List of Love Live! episodes

Love Live! is a television anime series produced by Sunrise in collaboration with ASCII Media Works and Lantis as part of the Love Live! School Idol Project. The series follows a group of school girls who form an idol group in order to save their school from being shut down. The first season aired 13 episodes on Tokyo MX between January 6 and March 31, 2013. The opening theme is "Bokura wa Ima no Naka de" (僕らは今のなかで^?, lit. "We're Living in the Moment") and the ending theme is "Kitto Seishun ga Kikoeru" (きっと青春が聞こえる^?, lit. "Surely Our Youth Will Hear Us"); both are performed by μ's (Emi Nitta, Aya Uchida, Suzuko Mimori, Yoshino Nanjō, Pile, Riho Iida, Aina Kusuda, Yurika Kubo and Sora Tokui). A second season aired between April 6 and June 29, 2014. The opening theme is "Sore wa Bokutachi no Kiseki" (それは僕たちの奇跡That Is Our Miracle^?) and the ending theme is "Donna Toki mo Zutto" (どんなときもずっと Always No Matter What^?); both are performed by μ's. Both seasons are licensed in North America by NIS America and are simulcast by Crunchyroll.mAn animated film has been greenlit for production.

Season 1

<tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep01" style="text-align: center; font-weight: normal; background:#F2F2F2">01</th> <td class="summary" style="text-align: left;">"Come True! Our Dreams!"

"Kanae! Watashitachi no Yume"(叶え!私たちの夢――) </td><td>"Susume→Tomorrow" (ススメ→トゥモロウSusume→Tumorō^?, lit. "March Forward to Tomorrow") by Honoka Kōsaka (Emi Nitta), Kotori Minami (Aya Uchida) and Umi Sonoda (Suzuko Mimori)

"Private Wars" by A-Rise (Tsubasa Kira (Megu Sakuragawa), Erina Tōdō (Maho Matsunaga) and Anju Yūki (Ayuru Ōhashi))</td><td>January 6, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> Otonokizaka Academy is announced to be closing down after everyone graduates due to lack of applicants, which comes as a shock to second year student Honoka Kōsaka, who loves the school. As she and her classmates Umi Sonoda and Kotori Minami try to think of a way to make the school more attractive to prospective students, Honoka is surprised that her younger sister Yukiho wants to go to a different school, UTX, instead of Otonokizaka. As Honoka decides to investigate UTX, she finds it attracts a lot of students due to a school idol group known as A-Rise. Honoka decides that the best way to save her school is to start an idol group themselves, although Umi isn't keen on the idea. As Honoka becomes downhearted, she spots a talented singer and pianist and redoubles her efforts, while Umi inevitably gives in and joins Kotori in partaking in Honoka's idea. Despite their request to start an idol club being rejected by the student council president, Eli Ayase, Honoka remains determined to make it work. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep02" style="text-align: center; font-weight: normal; background:#F2F2F2">02</th> <td class="summary" style="text-align: left;">"Let's Start Being Idols!"

"Aidoru o Hajimeyō!"(アイドルを始めよう!) </td><td>"Start:Dash!!" by Maki Nishikino (Pile)
"Private Wars" by A-Rise</td><td>January 13, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> Honoka manages to get permission to use the auditorium for a concert in a month's time, although Umi has doubts that they'll be ready in time. Unable to think of a name for their group, Honoka holds a contest for the name instead before realizing they do not actually have a song to practice. As Umi reluctantly agrees to write lyrics, she puts Honoka and Kotori on an exercise regime to build up their stamina. As Honoka unsuccessfully attempts to get the girl from before, Maki Nishikino, to compose songs for them, Eli warns her that her idol plan could backfire and damage the school's reputation further. Honoka perks up when she hears the class is supporting her, as well as receiving a name for their group, μ's. Honoka once again approaches Maki, showing her how serious an idol's work is and giving her the lyrics Umi wrote. After a little advice from the student vice-president, Nozomi Tojo, she sends Honoka a CD of the song she composed from Umi's lyrics whilst also anonymously voting for μ's on an idol ranking site. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep03" style="text-align: center; font-weight: normal; background:#F2F2F2">03</th> <td class="summary" style="text-align: left;">"First Live"
"Fāsuto Raibu"(ファーストライブ) </td><td>"Start:Dash!!" by Honoka Kōsaka (Emi Nitta), Kotori Minami (Aya Uchida) and Umi Sonoda (Suzuko Mimori)

"Private Wars" by A-Rise</td><td>January 20, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> On the day before the concert, Umi starts to get cold feet about performing in front of others. To get her used to other people, Honoka and Kotori get Umi to hand out fliers to help build her confidence. Umi has some reservations about the costume, but Honoka manages to convince her to make sure all their efforts don't go to waste. However, on the day of the concert, no one shows up as all the freshmen are busy checking out the other clubs. Just as Honoka is about to break down over her wasted efforts, a nervous girl named Hanayo Koizumi shows up to see the concert, prompting Honoka to go ahead with the performance, which soon attracts a small, but very much appreciated crowd. When asked by Eli about what they'll do next, Honoka insists they'll keep on performing so they can eventually fill up the auditorium. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep04" style="text-align: center; font-weight: normal; background:#F2F2F2">04</th> <td class="summary" style="text-align: left;">"MakiRinPana"
"MakiRinPana"(まきりんぱな) </td><td>"Start:Dash!!" by Honoka Kōsaka (Emi Nitta), Kotori Minami (Aya Uchida) and Umi Sonoda (Suzuko Mimori)</td><td>January 27, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> As Hanayo debates whether she should join the idol club, which needs five members to become official, she asks her friend, Rin Hoshizora, if she would be willing to join with her, although she declines as she doesn't have enough confidence in her femininity. Later that day, Hanayo visits Maki's house to return her dropped Student ID, where Maki explains how she'll probably have to quit music in order to study medicine and inherit her family's hospital, but says she'll support Hanayo should she decide to become an idol. Afterwards, Hanayo comes across Honoka's shop, where Umi and Kotori are also visiting as they notice a recording of their performance has gained a fair amount of views on the internet. Mentioning that even they have their flaws, the girls formally invite Hanayo to join the club, asking her to think it over. Noticing her shyness in class, Maki decides to help Hanayo learn to speak loudly. After receiving a push from both Maki and Rin, Hanayo finally agrees to join the idol club, whilst Maki and Rin also decide to join. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep05" style="text-align: center; font-weight: normal; background:#F2F2F2">05</th> <td class="summary" style="text-align: left;">"Nico Strikes"

