whereas Core2 and PRS were not.
Furthermore, expression of DCore1- but not DCore2-Themis conferred dominant negative-type inhibition on T cell
development. Collectively, our current results indicate that PRS, NLS, CABIT1, and CABIT2 are all required for positive
selection, and that each of the CABIT domains exerts distinct functions during positive selection.
Citation: Okada T, Nitta T, Kaji K, Takashima A, Oda H, et al. (2014) Differential Function of Themis CABIT Domains during T Cell Development. PLoS ONE 9(2):e89115. doi:10.1371/journal.pone.0089115
Editor: Jon C. D. Houtman, University of Iowa, United States of America
Received October 18, 2013; Accepted January 15, 2014; Published February 21, 2014
Copyright: ! 2014 Okada et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by JSPS Grant-in-Aid for Scientific Research (B) 22390098, and grant for National Center for Global Health and Medicine (21111, 22-303). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: hsuzuki@ri.ncgm.go.jp. These authors contributed equally to this work.

deficient mice, functional requirements for Themis in Tregs could
be different between rat and mouse.
Themis is constitutively associated with Grb2 and is tyrosinephosphorylated by Lck and ZAP-70 upon TCR stimulation [5-910]. Some groups reported constitutive association of Themis with
Vav1 [9], Itk, and PLC-c1 [5-10-11]. Furthermore, we and othergroups (Fig. S3) demonstrated association of Themis with PLC-c1,
LAT, and SLP76 [5-9-10-11] upon TCR-stimulation, indicatingthat Themis would be a component of the SLP76-LAT
signalosome. From these results, Themis is likely to be involved
in TCR-mediated signal transduction. Accordingly, TCR-dependent activation of ERK and NFAT, as well as production of IL-2
was significantly reduced in Themis knockdown Jurkat cells [10].
In the Themis-knockout mice, however, results were different. We
and other groups observed unaltered activation of ERK andcalcium influx in Themis deficient immature DP thymocyte uponanti-CD3 antibody stimulation [2-3-4], although one groupreported impaired activation of these signaling events [5].
Moreover, recent study showed that TCR-dependent activationof ERK, p38 and Vav1 were reduced in Themis deficient CD4SP
and CD8SP thymocytes [9]. After all, results were not consistent
between different groups possibly because of different experimental systems, and therefore no consensus has been reached about
the effect of Themis on TCR-mediated signal transduction.

Introduction
T cells develop through positive and negative selection in thethymus to become either class II MHC-restricted helper
CD4+CD82 [CD4-single positive (CD4SP)] or class I MHCrestricted cytotoxic CD42CD8+ (CD8SP) cells [1]. However, themolecular mechanisms of TCR-mediated selection in developing
T cells are not yet fully understood.
Themis (thymocyte-expressed molecule involved in selection)
was identified as a novel mandatory factor for positive selection by
five independent groups in 2009 [2-3-4-5-6]. We identified Themis(initially named Gasp) from a set of uncharacterized genes whose
expression was restricted to the thymus [2]. Themis knockout mice
exhibited significantly reduced numbers of CD4SP and CD8SP T
cells both in the thymus and periphery [2-3-4-5-6]. Inhibitory
effects of Themis deficiency on negative selection and T cell
activation were controversial among the reports [2-3-4-5],
nevertheless, they were much slighter than that for positive
selection. Thus, Themis is a unique molecule that is essential for
positive selection, but not for negative selection. Expression ofThemis was significantly lower in regulatory T cells (Tregs)
compared to conventional T cells [7], however, study of a natural
mutant rat revealed the importance of Themis on the function ofTregs [8]. Since Themis-deficient mice did not exhibit inflammatory bowel disease or autoimmune diseases observed in TregPLOS ONE | www.plosone.org

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Function of Themis CABIT Domain

washed, resuspended in RPMI-1640 complete medium, andstimulated with streptavidin (BECKMAN COULTER, 10 mg/mL) at 37uC for the indicated times. Ice-cold PBS or paraformaldehyde (final concentration of 2%) was added to stop stimulation.

