JUOUUKINAL OF
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`VOLUME 207 NUMBER 10 SEPTEMBER 27, 2010 www.jem.org
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`TNATIONAL
`LIERARY OF
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`PLASTICITY OF NATURAL KILLER CELL LICENSING
`Dendritic Cells Required for Th2 Responses
`NKX3.1 in Human T Cell Acute Lymphoblastic Leukemia
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`
`THE
`JOURNAL OF
`EXPERIMENTAL
`MEDICINE
`
`VOLUME207 NUMBER 10
`SEPTEMBER 27, 2010
`www.jem.org
`
`
`
`
`ON THE COVER
`Brief Definitive Reports from the
`teams of Raulet and Yokoyama
`illustrate the functional plasticity
`of mature NK cells. Dysfunctional
`NK cells from MHC class I-deficient
`mice gain effector function when
`transferred to MHC-sufficient hosts,
`and vice versa. Joseph Sun discusses
`these papers in the context of other
`recent findings in a Minireview.
`Watercolor image represents the
`altered responsive states of NK cells.
`Artwork by Emilie Clark (emilie@
`emilieclark.com).
`See page 2049
`
`Minireviews
`
`p53 connects tumorigenesis and reprogramming to pluripotency
`Natalia Tapia and Hans R. Schéler
`
`2049
`
`Re-educating natural killer cells
`Joseph C. Sun
`
`Reviews
`
`2053
`
`‘Type [ interferon: friend or foe?
`Giorgio Trinchieri
`
`Brief Definitive Reports
`
`2073
`
`2081
`
`2089
`
`2097
`
`OVS
`
`2127
`
`Maturenatural killer cells reset their responsiveness when
`exposed to an altered MHC environment
`Nathalie T. Joncker, Nataliya Shifrin, Frédéric Delebecque, and David H. Raulet
`See minireview on page 2049
`
`MHCclass I-deficient natural killer cells acquire
`a licensed phenotype after transfer into an MHC
`class I-sufficient environment
`Julie M. Elliott, Joseph A. Wahle, and Wayne M. Yokoyama
`See minireview on page 2049
`
`IL-10 inhibits transcription elongation of the human TNF
`gene in primary macrophages
`Tim Smallie, Giuseppe Ricchetti, Nicole J. Horwood, Mare Feldmann,
`Andrew R. Clark, and Lynn M. Williams
`
`CD11e depletion severely disrupts Th2 induction and
`development in vivo
`Alexander T. Phythian-Adams, Peter C. Cook, Rachel J. Lundie, Lucy H. Jones,
`Katherine A. Smith, Tom A. Barr, Kristin Hochweller, Stephen M. Anderton,
`Giinter J. Hammerling, Rick M. Maizels, and Andrew S. MacDonald
`
`Articles
`
`Inflammatory dendritic cells—not basophils—are necessary
`andsufficient for induction ofTh2 immunityto inhaled
`house dust mite allergen
`Hamida Hammad, Maud Plantinga, Kim Deswarte, Philippe Pouliot,
`Monique A.M. Willart, Mirjam Kool, Femke Muskens, and Bart N. Lambrecht
`
`The Ets-1 transcription factor controls the development
`andfunction ofnatural regulatory T cells
`Enguerran Mouly, Karine Chemin, Hai Vu Nguyen, Martine Chopin,
`Laurent Mesnard, Maria Leite-de-Moraes, Odile Burlen-defranoux,
`Antonio Bandeira, and Jean-Christophe Bories
`
`Mutant p53 facilitates somatic cell reprogramming and augments
`the malignant potential of reprogrammedcells
`Rachel Sarig, Noa Rivlin, Ran Brosh, Chamutal Bornstein, Iris Kamer, Osnat Ezra,
`Alina Molchadsky, Naomi Goldfinger, Ori Brenner, and Varda Rotter
`See minireview on page 2045
`
`