"Niko Shūrai"(にこ襲来) </td><td></td><td>February 3, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> Honoka and Kotori are confronted by a curious girl named Nico Yazawa, who tells them they should break up. Meanwhile, as the rainy season causes the club to think about where they can practice, Honoka realizes that they could try and form an official club now they have enough members. Just then, they catch Nico trying to steal their food and insult their lack of professionalism. The girls learn from Eli that they can't form their idol club as there is already an Idol Research Club, whose sole member turns out to be Nico. After Nico rejects their offer of merging the groups, student vice-president Nozomi explains how Nico had previously attempted to start her own school idol group, which fell apart due to setting the bar too high. After thinking things over with the group, Honoka comes up with the idea that they should all join the Idol Research Club together to learn how to improve μ's, inviting Nico to join the group as their coach. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep06" style="text-align: center; font-weight: normal; background:#F2F2F2">06</th> <td class="summary" style="text-align: left;">"Who'll Be the Center?"

"Sentā wa Dare da?"(センターは誰だ?) </td><td>"Korekara no Someday" (これからのSomeday^?, lit. "Someday in the Future") by Honoka Kōsaka (Emi Nitta), Kotori Minami (Aya Uchida), Umi Sonoda (Suzuko Mimori), Rin Hoshizora (Riho Iida), Maki Nishikino (Pile), Hanayo Koizumi (Yurika Kubo) and Nico Yazawa (Sora Tokui)</td><td>February 10, 2013</td></tr><tr><td class="description" 
style="border-bottom:3px solid #CCCCFF" colspan="4"> Whilst shooting interviews for each of the clubs, Nozomi suggests to the group that they shoot a new music video now that they have more members. During the interviews, Nozomi becomes curious as to why Honoka is the leader of μ's when she doesn't seem to do anything. Thus, the discussion soon turns to who should be the leader and center in the group, but they can't decide on who is the most worthy. Thus, Nico suggests that the decision be settled by a sing and dance contest at a karaoke place, though her plan to outshine everyone doesn't go very well as everyone ends up with similar scores. Honoka then suggests that they don't really need a leader and should just have everyone taking turns to sing, although the others secretly agree that Honoka is the most leader-worthy person. As the girls complete their new music video, Nozomi prompts to Eli that she should be the one to help them. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep07" style="text-align: center; font-weight: normal; background:#F2F2F2">07</th> <td class="summary" style="text-align: left;">"Elichika"

"Erīchika"(エリーチカ) </td><td>"Start:Dash!!" by Honoka Kōsaka (Emi Nitta), Kotori Minami (Aya Uchida) and Umi Sonoda (Suzuko Mimori)</td><td>February 17, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> Hanayo informs the gang of a school idol tournament called Love Live in which the top 20 school idol groups from across the nation compete against each other. Needing the school's permission to enter, the group go to ask the chairwoman for permission, and despite Eli's objection, she approves of their entry on the condition that none of them fail a subject in the upcoming exams. This puts a lot of pressure on Honoka, Rin, and Nico, who have generally bad grades. Umi and Kotori decide to help Honoka study whilst Hanayo and Maki help Rin and Nozomi helps Nico. Later that day, Umi comes across Eli's little sister, Alisa, a fan of μ's who reveals Eli shot a video of their live performance and uploaded it to the internet. When confronted about why she refuses to acknowledge μ's, she simply responds that she considers all school idols amateurs. As Umi asks Nozomi about why Eli would think that way, she reveals that Eli used to be a talented ballerina. Rather than being deterred by this, Umi becomes determined to ask Eli to teach the group how to dance like her. After the exams finish and everyone manages to pass, Honoka overhears the chairwoman tell Eli that the school will stop accepting new students next year and be shut down. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep08" style="text-align: center; font-weight: normal; background:#F2F2F2">08</th> <td class="summary" style="text-align: left;">"What I Want to Do is..."

"Yaritai koto wa"(やりたいことは) </td><td>"Korekara no Someday" (これからのSomeday^?, lit. "Someday in the Future") by Honoka Kōsaka (Emi Nitta), Kotori Minami (Aya Uchida), Umi Sonoda (Suzuko Mimori), Rin Hoshizora (Riho Iida), Maki Nishikino (Pile), Hanayo Koizumi (Yurika Kubo) and Nico Yazawa (Sora Tokui)

"Bokura no Live, Kimi to no Life" (僕らのLIVE 君とのLIFE Our Live, Your Life^?) by μ's</td><td>February 24, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> The chairwoman clarifies the school will be closed down if their upcoming open day is received negatively. As Eli tries to think of ways to attract potential students besides μ's, Umi suggests to the group that they should get Eli to teach them how to dance. Eli eventually agrees to help them out and starts putting them through some intense training, surprised to find them willing to put through with it despite the roughness of it all. As Eli becomes conflicted by what she should do, Nozomi tells her that should do what she wants to do instead of what people expect her to do. Eli breaks down and says she wants to join μ's but doesn't feel she is able to do so. Nozomi informs the others, who formally invite her to join μ's as one of their members. Eli finally becomes honest with herself and joins the group alongside Nozomi, who reveals she was the one who came up with the group's name. The complete group performs at the open day to a pleased crowd. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep09" style="text-align: center; font-weight: normal; background:#F2F2F2">09</th> <td class="summary" style="text-align: left;">"Wonder Zone"
"Wandā Zōn"(ワンダーゾーン) </td><td>"Wonder Zone" by μ's</td><td>March 3, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> The success of the open day helps bring the faculty to reconsider the closing of the school, as well as earning the group an expansion to their clubroom. After the group manages to reach 50th in the rankings, they visit Akihabara where they find they are already being featured on merchandise. Upon discovering a photograph of Kotori in a maid's outfit among the goods, they learn that Kotori has been secretly working part-time at a maid café after school to build her confidence. Eli decides Akihabara would be the perfect place to hold a live performance, assigning Kotori to write a song about Akiba culture. However, Kotori struggles with coming up with lyrics, which soon has an impact on her studies. In order to help her out, Honoka and Umi join her at the maid café. Recalling the feelings she gets whenever she works in Akiba, Kotori is able to come up with some lyrics which help make the performance a success. As Honoka assures Kotori that they'll be together forever, an air mail letter arrives in Kotori's postbox the next morning. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep10" style="text-align: center; font-weight: normal; background:#F2F2F2">10</th> <td class="summary" style="text-align: left;">"No "Senpai" Allowed!"
"Senpai Kinshi!"(先輩禁止) </td><td></td><td>March 10, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> Honoka suggests that the group go on a training camp to the beach, with Maki coerced into letting them use her family's vacation home. Wanting to break down the barriers between underclassmen and upperclassmen, Eli puts in a rule that no one is allowed to call their upperclassmen 'senpai' for the duration of the trip. The girls spend the day playing around on the beach, though Eli and Nozomi are quick to notice Maki doesn't seem to be participating. After dinner and a bath, Nozomi helps Maki become more sociable by initiating a pillow fight. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep11" style="text-align: center; font-weight: normal; background:#F2F2F2">11</th> <td class="summary" style="text-align: left;">"The Greatest Live Performance"