Themis contains two novel cysteine-based CABIT (cysteinecontaining, all beta in Themis) domains [4], a bipartite type
nuclear localization sequence (NLS), and a proline-rich sequence(PRS). The CABIT domain is a newly designated domain
structure conserved among metazoans, and it could adopt an
all-beta-strand structure with at least 12 strands, which suggests
either an extended beta-sandwich-like fold or a dyad of sixstranded beta-barrel units [4]. In mammals, the CABIT domains
are conserved among three Themis family proteins (Themis/
Themis1, ICB1/Themis2 and 9130404H23Rik/Themis3) harboring two tandemly-repeated CABIT domains (CABIT1 and
CABIT2) and two Fam59 proteins (Fam59a and b) containing one
CABIT domain [4]. Although a number of proteins containing
CABIT domains have been identified, their function is totally
unknown. Therefore, elucidation of the function of CABIT
domains has long been awaited.
In order to reveal the function of Themis in positive selection,
we investigated the function of each structural domain and motifin Themis. In the present study, we generated a series of transgenic(Tg) mice expressing mutant Themis proteins lacking each domain
on a Themis2/2 background. Deleted motifs were the PRS andthe highly conserved cysteine-containing Core motif of the
CABIT1 or CABIT2 domain (Core1 and Core2, respectively).
We found that the PRS, Core1, and Core2 motifs were all
required for Grb2-binding and TCR-dependent tyrosine-phosphorylation, as well as for positive selection in the thymus.
Interestingly, the Core1 and NLS motifs were required for nuclear
localization of Themis, whereas Core2 and PRS were not.
Furthermore, Core1- but not Core2-deleted mutant exhibited adominant negative inhibitory effect on T cell development even in
the presence of wild-type Themis. These results indicate that eachstructural motif in Themis exerts an essential but distinct role in T
cell development, and that the two CABIT domains in Themis
have distinct functions.

Immunoprecipitations and Western Blot Analysis
Cells were lysed with lysis buffer (50 mM Tris-HCl [pH7.5],
150 mM NaCl, 10 mM MgCl2, 0.5% Nonidet P-40) containing
protease and phosphatase inhibitor cocktail (Thermo Scientific)).
Cell lysates were immunoprecipitated with antibodies conjugatedto protein G-sepharose beads (GE). Antibodies used for immunoprecipitation were anti-Themis pAb (06-1328, Millipore) and antiThemis mAb 2E7. 2E7 is a rat monoclonal antibody produced
against recombinant full-length Themis from mice [2]. Antibodies
used for western blotting are as follows: anti-Grb2 (MS-20-3,MBL), anti-phospho-tyrosine 4G10 (05-321, Millipore), antiPLCc-1 (2822, Cell Signaling), anti-LAT (9166, Cell Signaling),
anti-CRK (610035, BD), anti-PARP (ab6079, Abcam), antiSOS1/2 (SC-259, SantaCruz), and anti-HA (3F10, Roche).
Horseradish peroxidase-conjugated anti-IgG secondary antibodies
against rabbit, rat, or mouse IgG (GE) were used with Lumigo
substrate (Cell signaling).

Flow Cytometry
For cell-surface staining, the following Abs were used: antiCD25 (PC61), anti-CD62L (MEL-14), anti-CD4 (RM4-5 andGK1.5), anti-CD8a (5H10-1 and 53-6.7), anti-Foxp3 (FJK-16a),
anti-TCRb (H57-597), anti-CD69 (H1.2F3), anti-CD44 (IM7)
Abs. All antibodies were purchased from eBioscience or Biolegend.
To examine ERK phosphorylation, cells were fixed andpermeabilized with 90% methanol and stained with antiphospho-ERK (197G2, Cell Signaling) Ab. Foxp3 staining was
performed using the FOXP3 staining kit (00-5523-00, eBioscience)
according to the manufacturer’s protocol. Cells were analyzed
using a FACS CantoII (Becton Dickinson), and the data wereanalyzed using FlowJo software (Tree Star).

Materials and Methods
Mice

Themis2/2 mice have been previously described [2]. For
generation of Themis transgenic mice, PCR-cloned cDNA
fragments encoding WT or DPRS,DNLS, DCore1, DCore2, or
CAB2-1 mutants of Themis were inserted into the hCD2-VA
vector [12]. The transgenes were purified and microinjected into
the pronuclei of fertilized eggs from mixed background mice
[BDF1 (SLC)6Themis2/2 mice on a C57BL/6 background]using standard procedures. The embryos were transferred to theoviducts of pseudopregnant ICR female mice. Previous studies
have shown the human CD2 promoter/enhancer directs theexpression of transgenes in mice to the T cell lineage [12]. The
DPRS, DCore1, and DCore2-Tg mice used in the study were
homozygous for transgene (DPRS-Tg+/+, DCore1-Tg+/+, DCore2Tg+/+) with the exception of DCore1-Tg+/2 in the Fig. S4.
Instead, the DNLS-, WT, and CAB2-1-Tg mice were heterozygous for transgene (DNLS-Tg+/2, WT-Tg+/2, CAB2-1-Tg+/2).
All mice were housed under specific pathogen-free conditions. All
animal experiments were approved by the Animal Care and Use
Committee of the National Center for Global Health andMedicine (NCGM) Research Institute, and conducted in accordance with institutional procedures.