`

`
`
`NKX3.1 is a direct TAL1 target gene that mediates proliferation
`of TAL1-expressing human T cell acute lymphoblastic leukemia
`Sophie Kusy, Bastien Gerby, Nicolas Goardon, Nathalie Gault, Federica Ferri,
`Delphine Gérard, Florence Armstrong, Paola Ballerini, Jean-Michel Cayuela,
`André Baruchel, Francoise Pflumio, and Paul-Henri Roméo
`
`Conversion of Helicobacter pylori CagA from senescence inducer
`
`to oncogenic driver through polarity-dependentregulation
`
`FceRI* DCs initiate Th2 responses to
`house dust mite allergen.
`See page 2049
`
`of p21
`Yasuhiro Saito, Naoko Murata-Kamiya, Toshiya Hirayama, Yusuke Ohba,
`and Masanori Hatakeyama
`;
`:
`:
`:
`= 5
`Upregulation of Tim-3 and PD-1 expression is associated
`with tumor antigen-specific CD8* T cell dysfunction in
`melanoma patients
`Julien Fourcade, Zhaojun Sun, Mourad Benallaoua, Philippe Guillaume,
`Immanuel F. Luescher, Cindy Sander, John M. Kirkwood, Vijay Kuchroo,
`and Hassane M. Zarour
`
`Targeting Tim-3 and PD-1 pathways to reverse Tcell exhaustion
`and restore anti-tumor immunity
`Kaori Sakuishi, Lionel Apetoh, Jenna M. Sullivan, Bruce R. Blazar, Vijay K. Kuchroo,
`and Ana C. Anderson
`
`2141
`
`21 at
`
`2187
`
`2195
`
`2207
`
`2225
`
`IAP inhibitors enhance co-stimulation to promote tumor immunity
`Michael Dougan, Stephanie Dougan, Joanna Slisz, Brant Firestone,
`Matthew Vanneman, Dobrin Draganov, Girija Goyal, Weibo Li, Donna Neuberg,
`Richard Blumberg, Nir Hacohen, Dale Porter, Leigh Zawel, and Glenn Dranoff
`
`Lnk-dependent axis of SCF-cKit signal for osteogenesis in bone
`fracture healing
`Tomoyuki Matsumoto, Masaakili, Hiromi Nishimura, Taro Shoji, Yutaka Mifune,
`Atsuhiko Kawamoto, Ryosuke Kuroda, Tomoaki Fukui, Yohei Kawakami,
`Tomoya Kuroda, Sang Mo Kwon, Hiroto Iwasaki, Miki Horii, Ayumi Yokoyama,
`Akira Oyamada, Sang Yang Lee, Shinya Hayashi, Masahiro Kurosaka,
`Satoshi Takaki, and Takayuki Asahara
`
`Somatic hypermutation as a generator ofantinuclear antibodies
`
`Polarity-dependent oncogenesis of H.
`pylori CagA.
`See page 2157
`
`in a murine model of systemic autoimmunity
`Wenzhong Guo, Diana Smith, Katja Aviszus, Thiago Detanico, Ryan A.Heiser,
`and Lawrence J. Wysocki
`
`y6 T cells protect against lung fibrosis via IL-22
`Philip L. Simonian, Fabian Wehrmann, Christina L. Roark, Willi K. Born,
`RebeccaL. O'Brien, and Andrew P. Fontenot
`
`2255
`
`Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic
`skin inflammation
`Reto Huggenberger, Stefan Ullmann, Steven T. Proulx, Bronislaw Pytowski,
`Kari Alitalo, and Michael Detmar
`
`

`

`
`
`22371
`
`2283
`
`Engulfment of cerebral apoptotic bodies controls the course
`ofprion disease in a mouse strain—dependent manner
`Jan Kranich, Nike Julia Krautler, Jeppe Falsig, Boris Ballmer, Shulei Li,
`Gregor Hutter, Petra Schwarz, Rita Moos, Christian Julius, Gino Miele,
`and Adriano Aguzzi
`
`Corrections
`
`Soluble HIV-1 Env trimers in adjuvantelicit potent and diverse
`functional B cell responses in primates
`Christopher Sundling, Mattias N.E. Forsell, Sijy O'Dell, Yu Feng, Bimal Chakrabarti,
`Srinivas S. Rao, Karin Loré, John R. Mascola, Richard T. Wyatt, lyadh Douagi,
`and Gunilla B. Karlsson Hedestam
`
`
`
`Fracture healing is accelerated in
`mice lacking the adaptor protein Lnk.
`See page 2207
`
`