"Saikō no Raibu"(最高のライブ) </td><td>"No Brand Girls" by μ's</td><td>March 17, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> μ's manages to make it to rank 19, making them eligible to apply for the Love Live! tournament, putting pressure on them to keep their position. However, things don't go their way when they are unable to secure a spot on the auditorium for the upcoming school festival. Honoka suggests that they instead hold their performance on the school rooftop where they train, which the others agree too. Meanwhile, Kotori seems to be preoccupied by the letter she received, not being able to find an opportunity to discuss it with the others. As Kotori calls Umi on the night before the festival to tell her what's bothering her, Honoka ends up overexerting herself, ending up with a fever on the day of the festival, but she forces herself to come to school anyway. Despite the rain, the concert goes ahead, but after the first song finishes, Honoka suddenly collapses due to her fever. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep12" style="text-align: center; font-weight: normal; background:#F2F2F2">12</th> <td class="summary" style="text-align: left;">"Friends"

"Tomodachi"(ともだち) </td><td></td><td>March 24, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> As Honoka recovers from her fever, she becomes depressed when the others tell her they've withdrawn their entry from Love Live, which is inevitably won by A-Rise. After Honoka recovers and is eventually cheered up by the others, the group are pleased to learn that the school has gathered enough applicants to stay open for another year. Meanwhile, Kotori still hasn't brought herself to tell Honoka about the decision she had made. Unable to let her keep it a secret any further, Umi announces to the others that Kotori will be leaving in two weeks to study fashion abroad. As Honoka questions Kotori about why she never told her, she learns she was intending to tell her following the live performance, but got put off following her accident. Honoka then becomes deperessed, feeling guilty for not noticing her feelings due to her focus on Love Live. The group suggest putting on one more live performance before Kotori leaves, but Honoka doesn't see the point in continuing and says she wants to quit being a school idol, prompting Umi to become angry and slap her. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep13" style="text-align: center; font-weight: normal; background:#F2F2F2">13</th> <td class="summary" style="text-align: left;">"μ's Music Start!"

"Myūzu Myūjikku Sutāto!"(μ'sミュージックスタート!) </td><td>"Susume→Tomorrow" (ススメ→トゥモロウSusume→Tumorō^?, lit. "March Forward to Tomorrow") by Honoka Kōsaka (Emi Nitta), Kotori Minami (Aya Uchida) and Umi Sonoda (Suzuko Mimori), "Start:Dash!!" by μ's</td><td>March 31, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> With Honoka and Umi still not talking to each other, Eli puts μ's on indefinite hiatus so they can think rationally about the situation. As Honoka's friends take her out to cheer her up, Umi visits Kotori, who has also yet to talk with Honoka, trying to see what she truly wants. Honoka later runs into Nico training alongside Rin and Hanayo, stating that even if μ's is on hiatus, she still wants to be an idol because she loves it. Later, Eli visits Honoka, who reminds her of the words that saved her when she was down. Realising her true feelings, Honoka apologises to Umi, stating she still wants to become an idol. Making up with her, Umi sends Honoka to get to Kotori before she leaves Japan and convince her to stay, helping her realise her own desire to stay as well. With all nine members reassembled, μ's is reformed and perform together to a filled school auditorium, vowing to keep on pushing forward. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="epOVA" 

style="text-align: center; font-weight: normal; background:#F2F2F2">OVA</th> <td class="summary" style="text-align: left;">  </td><td>"Music S.T.A.R.T!!" by μ's</td><td>November 27, 2013</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> After hearing about a dream from Hanayo, Maki ends up absent from school the next day. As the others decide to go visit her, Eli returns to the school to pick up a textbook and follows a mysterious shadow, only to wind up back outside with the others, where it is suddenly nighttime. There, they are approached by a younger version of Maki, who expresses how she's always known of the others, before returning to the normal-aged Maki. </td></tr>

Season 2
<tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep01" style="text-align: center; font-weight: normal; background:#F2F2F2">01</th> <td class="summary" style="text-align: left;">"Love Live Once Again!"

"Mōichido Rabu Raibu!"(もう一度ラブライブ！) </td><td>"Koremade no Love Live! (Musical ver.)" (これまでのラブライブ! 〜ミュージカルver.〜 Koremade no Raburaibu! Myujikaru ver.^?, Previously on Love Live! (Musical ver.)) by μ's, Hideko, Fumiko, and Mika.</td><td>April 6, 2014</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> With a new school year approaching, Honoka succeeds Eli as student council president, with Umi and Kotori as her aides. Word soon reaches the gang that Love Live is taking place yet again on a larger scale then last year. However, since the format is now changed to include regional preliminaries, they soon realise that in order to qualify for the tournament, they would have to beat A-Rise in their regional preliminary. The gang is taken aback when Honoka suggests they don't need to enter Love Live, feeling it is not something she would say. Later that night, Yukiho informs Honoka that the opening date of Love Live is at the end of March, when the new school year begins. The next day, as Nico challenges Honoka to a race to decide if μ's should enter Love Live or not, Honoka comes to understand the meaning of Yukiho's words, as come March, Eli, Nozomi, and Nico will all graduate and no longer be school idols, making this year's Love Live the last opportunity for μ's to perform together as a group of nine. With the others easing her worries, Honoka states her true desire to enter Love Live and aim for victory. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep02" style="text-align: center; font-weight: normal; background:#F2F2F2">02</th> <td class="summary" style="text-align: left;">"Aim for Victory"