Subcellular Protein Fractionation
Nuclear and cytoplasmic fractions were isolated from total
thymocytes or sorted DP thymocytes using the Subcellular Protein
Fractionation Kit (Pierce) following the manufacturer’s protocol.

Immunofluorescence Confocal Microscopy
Thymocytes were fixed with 2% paraformaldehyde andpermeabilized with 0.1% saponin, and stained with anti-Themis
antibody (06-1328, Millipore) or isotype control antibody, followed
by staining with Alexa-fluor 647-conjugated anti-rabbit IgG(A21246, Molecular Probes, 5 mg/mL) and DAPI (0.2 mg/mL).
Images were collected with an Olympus FV-1000 multitracking
laser scanning confocal microscope (Olympus Japan) with a 100 6
oil objective (NA 1.4), giving a resolution of 0.9 mm in the X, Y –plane.

Statistical Analysis
All data are represented as means 6 SEM. Flow cytometricdata were analyzed using the Unpaired T test (GraphPad Prism
version 5.0). Asterisks in all figures are as follows: *P,0.05; **P,
0.01; ***P,0.001. N.S.; not significant.

TCR Stimulation
Thymocytes were pre-treated with biotin-conjugated anti-CD3e(145-2C11, Biolegend) and anti-CD4 (RM4-4, eBioscience)
antibodies (10 mg/mL each) at 4uC for 30 min. Cells were then
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Function of Themis CABIT Domain

Results[2-3-4-5-6]. Although the transgenic expression of WT Themis
completely restored T cell development in Themis2/2 mice
(Fig. 2A), none of the mutants (DPRS, DNLS, DCore1, DCore2,
CAB2-1) could restore positive selection. In the thymus, generation of CD4-SP in these mutants was the same or even less than
the knockout (Fig. 2A–B, Fig. S2A) with the exception of the NLS
mutant which showed some recovery of CD4-SP thymocytes(Fig. 2A–B). In the DPRS mutant, peripheral CD4SP cells weresignificantly recovered despite the defective positive selection in
the thymus, suggesting that PRS might not be critical in peripheral
expansion and survival of mature T cells. The NLS mutant also
showed some recovery of CD4-SP in the periphery, possibly
because of the milder defect of positive selection in the thymus.
Interestingly, DCore1 mutant mice exhibited a severer inhibition
of T cell development than the Themis deficient mice. These micealso showed decreased numbers of thymic DP, which was not seenin Themis knockout mice. Also, post-selected (CD69+, TCRhi) DP
thymocytes were significantly fewer in the DCore1 mutant mice
(Fig. S2C). Indeed, all of the T cell subsets in DCore1 Tg mice
were fewer than the knockout (Fig. 2B). From these results, loss of
the Core1 motif in Themis resulted in a severer phenotype thanthe loss of entire Themis. Finally, complete loss of positive
selection in CAB2-1 mutant indicates that even the order of the
two CABIT domains is critical for its function. Collectively, the
PRS, NLS, Core1, and Core2 are all required for Themis’s
function, and each motif may have different roles.

Establishment of Mutant Themis Transgenic Mouse Lines
To determine whether the structural motifs of Themis
contribute to its function, we generated a series of transgenic
mice expressing mutant Themis proteins lacking each domain(Fig. 1A, Fig. S1). The Themis mutants used in the present study
were as follows: DPRS (lacking the PRS: aa 555-563), DNLS
(lacking the NLS: aa 345-349), DCore1 (lacking the Core region of
CABIT1 domain: aa 150-174), DCore2 (lacking the Core region of
CABIT2 domain: aa 411-434), and CAB2-1 (swapping entire
region of CABIT1 [aa 1-261] and CABIT2 [aa 262-521]). Thesemutant proteins were expressed under the control of the humanCD2 promoter [12]. Each Tg line was then bred with Themis2/2
mice to replace endogenous wild-type Themis with each mutant
Themis. As shown by Western blotting analysis in Fig. 1B,
expression of mutant Themis protein in each Tg line was almost
comparable to the endogenous Themis+/+ level, although expression of DCore2 and CAB2-1 were more comparable to Themis+/2
level. As has been reported [9], introduction of a CD2-driven wildtype Themis transgene into Themis2/2 mice successfully recovered defective positive selection in the knockout (Fig. 2A), provingthat this transgenic approach is useful to evaluate the function ofThemis mutant proteins in vivo.