`

`Article
`
`Targeting Tim-3 and PD-1 pathways
`to reverse T cell exhaustion and restore
`anti-tumor Immunity
`
`Kaori Sakuishi,' Lionel Apetoh,' Jenna M. Sullivan,' Bruce BR. Blazar,-
`Vijay K. Kuchroo,! and Ana C. Anderson!
`
`Center for Neuroloqiea! Diseases, Brigham and Women's Hospiral and Harvard Mecheal Sehoal, Bastan, MA G2 11S
`Departrent of Pediatnes and Division al Blood and Marrow Transplar tation, University.ot Minnesota, Minneapolis, MM 554.55
`
`The immune response plays an important role in staving off cancer; however, mechanisms
`of immunosuppression hinder productive anti-tumor immunity. T cell dysfunction or
`exhaustion in tumor-bearing hosts is one such mechanism. PD-1 has been identified as
`a marker of exhausted T cells in chronic disease states, and blockade of PD-1—PD-1L
`interactions has been showntopartially restore T cell function. We have found that T cell
`immunoglobulin mucin (Tim) 3 is expressed on CD8* tumor-infiltrating lymphocytes (TILs)
`in mice bearing solid tumors. All Tim-3" TILs coexpress PD-1, and Tim-3"PD-1' TILs repre-
`sent the predominantfraction of T cells infiltrating tumors. Tim-3'PD-1* TILs exhibit the
`most severe exhausted phenotypeas defined by failure to proliferate and produce IL-2,
`TNF, and IFN-y. We further find that combined targeting of the Tim-3 and PD-1 pathways
`is more effective in controlling tumor growth than targeting either pathway alone.
`
`The HH POrldney of the maniune SVSECHE IE pro
`COCO) Pest Cane er Was orteially proposed
`i the theory of cancer Hmimunosurvellince:
`Phis theory holds that the mamune systent etn
`recopmize cancerous cells ts they arise and can
`mount both ammnate aod adapove manune re-
`sponses to climate then. In support of cancer
`Hmunosurverlance as
`the fer that beth im
`Munodeficient Or Tamunesuppressed paticnts
`and experimental animals are nore susceptible
`ro tumor development (lor reviews see Dun
`eral, 2004: Zatwogel eral. 2006: Swann and
`Siyeh, 2007), Counterto the role of the immune
`system)
`In staving, OFF cancer
`ts the ability of
`tu
`iors to esdape the amamane system by engen
`dering a state et PN OStIppresston (for review
`see Zitvogel et al, 2006). One example of a
`mechaisiny OF WmunOsuppression prescuit 1)
`himor-bearing hosts is che prarmouan of
`T cell
`dystunction or extiustion.
`Pocell exhaustion describes state of “T vel
`dystunetian that was initially observed dun
`ing chrome lWiphoeyar Chorporye vigils Varls
`(LOMYV) infection a mice (Zajac etal, 1998).
`Exhausted PE
`cells Gal
`to proliferate and exert
`elector
`funetiens such as
`EN TOTONTCTEN and
`
`KoSvkuisb et]
`
`Apetobh cornered eqqitatl:
`
`to dtins pager
`
`cytokine secretion i response TO UItLAcH STI
`ulation, Purther studies denoted that exhausted
`I cells ave characterized by stistamed expression
`of the inhibitory molecule PI} 1 (programmed
`cell death b) and that blo kade of PID-
`1 and
`P-L) (PI-1 head) interictions can reverse
`T cell exhaustion ane restore suitienespee tte
`I cell responses in LOM V-intected nice (Barber
`etal. 2000),
`T cell exhaustion also occurs dur
`iiss Chron mifections tn burns (lor reviews
`see Klenerman and PAIL 2005). C18)
`1 cells an
`Homans chronically miteeted wach PLY (Das
`etal, 2006: Petrovas ctl, 2000. Draucnnin
`etal, 2006), hepatitis B virus (Boettler et al.
`2006), and hepacies C virus (EEOV: Urban eral,
`2006) express high levels of PIT, and block
`img oF PD-I-PD-L interactions can restore |
`cell function i vitro.
`Several lines of evidence alsa tmpheate the
`PII PD-L pathway a Poeell exhaustion in
`cancer, Firse, PID-T expression is found on timo
`infiltmony CIS! PE cells in multiple solid tuners
`(Blank et al, 2000; Abnadvadeh etal, 2009;
`
`2187
`
`This material maybeprotected by Copyright law (Title 17 U.S. Code)
`
`
`
`CORRESPONDENEC
`Vijay KE Kuchton
`JETER Ores, Ga Pyare
`(yf
`‘ia Arilerscn
`Winders nes 0h beaver. ed
`
`Abbreyviiiii dived, PROV,
`hrepaened
`vnniay DMM
`HCP re Ghee TG oul ibe iinet
`Hvis,
`EEL,
`trvteercantalerateene
`Woophocvie:
`TIM, Pell
`‘oereeoerenpelood oar line denen
`
`