"Yūshō o Mezashite"(優勝をめざして) </td><td></td><td>April 13, 2014</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> A new rule is introduced to Love Live in which contestants in the preliminary rounds must perform songs that haven't debuted yet, so μ's decide to hold another training camp at one of Maki's villas in the mountains in order to come up with a new song. As Maki, Umi, and Kotori find themselves in a creative slump because of the pressure, the girls form camping groups of three to help them come up with ideas for music, lyrics, and costumes, but even then they have trouble keeping focused. However, after hearing some encouragement from their friends, Maki, Umi, and Kotori gain the drive to come up with a new song overnight. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep03" style="text-align: center; font-weight: normal; background:#F2F2F2">03</th> <td class="summary" style="text-align: left;">"Door of Dreams"

"Yume no Tobira"(ユメノトビラ) </td><td>"Shocking Party" by A-Rise
"Yume no Tobira" (ユメノトビラ^?, Door of Dreams) by μ's</td><td>April 20, 2014</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> With various alternatives to performing on one of the main stages for the Love Live preliminaries, the girls consider streaming from within their school, though realise it'll be harder to appeal to voters that way. With help from the broadcasting club, Honoka, Umi, and Hanayo use the school's broadcast to both spread word of their performance and give themselves more practise. Whilst trying to find a suitable location to perform where they haven't already performed before, the girls are brought to UTX Academy by the members of A-Rise, who have been keeping a close eye on each of μ's' members. After stating their intention to not lose against them, A-Rise offers to let μ's perform on their stage atop of UTX Academy's roof for the preliminaries. As the girls feel doubtful after seeing A-Rise's performance, Honoka pumps up the other girls whilst the other students come to show their support as μ's performs the new song they created together. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep04" style="text-align: center; font-weight: normal; background:#F2F2F2">04</th> <td class="summary" style="text-align: left;">"No. 1 Idol in the Universe"

"Uchū Nanbā Wan Aidoru"(宇宙No.1アイドル) </td><td></td><td>April 27, 2014</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> After an agonising wait for the results, the girls are delighted to discover that μ's, along with A-Rise, has qualified the first preliminary round. As the girls strive to practise hard for the next round, Nico seems to be preoccupied by something, so the others decide to follow her, though she ends up spotting them and escapes. Whilst wondering what Nico is up to, they end up meeting her younger sister, Kokoro, who, along with her siblings Kotaro and Koko, has been led to believe that Nico is the star of μ's and the others are merely her backup dancers. After finally being pinned down by the understandably bemused girls, Nico explains she has been skipping practise to look after her siblings whilst their parents are away, stating that she has been telling them she was a super idol from the beginning. Nozomi believes she has kept this front up even after her attempt at being an idol during her first year fell apart, as she didn't want to destroy their vision of her. Upon hearing about this, the girls arrange for Nico to give a special performance for her siblings, claiming it to be her last performance as a 'solo idol' before joining her fellow idols as equals in μ's. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep05" style="text-align: center; font-weight: normal; background:#F2F2F2">05</th> <td class="summary" style="text-align: left;">"A New Me"

"Atarashī Watashi"(新しいわたし) </td><td>"Love Wing Bell" by Rin Hoshizora (Riho Iida), Maki Nishikino (Pile), Hanayo Koizumi (Yurika Kubo), Eli Ayase (Yoshino Nanjō), Nozomi Tojo (Aina Kusuda) and Nico Yazawa (Sora Tokui)</td><td>May 4, 2014</td></tr><tr><td class="description" 
style="border-bottom:3px solid #CCCCFF" colspan="4"> While Honoka, Umi, and Kotori are on a field trip to Okinawa, Rin is assigned as temporary leader of the group as they prepare for a fashion event at the weekend. However, Rin isn't confident in herself and struggles with the responsibilities. As Rin feels she is unfit to be leader, thinking she isn't cute enough, Hanayo tells Maki about how Rin has a complex about her boyish looks, which she was often teased about during elementary school. With bad weather leaving Honoka's group stranded until after the fashion event, Rin feels she isn't cute enough for the fancy dress given to the center and passes on the role to Hanayo, though the others can tell she is bothered about it. After Hanayo speaks with Honoka, the group arranges for Rin to wear the dress whilst everyone else wears butler outfits, feeling it fits her the most. The fashion event turns out to be a success and Rin ends up being more confident about expressing her femininity. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep06" style="text-align: center; font-weight: normal; background:#F2F2F2">06</th> <td class="summary" style="text-align: left;">"Happy Halloween"

"Happī Harowīn"(ハッピーハロウィーン) </td><td>"Dancing stars on me!" by μ's</td><td>May 11, 2014</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> Both μ's and A-Rise are invited to appear in a Halloween event in Akihabara, with μ's hoping to make a big impact to gain support for the Love Live preliminaries. Thinking they need a new look to provide that impact, the group tries various methods to accomplish, such as changing their outfits, impersonating each other, and even trying out a rock look, but nothing seems to work out. On the day of their performance, Honoka realizes they don't need to change themselves, as they already have quite varied members themselves, and they perform together with their natural abilities. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep07" style="text-align: center; font-weight: normal; background:#F2F2F2">07</th> <td class="summary" style="text-align: left;">"We Have to Do Something!"