The PRS, NLS, Core1, and Core2 Motifs in Themis are all
Required for Positive Selection
First, we analyzed positive selection using these mutant mice.
Themis2/2 mice showed a marked reduction of CD4SP and
CD8SP thymocytes and splenocytes (Fig. 2A) as has been reported

PRS, NLS, CABIT1, and CABIT2 are Required for Tyrosinephosphorylation and Grb2-association
We and other groups previously reported that Themis
constitutively associates with Grb2 [2,3,9-11], and is tyrosinephosphorylated upon TCR stimulation [5,9-11]. We theninvestigated whether the deletion of each motif affects Grb2association and tyrosine-phosphorylation. We observed that
endogenous wild-type Themis constitutively associated with
Grb2 in thymocytes, and was tyrosine-phosphorylated upon
stimulation with anti-CD3 plus anti-CD4 antibodies (Fig. 3A) as
previously reported [2-5,9-11]. In addition, we observed stimulation-dependent association of Themis with PLC-c1 and SOS (Fig.
S3A), suggesting a possible involvement of Themis as a componentof the SLP76/LAT-signalosome complex [11]. As shown in
Fig. 3A, we could successfully immunoprecipitate DPRS, DNLS,
DCore1, DCore2, and CAB2-1 Themis mutant proteins, but all ofthese mutant Themis proteins lost constitutive association with
Grb2. Additionally, all five mutants also lost tyrosine-phosphorylation upon TCR stimulation (Fig. 3A). These results indicatethat the PRS, NLS, Core1, and Core2 motif, as well as order of
the two CABIT domains are all critical for Grb2-association andtyrosine-phosphorylation of Themis.
Because a previous in vitro study reported that Themis binds to
Grb2 via its PRS motif [11], inability of DNLS, DCore1, DCore2
and CAB2-1 mutants to associate with Grb2 was quite surprising.
Therefore, we decided to reinvestigate the biochemical basis of theThemis-Grb2 interaction in thymocytes. We utilized our monoclonal antibody (mAb) against Themis (clone 2E7) [2], which
recognizes an epitope around the PRS (aa 555-563) (Fig. 3B). We
found that the 2E7 mAb binds only to Grb2-unbound Themis, but
not to Grb2-bound Themis by sequential immunoprecipitation
experiments (Fig. 3C). These results strongly indicate that 2E7
mAb and Grb2 compete for binding to the PRS, supporting direct
association of Grb2 with the PRS of Themis in thymocytes.

Figure 1. Generation of Themis2/2 mice expressing mutant
Themis proteins. (A) Schematic representation of the mutant Themis
proteins. (B) Analysis of Themis protein expression by immunoblot
using sorted CD692 DP thymocytes from Themis+/+, Themis+/2,
Themis2/2 and Themis2/2 mice expressing DPRS, DCore1, DCore2,
DNLS, CAB2-1 mutant, or WT Themis. Data are representative of morethan three independent experiments.
doi:10.1371/journal.pone.0089115.g001

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Function of Themis CABIT Domain

Figure 2. Expression of the Themis mutants failed to restore positive selection in Themis2/2 mice. (A) CD4 and CD8 expression profilesof thymocytes or splenocytes from Themis mutant mice. Numbers in plots show the frequency of cells in the indicated area. Proportion (%) ofindicated subsets of (B) thymocytes and (C) splenocytes (mean 6 SEM). (A-C) Data are representative of more than three independent experiments.
Only significant differences against 2/2 mice are noted in the graphs. *p,0.05, **p,0.01, ***p,0.001.
doi:10.1371/journal.pone.0089115.g002