"Nantoka Shinakya!"(なんとかしなきゃ!) </td><td></td><td>May 18, 2014</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> Upon reading the results of her physical exam, Honoka discovers she has put on weight, with Hanayo also shown to have gained weight too. Both Honoka and Hanayo are put on a diet whilst Umi and Kotori help sort out Honoka's student council duties. However, Umi soon discovers the two had been sneaking off during their runs to eat rice. Things get worse when a blunder by Kotori causes a budget request from the Art Club to be approved ahead of a budget meeting, becoming met with resistance when they attempt to withdraw it. Despite Eli offering to help, Honoka and the others decide to sort out the crisis themselves, apologising for the mix up and sorting out a budget draft for all the clubs. In the process, Honoka manages to get back to her old weight, with Eli and Nozomi left assured that the student council is in good hands. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep08" style="text-align: center; font-weight: normal; background:#F2F2F2">08</th> <td class="summary" style="text-align: left;">"My Wish"

"Watashi no Nozomi"(私の望み) </td><td></td><td>May 25, 2014</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> As the group decides to perform a new song at the Love Live preliminary finals, Nozomi suggests that they should go with a love song. However, the group struggles with this due to having little experience in the matter, and many of them feel they should just stick with one of their existing songs. Later, Maki follows Eli and Nozomi home, where she learns Nozomi's wish wasn't simply to write a love song, but to create a song that μ's wrote together. As Nozomi didn't have any friends at school before meeting Eli, the formation of μ's, which she played a strong hand in creating, allowed the nine girls to be connected and share the same feelings. Upon hearing this, Eli and Maki call the other members to join them in writing a song together, getting lyrical inspiration when it starts snowing outside. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep09" style="text-align: center; font-weight: normal; background:#F2F2F2">09</th> <td class="summary" style="text-align: left;">"Melody of the Heart"
"Kokoro no Merodi"(心のメロディ) </td><td>"Snow Halation" by μ's</td><td>June 1, 2014</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> The day of the Love Live preliminary finals arrives, with the girls overcoming the cold weather to get together to the stage. However, Honoka, Umi, and Kotori are held back by a delay on an address they are giving at the school and become snowed in. Determined not to let their efforts go in vain, the three girls decide to head to the stage on foot, finding that all of their fellow schoolmates have cleared the path for them. Rejoining the others just in time, the girls of μ's take on the feelings of all of their supporters and perform the song that they all wrote together. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep10" style="text-align: center; font-weight: normal; background:#F2F2F2">10</th> <td class="summary" style="text-align: left;">"μ's"

"Myūzu"(μ's) </td><td></td><td>June 8, 2014</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> On their way to the local shrine to celebrate the new year, the girls run into A-Rise, who congratulate them on winning the Love Live preliminaries. Meanwhile, Eli, Nozomi, and Nico, who are helping out at the shrine, worry about when it will be time to graduate. With μ's needing to make an impression to give them an advantage during Love Live, Hanayo suggests they come up with a group catchphrase for the official website that best represents the group. Later that day, Honoka is approached by A-Rise's leader, Tsubasa, who had been contemplating over how her group lost to μ's, leaving Honoka wondering the same thing. The next day, the girls get together at Honoka's store to make mochi for all of their supporters, though come no closer to thinking of a catchphrase. Later, upon encountering people's emas at the shrine giving their support for μ's, Honoka comes to understand the group's driving force came from everyone's support and decides on the catchphrase, "The story we all realize." </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep11" style="text-align: center; font-weight: normal; background:#F2F2F2">11</th> <td class="summary" style="text-align: left;">"That Which We Decided"
"Watashi-tachi ga Kimeta Koto"(私たちが決めたこと) </td><td></td><td>June 15, 2014</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> After she and Alisa pass the entrance exams for Otonokizaka, Yukiho asks Honoka about what will happen to μ's after Eli, Nozomi, and Nico graduate. With the girls divided between whether or not μ's should carry on with new members in the new year, Eli states that Honoka and the other younger members should be the ones to decide what the future of the group will be. Later, Alisa, who had originally wanted to join μ's upon entering Otonokizaka, tells Honoka that she instead intends to form her own idol unit with Yukiho, having realized that the reason she loves μ's is because of its nine members. Later that weekend, Honoka brings out the group to play, visiting all sorts of places that each of the members decide to go. Upon arriving at the beach, Honoka and the others announce their decision that μ's will disband after graduation, with everyone feeling the same way that a group with even one member missing is not μ's at all. After letting out all of their tears, the girls return home and make preparations for their final performance together at Love Live. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep12" style="text-align: center; font-weight: normal; background:#F2F2F2">12</th> <td class="summary" style="text-align: left;">"Last Live"
"Rasuto Raibu"(ラストライブ) </td><td>"KiRa-KiRa Sensation" by μ's

"Bokura wa Ima no Naka de" (僕らは今のなかで We're Living in the Moment^?) by μ's</td><td>June 22, 2014</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> Following the drawings for performance order for the Love Live finals, it is decided that μ's will be the last performers of the event. After finishing what could well be their final practice together, the girls find it difficult to part ways and instead decide to have a sleepover at the school. That night, the girls go up to the rooftop to see the city lights, feeling proud that they became school idols. The next day, the girls arrive at the venue for Love Live finals, stepping out on stage for their final performance, pleasing the fans so much that they call for an encore. </td></tr> <tr class="vevent" style="text-align: center; background:#F2F2F2"><th scope="row" id="ep13" style="text-align: center; font-weight: normal; background:#F2F2F2">13</th> <td class="summary" style="text-align: left;">"Come True! Everyone's Dreams"

"Kanae! Minna no Yume"(叶え!みんなの夢---) </td><td>"Happy Maker" by μ's
"Aishiteru Banzai! (Piano Mix)" (愛してるばんざーい!（Piano Mix） Hooray for I Love You (Piano Mix)^?) by μ's

"Oh,Love&Peace!" by μ's</td><td>June 29, 2014</td></tr><tr><td class="description" style="border-bottom:3px solid #CCCCFF" colspan="4"> Following μ's' victory at Love Live, Graduation Day finally arrives at Otonokizaka. Whilst Nico shows her family around, Honoka and the others work on preparations for the graduation ceremony. Honoka soon comes across Eli reminiscing in the student council room, and gives her thanks to all she has done for everyone. As the opening ceremony gets underway, Honoka presents her commemorative speech, which turns out to be a special song she and the others had prepared for the third years. After the ceremony, Nico assigns Hanayo as the next president for the Idol Research Club, with Maki nominated for vice-president, before the group take a reminiscing tour around the school. Arriving at the rooftop where everything began, Honoka gives a proper sendoff to the name of μ's. Before the girls can get a chance to part ways, Hanayo receives some 'big news', prompting everyone to rush back to the club room. </td></tr>

In vivo leukemogenic potential of an interleukin 7 receptor α chain mutant in hematopoietic stem and progenitor cells

Key Points

• Gain-of function mutation of IL7Rα induces lymphoid leukemia as well as myeloproliferative disease.
• In vivo oncogenicity of mutant IL7Rα is influenced by the differentiation stage at which it occurs.