The Core1 but not Core2 Motif is Required for Nuclear
Localization

Dominant Negative Inhibitory Effect of DCore1 Mutant
on T Cell Development

We observed that DCore1 mutant Tg mice in Themis2/2
background showed severer and distinct defects on T cell
development when compared to Themis2/2 mice (Fig. 2A).
These results indicate that DCore1 mutant protein could affect T
cell development in a dominant negative fashion. To confirm thispossibility, we investigated the phenotype of DCore1 Tg mice on a
Themis+/+ background. Indeed, positive selection of both CD4SP
and CD8SP thymocytes was significantly decreased in DCore1-Tg
Themis+/+ mice, whereas DCore2-Tg Themis+/+ mice did notexhibit any significant change (Fig. 5A). Absolute numbers of DP
thymocytes in DCore1-Tg Themis+/+ were also reduced, which is
totally different from the phenotype of Themis deficiency. In
addition, numbers of peripheral CD4SP and CD8SP cells wereseverely reduced in DCore1-Tg mice. In DCore1-Tg Themis+/+mice we also observed a strong reduction of the earliest postselected DP (CD69hi TCRhi) thymocytes, whereas the reduction
was slight in the DCore2-Tg Themis+/+ mice (Fig. 5B). Expressionof CD25 and CD44 on DP thymocytes was somehow increased in
DCore1-Tg, but not in DCore2-Tg Themis+/+ mice (Fig. 5C). In
the periphery of DCore1-Tg Themis+/+ mice, memory CD4SP
and CD8SP cells (CD44hi CD62Llo) were strongly increased with aconcomitant decrease of naive populations (CD44lo CD62Lhi),
possibly because of homeostatic expansion in lymphopenic mice
(Fig. 5D). Again, this increase of the memory population was

Although initial experiments using a GFP-fusion protein ofThemis exhibited cytosolic localization of Themis protein [2-4],
Western-blotting results by subcellular fractionation proved that
endogenous Themis exists in the nucleus as well as the cytosol [39]. In fact, we also confirmed the existence of endogenous Themis
protein in nuclear fractions by Western blotting (Fig. 4A).
Furthermore, existence of Themis in the nucleus was also
confirmed by immunostaining analysis by confocal fluorescencemicroscopy. As shown in Fig. 4B, Themis was visualized in cytosol,
as well as in the nucleus as punctate staining. Staining of DNA and
Themis appeared mutually exclusive, suggesting that Themis exists
unassociated with DNA in the nucleus. We next investigatednuclear localization of each mutant Themis protein in thymocytes
by subcellular fractionation. As shown in Fig. 4C-D, DPRS andDCore2 mutant Themis successfully translocated to the nucleus
similar to wild-type Themis, whereas DCore1 and DNLS mutant
existed only in the cytosol (Fig. 4C-D). Although the importance of
the NLS motif on nuclear localization of Themis protein has beenreported by another group [9], we further showed the Core1 motif
to be essential for nuclear translocation of Themis. Furthermore,
the order of the two CABIT domains was also critical for thetranslocation. From these results, we conclude that not only NLS
but also the Core motif in the CABIT-1 domain are important for
nuclear translocation of Themis.
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Function of Themis CABIT Domain

Figure 3. Themis mutants lack tyrosine-phosphorylation and Grb2-association. (A) Analysis of tyrosine-phosphorylation and protein
interactions of Themis mutants. Thymocytes from the indicated mutant mice were stimulated with anti-CD3 plus anti-CD4 antibodies. Proteins were
immunoprecipitated (IP) with anti-Themis antibody and analyzed by immunoblot (IB) with the indicated antibodies. Data are representative of four
independent experiments. (B) Anti-Themis monoclonal antibody (mAb) 2E7 binds to the PRS motif. Immunoprecipitated Themis proteins from
Themis+/+, Themis2/2 DCore1, Themis2/2 DCore2 and Themis2/2 DPRS thymocytes were immunoblotted with anti-Themis mAb 2E7. Data arerepresentative of three independent experiments. (C) Cell lysates from Themis+/+ thymocytes were sequentially immunoprecipitated with 2E7 andanti-Themis polyclonal antibody (pAb). Grb2 was co-precipitated with anti-Themis pAb but not with 2E7. Data are representative of three
independent experiments.
doi:10.1371/journal.pone.0089115.g003