Abstract

Somatic gain-of-function mutations in interleukin 7 receptor α chain (IL7Rα) have been described in pediatric T and B acute lymphoblastic leukemias (T/B-ALLs). Most of these mutations are in-frame insertions in the extracellular juxtamembrane-transmembrane region. By using a similar mutant, a heterozygous in-frame transmembrane insertional mutation (INS), we validated leukemogenic potential in murine hematopoietic stem/progenitor cells, using a syngeneic transplantation model. We found that ectopic expression of INS alone in hematopoietic stem/progenitor cells caused myeloproliferative disorders, whereas expression of INS in combination with a Notch1 mutant led to the development of much more aggressive T-ALL than with wild-type IL7Rα. Furthermore, forced expression of INS in common lymphoid progenitors led to the development of mature B-cell ALL/lymphoma. These results demonstrated that INS has significant in vivo leukemogenic activity and that the lineage of the resulting leukemia depends on the developmental stage in which INS occurs, and/or concurrent mutations.

Introduction

Interleukin 7 (IL7) is essential for T-cell development and homeostasis.1 Its cognate receptor (IL7R) forms a heterodimer composed of the α chain (IL7Rα) and common γ chain; binding of IL7 to IL7R triggers activation of Janus kinase (JAK)/signal transducer and activator of transcription signaling and the PI3K/v-akt murine thymoma viral oncogene homolog 1 (Akt) pathways.

Accumulating evidence has demonstrated that dysregulation of the IL7 signaling axis may be implicated in lymphoid malignancies. For example, IL7 transgenic mice develop T- and B-cell lymphomas, and human primary T-cell acute lymphoblastic leukemia (T-ALL) cells respond to IL7 in vitro and in vivo. Moreover, recent findings describing IL7Rα gain-of-function mutations in pediatric ALL and a T-ALL cell line have provided direct evidence that the IL7-IL7R axis plays a crucial role in the pathogenesis of human ALL.

Although the gain-of-function properties of these mutants have been precisely studied in vitro, their leukemogenic potential in vivo has not been well studied. One study reported that T-cell leukemogenesis was triggered by an IL7Rα mutant. However, they used murine IL7-dependent D1 progenitor T-cell lines derived from p53-knockout mice, which spontaneously develop T-cell lymphoma, and this specific animal model may not be generally applicable.

To extend these observations, we demonstrate the in vivo leukemogenic potential of such a mutant when expressed in primary hematopoietic stem and progenitor cells by using a IL7Rα mutant, which was previously identified in a T-ALL cell line.

Methods

Mice

Six- to 12-week-old Balb/c mice were used for all experiments. Lineage depletion of bone marrow (BM) or embryonic day 14.5 (E14.5) fetal liver was performed by the EasySep Mouse Hematopoietic/Progenitor Cell Enrichment Kit (StemCell Technologies). Via tail vein injection, 1 × 106 Lineage− BM/fetal liver cells (lin− cells), pro-B, or Thy1+T cell progenitors were injected into lethally (8 Gy) or sublethally (4 Gy) irradiated recipients. Mice were maintained in accordance with institutional animal care guidelines (Institute of Medical Science, University of Tokyo). Detailed methods are provided in the supplemental Methods.

Results And Discussion

In vitro transforming activity of the mutant IL7Rα, INS

Consistent with previous report, sequencing analysis of exon 6 of the IL7Rα gene, mainly encoding the transmembrane domain, identified a heterozygous in-frame transmembrane insertional mutation (INS) in the T-ALL cell line DND-41.6 Forced expression of INS exerted transforming activity in Ba/F3 cells, as revealed by acquisition of cytokine-independent growth (supplemental Figure 1A-C, found on the Blood website) as well as the autonomous phosphorylation of Stat1, Stat3, Stat5, and Akt (supplemental Figure 1D). In addition, transient expression of IL7Rα in human embryonic kidney 293 cells leads to autonomous tyrosine phosphorylation of Jak1 only in those expressing INS (supplemental Figure 1D, left), suggesting that INS constitutively activated IL7R downstream signals via Jak1. As INS falls within the same category of reported mutation of IL7Rα, we decided to use INS as a representative gain-of-function IL7Rα mutation for further experiments.

INS in stem/progenitor cells caused myeloproliferative disorders

The leukemogenic activity of INS and wild-type (WT) IL7Rα was assessed by retroviral transduction of Lin− cels. Within 6 to 9 weeks after transplantation, recipient mice transplanted with INS Lin− cells, but not WT Lin− cells, developed myeloproliferative disorders (MPDs) characterized by splenomegaly, leukocytosis, and polycythemia (Figure 1A-C, right; supplemental Figures 1D and 3C-D). Fluorescence-activated cell sorter (FACS) and morphological analysis revealed a marked increase in Mac1++Gr-1++ mature myeloid cells in the peripheral blood (PB), spleen (SP), and BM (Figure 1C-D; supplemental Figures 2 and 4). An increase of Ter119+ CD71+immature erythroblast was also noted in SP and BM (supplemental Figure 4, SP; data not shown). INS-induced MPD was oligoclonal, as evidenced by Southern blot analysis (supplemental Figure 5, left). A similar disease phenotype was also observed in mice transplanted with Lin−c-Kit++Sca1++(KSL) fractions transduced with INS (supplemental Figures 3A-B,E and 4). Both B- and T-cell development were severely perturbed in INS recipient mice (supplemental Figure 4). As transplantation of INS-transduced KSL cells resulted in preferential expansion of myeloid progenitor–enriched Lin−c-Kit++Sca1− fraction (supplemental Figure 6), we speculate that in vitro transforming activity of INS skewed myeloid progenitor expansion at the expense of common lymphoid progenitor Lin–c-kitlowSca1+ IL7Rα+ (CLP) expansion, through which normal lymphopoiesis might be perturbed. This was also supported by the fact that INS exerted transforming activity in input KSL cells, as well as resultant myeloid progenitors ex vivo, as revealed by colony-forming cell assay (supplemental Figure 7). It was previously reported that forced expression of wild-type murine IL7Rα into IL7Rα knockout BM progenitors induces a very similar MPD phenotype, including splenomegaly resulting from neutrophilia. Consistent with this report, transduced WT appeared to induce some degree of increase in myeloid fraction and neutrophilia in PB and SP compared with that of mock increase of myeloid fraction and neutrophilia in PB (supplemental Figure 4). Importantly, the magnitude was quite different, as we could not find a statistically significant difference of SP weight in mock and WT (n = 4 each; P = .61, 1-way analysis of variance; supplemental Figure 3C). This is in contrast to the difference of INS (n = 4) and WT (P < .01; supplemental Figure 3C). WT-induced mild myeloid expansion was accompanied by concomitant increase in lymphoid subset in PB, SP, and BM, specifically CD19+ B-cell fractions (supplemental Figure 4). Considering the fact that the phenotype of WT-recipient mice was different from that of mock (supplemental Figure 4), it should be mentioned that we could not rule out the possibility that the phenotype elicited by INS is in part a result of the effect of IL7R overexpression per se, irrespective of its mutational status. The major difference from the previous report was that they rescued the loss-of-function phenotype of IL7Rα by ectopic expression of IL7Rα.

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Figure 1

In vivo transforming activity of INS. (A-D) Lin− cells were retrovirally transduced with mock vectors (mock), WT, or INS, followed by injection into lethally irradiated congenic mice. (A) May-Giemsa staining of PB smears at day 40, showing marked leukocytosis consisting predominantly of mature myeloid cells. (B) White blood cell count at day 40. *P < .05 (analysis of variance; INS vs WT or mock recipient mice). (C) FACS of the PB and SP at day 40, showing an increase in Mac-1+/Gr-1+ myeloid cells. (D) Immunohistochemical analysis (IHC) of SP and BM specimens by anti-myeloperoxidase, indicating an increase in the number of myeloid cells in INS recipient mice. Bars represent (A) 10 μm and (D) 20 μm. (E) Demonstration of in vivo reconstitutive capacity of hIL7R (WT/INS) transduced T-cell progenitors. Lin− kit+ stem/progenitor cells were cultured on OP9-DL1 stromal layer for 7days, supplemented with mIL7+ human Fms-like tyrosine kinase 3-ligand, which allowed them to differentiate into Thy1+CD25−CD44+DN1 immature T-cell progenitor fractions. These cells were retrovirally transduced with WT/INS vector. The resultant cells were allowed to expand on OP9-DL1 stroma for additional 7 to 10 days, and developed into CD25+CD44−DN3 immature T-cell progenitor fractions. These Thy1+ cells were green fluorescent protein (GFP)-sorted and intravenously injected into sublethally irradiated mice. The resultant GFP+ thymic seeding progenitors (denoted as “input”) in recipient mice of WT and INS at day 52 was shown (denoted as “output”).

Neither of these recipient mice developed overt leukemia throughout the median follow-up period of 5 months (WT, n = 28; INS, n = 22), suggesting that additional transforming events are required for clonal evolution to aggressive leukemia. Considering the fact that recipient mice for hematopoietic stem cells transduced with constitutively active Akt or signal transducer and activator of transcription-5 also developed similar diseases together, this MPD phenotype is likely to be induced by stem cells ectopically expressing INS.

Nononcogenic consequence of INS in T-cell progenitors

Next, we wished to test the effect of INS on T-cell precursors. Toward this aim, we cocultured Lin− kit+ stem/progenitor cells for 7 days, which allowed the emergence of Thy1+CD25−CD44+DN1 immature T-cell-progenitor fractions (data not shown). These cells were transduced by retroviral transduction of the WT/INS vector. The resultant transduced cells were allowed to expand on OP9 expressing the Notch ligand Delta-like 1 (OP9-DL1) stroma for an additional 7 to 10 days, which allowed them to develop Thy1+CD4−CD8−CD25−CD44+DN1 to DN3 CD25+CD44−DN3 immature T-cell-progenitor fractions (Figure 1E). The resultant Thy1+ cells were GFP sorted and injected into sublethally irradiated mice. As a result, we could detect stable engraftment of GFP+ T-cell progenitors in recipient mice from day 40 to day 50 in thymus (Figure 1E) and CD4 or CD8 single-positive cells in the periphery, such as SP or PB (data not shown). Neither of these recipient mice developed overt leukemia throughout the median follow-up period of 106 days (WT and INS, n = 12 each). We speculate that this might be partly attributable to the limited engraftment of WT/INS-transduced T-cell progenitors in thymus (data not shown).

INS exacerbates the in vivo oncogenic activity of Notch1

INS-like mutations were reported to occur in 10% of T-ALL patients. In contrast, Notch1 mutations were more frequently found in T-ALL patients and were equally distributed between patients with WT and INS. The DND-41 cell line carries both INS and Notch1 mutations. Moreover, IL7 signaling coordinates with Notch1 in proper T-cell developmental programming. We hypothesized that INS may cooperate with active Notch1 mutants in T-cell leukemogenesis. Therefore, Lin− cells were transduced with mock or IL7Rα-WT/INS along with an active form of intracellular Notch1 (ICN1), followed by syngeneic transplantation. As reported previously, within 4 to 6 weeks after transplantation, all mice developed T-ALL, characterized by extrathymic expansion of leukemic cells (Figure 2A-B; supplemental Figure 8B). Clonality of INS/ICN1-induced leukemia was confirmed by Southern blot analysis around day 35 (supplemental Figure 5, right). Despite similar immunophenotypes (CD3+CD4+CD8+TCR-β+) between WT/ICN1 and INS/ICN1 cells (Figure 2A,Upper; supplemental Figures 8A and 9), histological examinations of the liver, SP, and BM in recipient mice revealed that systemic expansion of INS/ICN1-Lin− cells was much more aggressive than that of WT/ICN1− and mock/ICN1−Lin− cells (Figure 2A, Lower, and 2B). Furthermore, the median survival time of INS/ICN1 mice (44 days; n = 54) was significantly shorter than that of mock/ICN1 (60 days; n = 44) and WT/ICN1 (57 days; n = 42) mice (P < .001 by log-rank test; Figure 2C). Taken together, INS clearly exaggerated ICN1-induced T-ALL.