observed only in DCore1-Tg, but not in DCore2-Tg Themis+/+mice. Since DCore1-Tg Themis+/+ mice showed a strong defect inpositive selection, we investigated TCR-dependent activation of
ERK [13], which is critical in the process. As shown in Fig. 5E,
anti-CD3 plus anti-CD4 mAb stimulated phosphorylation of ERK
was strongly inhibited in the presence of DCore1 mutant Themis
but not of DCore2 mutant. Therefore, decreased positive selectionin DCore1-Tg mice could partly be due to the impaired ERK
activation. We also found that the absolute number ofCD25+Foxp3+ natural regulatory T cells (nTreg) in the thymus
was significantly reduced in DCore1-Tg, but not in DCore2-Tg
Themis+/+ mice (Fig. 5F). Differentiation of NKT and cd-T cells
in Themis+/+ DCore1 mice was not changed (data not shown).
Collectively, expression of Core1-deleted Themis in immature
thymocytes caused dominant-negative type inhibition of T cell
development, possibly by interfering with signaling related to ERK
activation. All of these analyses were carried out using transgene
homozygous (DCore1-Tg+/+ or DCore2-Tg+/+) mice. Because
expression levels of mutant protein in DCore2-Tg+/+ weresignificantly less than that of DCore1-Tg+/+ mice, one can assume
that the absence of dominant negative effects in DCore2-Tg mice
was simply due to the fewer expression of less DCore2 mutant
protein. Therefore, we compared transgene heterozygous(DCore1-Tg+/2) mice with homozygous (DCore2-Tg+/+) mice.
As shown in Fig. S4A, protein expression level of mutant Themis
protein in DCore1-Tg+/2 mice and DCore2-Tg+/+ mice were thesame, which were equivalent with that of Themis+/2 mice.
Although expression of the two mutant proteins were the same
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level, only DCore1-Tg showed dominant negative effects (Fig.
S4B-C), proving that the effect is specific to the DCore1 mutant
protein. Since the dominant negative inhibitory effects were only
observed in DCore1-Tg but not in DCore2-Tg, our current results
clearly indicated distinct functions for CABIT1 and CABIT2
domains.

Discussion
Themis contains an NLS, PRS, and two CABIT domains. The
CABIT domain, which was identified by Schwartz’s group, isconserved through protozoa and is predicted to form either an
extended beta-sandwich-like fold or a dyad of 6-strand beta-barrel
structures [4]. Themis family proteins (Themis, Themis2, andThemis3) contain two CABIT domains and a PRS. A recent
report demonstrated that Themis and Themis2 were functionallyinterchangeable in terms of T cell development [9], indicating theimportant function of CABIT domains. Therefore, in the present
study, we focused on the function of CABIT domains, especiallyon differences in the two CABIT domains in Themis.
To elucidate the involvement of each domain/motif in Themis
on its function in positive selection, we established several mutant
Themis Tg lines. Because Themis+/2 mice showed an intermediate effect on positive selection between Themis+/+ andThemis2/2 mice [2], it is obvious that the expression level ofThemis protein in thymocytes critically affects the result of positive
selection. Therefore, the expression level of mutant protein inthese Tg mice is important for evaluating their function. We
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Function of Themis CABIT Domain

Figure 4. The CABIT1 domain of Themis is important for nuclear localization. (A) DP-thymocytes from Themis+/+ mice were fractionatedinto cytoplasm, membrane and nuclear fractions and immunoblotted with indicated Abs. The CRK and PARP proteins were used as purity controls forthe cytoplasmic and nuclear fractions, respectively. Data are representative of more than four independent experiments. (B) Representative
micrograph showing localization of Themis (white) in cytoplasm and nucleus stained with DAPI (blue), together with BF (bright field). Data arerepresentative of three independent experiments. (C) Localization of Themis mutants was analyzed by Western blot in cytoplasmic and nuclear
extracts. Thymocytes from Themis mutant mice were fractionated into the cytoplasm and nucleus. (D) Bar graph shows that the nuclear/cytoplasmic
Themis protein ratio compared with Themis+/+ mice. Data are representative of more than three independent experiments. Significant differences arenoted in the graphs. *p,0.05.
doi:10.1371/journal.pone.0089115.g004

thymic nTregs, and an increased memory population (CD44hi
CD62Llo) of peripheral mature T cells. The DP thymocytes from
DCore1-Tg showed decreased ERK activation upon TCR
stimulation. These phenotypes were not observed in Themis2/2
mice, suggesting that the DCore1 mutant inhibits T cell
development in a Themis-independent manner. Instead, theDCore1 mutant may perturb the function of unidentified protein(s)that compensate the residual T cell development in Themis2/2
mice. Lastly, and most importantly, the dominant negative
inhibitory effects were not observed in either the DCore2-Tg
mice or the wild-type Themis-Tg mice, indicating that the loss of
the Core motif of either CABIT1 or CABIT2 domains induces
qualitatively different effects. It is possibly explained by differential
protein association with DCore1 and DCore2, or because of thelack of nuclear interaction of DCore1 with another protein.
Because two independent lines of DCore1 Tg mice showed theidentical phenotype, the phenotype must not be an artifact causedby the destruction of other genes by the insertion of Tg vector.
The dominant negative effects were not due to higher protein
expression of the DCore1 versus the DCore2 mutant, because
heterozygous DCore1-Tg+/2 mice, having equivalent protein
expression with homozygous DCore2-Tg+/+ mice, also showed
the same dominant negative phenotypes (Fig. S4A–C). Collectively, in the present study, we clearly demonstrated differentialfunctions for CABIT1 and CABIT2 domains.