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Figure 2

INS synergized with active Notch1 (A-C) and exerted transforming activity in CLPs (D-F) in vivo. (A-C) Lin− cells were cotransfected with vectors encoding the ICN1 gene (mock, ICN1) and the hIL7R gene (mock, WT, INS), followed by injection into lethally irradiated congenic mice. (A, upper) FACS analysis of the SP from WT/ICN1 or INS/ICN1 recipient mice at day 40. Data were obtained from GFP+ (marker for the ICN1 gene) and rat CD2+ (marker for the hIL7R gene) fractions. (A, lower) IHC of SP specimens using anti-CD3 antibodies from WT/ICN1 (left) and INS/ICN1 (right) recipient mice. Bar represents 20 μm. (B) Histological findings of liver (left 2 panels) and BM (right 2 panels) from WT/ICN1 (upper) and INS/ICN1 (lower) recipient mice (hematoxylin and eosin stain). Bar represents 50 μm. (C) Survival curves of recipient mice (mock/mock, n = 22; mock/ICN1, n = 44; WT/ICN1, n = 42; INS/ICN1, n = 54) **P < .01 (log-rank test). (D-F) CLPs transfected with hIL7R constructs were expanded in vitro for 18 days and injected into sublethally irradiated congenic mice. (D, top 2 panels) FACS analysis of the BM: WT recipient mice at day 60 (WT) and INS recipient mice at day 60 (INS). Data are obtained from GFP+ gated fractions. Open histogram, isotype control; shaded histogram, specific staining. (D, lower) Splenomegaly and lymphadenopathy developed in CLPs-INS recipient mice at day 60 (denoted as “INS”). (E) Survival curves of recipient mice (n = 11 for each condition). **P < .01 (log-rank test, INS vs WT or INS vs INS2nd). (F) Histological findings: BM specimens from WT recipient mice (upper left) and INS recipient mice at day 60 by hematoxylin and eosin stain (lower left) or by IHC of B220+ cells (upper right). Lymph node cytospin from INS recipient mice at day 60 by May-Giemsa stain (lower right). Bar represents 20 μm. WT, WT primary recipients; INS, INS primary recipients; WT-2nd, WT day 30 BM secondary recipients; INS-2nd, INS day 30 BM secondary recipients.

Forced expression of INS in B-cell progenitors caused mature B-ALL/lymphoma

Because the IL7Rα gene is transcriptionally active in common lymphoid progenitors (CLPs; Lin−c-kitlowSca1+ IL7Rα+) and their progenies and not expressed in stem cell compartments, the INS allele could target the same cell populations. Then, CLPs were transduced with INS or WT IL7Rα and cultured on the OP9 stromal layer with a cytokine cocktail for 18 days, followed by transplantation of resulting pro-B cells into syngeneic recipient mice (supplemental Figure 10A). All but 1 of the INS-CLP recipients died of mature B-ALL/lymphoma, whereas no WT-CLP recipients died (P < .01;Figure 2D-E). Autopsy specimens revealed massive infiltration of B220+leukemic blasts into the BM, SP, and lymph nodes (Figure 2F; data not shown). This mature B-cell ALL/lymphoma was transplantable to secondary recipients, resulting in more aggressive mature B-ALL/lymphoma with much shorter survival periods (Figure 2E). INS-induced mature B-ALL/lymphoma was biclonal, as evidenced by Southern blot analysis (supplemental Figure 5,right). Under these experimental conditions, INS-CLPs had already committed to the cytokine-independent clonogenic pro-B cells before transplantation (supplemental Figure 10B-C; data not shown).

Finally, we wished to identify the downstream signals involved in INS-induced leukemogenesis. Using microarray analysis (Gene Expression Omnibus accession number GSE51211) of the resultant transformed cells in vitro and in vivo, we performed a comparative analysis of gene expression profiles from WT and INS-transduced hematopoietic stem/progenitor cells, as well as resultant leukemia cells that developed in vivo. As a result, we found a list of candidate genes (n = 6133) that were up- or downregulated by INS in comparison with WT. Among those genes, by reviewing hierarchical clustering analysis, several genes could be candidate mediators downstream of INS in comparison with WT, including hairy and enhancer of split-1 (HES1) for MPD, proviral insertion site in Moloney murine leukemia virus 1 (PIM1) for B-ALL, and insulin-like growth factor 1 receptor (IGF1R) for T-ALL (supplemental Figure 11). Quantitative RT-PCR verified their differential expression in comparison with WT (supplemental Figure 12). Putative involvement of these genes in INS-induced leukemogenesis was supported by previous data reporting the significance of HES1 overexpression reported in advanced chronic myelogenous leukemia, PIM1 activation involved in pre-B-cell transformation, PIM1 overexpression reported in B-ALL, and high-level expression of IGF1R in T-ALL. In addition, we performed gene set enrichment analysis to find significant overlaps between INS/ICN1 (in comparison with WT/ICN1) gene expression signature and gene sets present in the public database (supplemental Discussion). As a result, we found that in vivo INS/ICN1 was characterized by overexpression of interferon (IFN)-stimulated genes23 and IGF1-signal-related genes,24 suggesting constitutive activation of IFN− as well as the IGF1− signal pathway (supplemental Figures 13 and 14; supplemental Tables 2 and 3). These are consistent with the previous report that JAK1-mutated T-ALL samples were characterized by the IFN-pathway signature, as well as our findings of a higher IGF1R transcript level in INS/ICN1 cells compared with that of WT/ICN1 (supplemental Figures 11 and 12).

In conclusion, we provided evidence that INS has significant in vivo leukemogenic activity and that determination of the lineage of resulting leukemias depends on the developmental stage during which they occur and/or concurrent mutations. In addition, as far as we know, this is the first report in which transformation of CLP leading to in vivo malignancy is shown. This is also of general relevance for the field of lymphoid malignancies. Given that either IL7Rα or Jak1 gain-of-function mutations have been found in approximately 10% or 19%25 of T-ALL patients and that IFN- pathway signatures have been associated with Jak1-mutated T-ALL, it is fairly certain that IFN-pathway signatures induced by aberrant IL7R/Jak1 axis might substantially contribute to the pathogenesis of T-ALL in close association with activating mutations in the Notch pathways.

Authorship

Contribution: K.Y., N.Y., and K.I. performed experiments; K.Y. wrote the manuscript; A.H., A.K., and K.H. provided vital reagents; and A.T. supervised the research.

Conflict-of interest disclosure: The authors declare no competing financial interests.

Correspondence: Arinobu Tojo, Department of Hematology-Oncology, Research Hospital, Institute of Medical Science, the University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; e-mail: a-tojo@ims.u-tokyo.ac.jp.