screened a large number of founder lines of each mutant transgeneand managed to establish lines with comparable protein expression(Fig. 1B).
Very recently, the PRS motif was reported to be important for
Grb2-association, tyrosine-phosphorylation, and positive selection,using irradiation-mediated chimeric mice reconstituted withretrovirally transduced bone marrow cells [11]. It would be
difficult to evaluate the expression level of mutant Themis protein
in reconstituted thymocytes, however, our present results using
DPRS-Tg mice with equivalent protein expression with endogenous Themis (Fig. 1B) strongly supported their observations.
Despite strong inhibition of positive selection in the thymus of
DPRS mice, the number of CD4SP in the periphery wassignificantly increased compared to Themis2/2 mice, suggestingthat the PRS motif in Themis is critical in positive selection in thethymus, but may not be important in peripheral survival andmaintenance of mature T cells.
We firstly demonstrated that the consensus motif of CABIT2
domain (Core2) was crucial for positive selection. Furthermore,deletion of the Core1 motif resulted in a much severer inhibition of
T cell development than seen in Themis2/2 mice. Therefore, we
next investigated the phenotype of DCore1- and DCore2-Tgs on awild-type background. The DCore1-Tg mice in Themis+/+background showed decreased numbers of DP thymocytes, strong
inhibition of CD8SP thymocyte development, reduced numbers of
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Function of Themis CABIT Domain

Figure 5. The CABIT1 and CABIT2 in Themis have different functions. (A) CD4 and CD8 profiles of thymocytes and splenocytes of Themis+/+,Themis+/+ DCore1, and Themis+/+ DCore2 mice. Numbers in plots show the frequency of cells in the indicated area. Bar graphs show absolute numberof total thymocytes and thymocyte subsets from Themis+/+, Themis+/+ DCore1, and Themis+/+ DCore2 mice (mean 6 SEM). Data are representative offour independent experiments. (B) Proportion of post-selected CD69+ TCRhi cells in CD4+CD8+ (DP) thymocytes. Data are representative of four
independent experiments. (C) Surface expression of CD25 and CD44 on gated DP thymocytes. Data are representative of four independent
experiments. (D) CD44 and CD62L profile of splenic CD4SP and CD8SP cells. Data are representative of four independent experiments. (E)Representative histogram overlays of phosphorylation of ERK. DP thymocytes were stimulated with anti-CD3 plus anti-CD4 Abs for 1min, thenintracellular staining of phosphorylated-ERK antibody was performed (solid line). The shaded line is without stimulation. Data are representative of
three independent experiments. (F) Absolute number of thymic CD4+25+Foxp3+ Treg cells (mean 6 SEM). Data are representative of four
independent experiments. Significant differences are noted in the graphs. *p,0.05, **p,0.01. N.S. = not significant.
doi:10.1371/journal.pone.0089115.g005

selection. Involvement of Sos in positive selection was also recently
shown to be nonobligatory because Sos1/Sos2 DKO exhibited
normal positive selection [18]. Therefore, Themis and Grb2 may
be cooperatively required for positive selection. Further analysis ofThemis/Grb2 DKO would clarify whether Themis and Grb2 arefunctionally complementary during positive selection.
We demonstrated that the PRS, NLS, Core1, and Core2 motifs
were all required for Grb2-association, tyrosine-phosphorylation,and positive selection, whereas NLS and CABIT1 are required fornuclear translocation. The CABIT1 domain might be important
for the interaction of Themis with nuclear transporter proteins. So
far, the causal relationship between positive selection, Grb2association, and tyrosine-phosphorylation is not yet understood,
however, our results demonstrated that all of these are tightly
correlated to one another. On the contrary, the current study
demonstrated that positive selection and nuclear translocation ofThemis are not directly correlated. As a matter of fact, it should be
noted that deletion of the NLS motif exhibited the least effect on
positive selection compared to the deletion of other motifs,
indicating that nuclear translocation of Themis may be lessimportant for positive selection.
Finally, this is the first report that describes the significance of
the CABIT domain in cellular events. Furthermore, two CABIT
domains exist in Themis are functionally different and notinterchangeable. Although the structural basis for the function of
CABIT domains remains to be elucidated, our study clearly

We demonstrated that Themis constitutively associates with
Grb2 in thymocytes. Themis2 also associates with Grb2 [9],therefore Grb2-association must be an important feature ofThemis family proteins. In the recent peptide inhibition study,
the PRS motif of Themis was shown to directly interact with the
C-terminal SH3 motif of Grb2 [11]. The result from immunoprecipitation experiments using our own anti-Themis monoclonal
antibody (2E7) also supported direct binding of Grb2 to the PRS
motif of Themis (Fig. 3B). As has been recently reported [9], we
also observed defective association of the DPRS mutant Themis
with Grb2 in thymocytes (Fig. 3A). More surprisingly, not only
DPRS, but also all other mutants lost the ability to associate with
Grb2 (Fig. 3A). These results suggest that the intra-molecularinteraction of these domains is crucial to form the proper threedimensional structure to associate with Grb2. Moreover, all of thefive mutants in the present study lost not only Grb2 binding, butalso tyrosine-phosphorylation. It should be noted that these fivemutants also lost weak basal phosphorylation of the protein in
unstimulated thymocytes (Fig. 3A).
From our sequential immunoprecipitation experiments, a half ofThemis binds to Grb2 (Fig. 3C, Fig. S3C), and about one tenth ofGrb2 binds to Themis (Fig. S3B). A recent study with T cellspecific Grb2-deficient mice revealed that Grb2 is critical for
positive selection [14]. Although Grb2 has been reported toassociate with several molecules (e.g. Sos, Shc, FAK) other than
Themis [15-17], deficiency of Shc or FAK does not inhibit positive

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Function of Themis CABIT Domain

demonstrated that the two CABIT domains in Themis are pivotal
and serve distinct roles in its function for driving T cell
development.

ed with 2E7 and anti-Themis Ab. Bar graphs show the relative
density of the immunoblot Themis bands (mean 6 SEM). Results
are representation of three independent experiments. The relative
densities are expressed as mean 6 SEM.
(EPS)

Supporting Information

Figure S4 Dominant-negative inhibitory effect observed in
Themis+/+ DCore1-Tg+/2 mice. (a) Comparison of protein
expression by immunoblot between thymocytes from Themis+/+DCore1-Tg+/2 and Themis+/+ DCore2-Tg+/+. (b) CD4 and CD8
expression profiles of thymocytes from Themis+/+ and Themis+/+mice expressing heterozygous DCore1-Tg+/2 and Themis
DCore2-Tg+/+. Numbers in plots show the frequency of cells in
the indicated area. Results are representative of three independent
experiments. (c) Proportion of post-selected CD69+TCRbhi DP
cells in each mutant. Results are representative of three
independent experiments.
(EPS)

Figure S1 The amino acid sequence used for generation ofmutant Themis transgenes.
(EPS)
Figure S2 Characterization of Themis transgenes lacking theCore1, Core2, PRS, or NLS motif in Themis2/2 backgroundmice. Bar graphs represent numbers of (a) thymocyte and (b)splenocyte subsets (mean 6 SEM). (c) Proportion of post-selected
CD69+TCRbhi DP cells in each mutant. Results are representativeof more than three independent experiments. Only significant
differences in mutant-Tgs against 2/2 are noted in the graphs.
*p,0.05, **p,0.01, ***p,0.0001.
(EPS)
Figure S3 Themis interacts with the LAT signalosome complex
in thymocytes. (a) Thymocytes from Themis+/+ and Themis2/2
mice were stimulated with anti-CD3/anti-CD4/anti-CD8 Abs forindicated times at 37uC. Proteins were immunoprecipitated with
anti-Themis pAb and analyzed by immunobloting with theindicated Abs. (b) Immunoprecipitates with anti-Themis Ab from
Themis+/+ thymocytes were immunoblotted with anti-Grb2. Bargraphs show the relative density of the immunoblots of Grb2 fromtotal lysate, immunoprecipitates with anti-Themis (IP), and thesupernatant after the immunoprecipitation (Sup). (c) Cell lysatesfrom Themis+/+ thymocytes were sequentially immunoprecipitat-

Acknowledgments
We would like to thank Masashi Yoshida and Sachiko Nitta for expert
technical assistance.

Author Contributions
Conceived and designed the experiments: HS. Performed the experiments:
T. Okada TN HO AT MSP KK NT. Analyzed the data: T. Okada TN
HS. Contributed reagents/materials/analysis tools: NT MG T. Okamura.
Wrote the paper: T. Okada TN HS.

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