`

`
`
`Balbo C126
`
`an
`
`CS57BL6
`
`B16
`
`B =r
`
`a=oO=woarT
`
`2iC
`
`a
`
`=oO&aa
`
`o
`pe
`&E=
`
`A
`
`cps
`
`cpa
`
`PMO
`(Timed)
`
`
`
`
`
`FMO
`(PD)
`
`iE
`
`PD
`
`PD-1 and Tim-3 expression in TiLs. BALB/c mice were implanted with CT26 colon adenocarcinoma or 47] mammary adenocarcinoma.
`Figure 1.
`CS7BL/6 mice were implanted with BIGEIO melarioma. TILs were harvested and stained with 7AAD to exclude dead cells and antibodies against CDS, CD4,
`Tim-3, and PD-1, (A) Expression of Tim and PD-
`1 on gated CD4° and CDB* TILs from a BALB/c mouse bearing CT26 tumor. FMO, fluorescence minus one
`controls for Tim-3 and PD-1 staining, Data shown are representative of more than five independent analyses. (B) Frequeney of CD8° cells in Thks express
`ing Tim-3 and PO-1 from tumor-bearing mice The horizontal bars indieate means, "P< O.0010" P
`0.05, one-way ANOVAfollowed by Tukey's multiple
`carnpansan test, CT26 (7
`<5) 477 (A
`6), and Ble tn
`HAC) Prequenty of CDR Tim-3" cells in spleens of tumor-bearing mice compared with spleens of
`naive tumor-free mice, The harigontal hry indieale meany "2
`unpaired
`(00), one-way ANOVA, Lukey's multiple comparison test: **, P = 0.0188
`10
`1 CT26, ne B47, he
`FECR7 ALG ne
`& BTS
`Student's t test, BALB/e, A
`
`specie © 18 fb cells un
`Gehring etal, 2009) aibon wniticen
`hosts with nonsold tainor (Yortireto et
`ah
`2008) Miinapeec ha
`etabk, 2009)) Second,
`these PRP
`TP
`ceths
`ane
`chy stu ranial
`Phir, PIL} is expressed ait Taele levels
`ta seven) differena
`cancers (Latchmian etal, 200). Done eraho
`T0024) Prawn eral
`F003), and lnveh expression ol PP TT Or Perera is
`Sironagely
`assoctated wath poor progiiasis CP homipsan er
`al.
`06)
`bourth, interference wil PI | PIT suenaliae. cither Creel
`antibody blockade or PRT detereney.
`tas been slow te
`nnprove chineal outcome and restore
`tunetonal Pocell
`re
`sponses i severitounoers (Blink etal. 2006. Yournarneto otal
`TOO), Plowever,
`2008; Mumprecheceal. 2009) 4 bane et ak,
`targeting: the PDE PD ATE parkway does not ahways resaltain
`reversalot
`TP celbexhaustiom (Bhikburn et ah,
`2008: Celine
`etal, 2009) and PII expresslon is bot always assocnined wae
`exhausted phenotype (Petrovas etal, 2006; bourcade etal,
`2009), Indicate that orher molecules are likely mivolved in
`I cell exhaustion.
`A recent study tn patients with FIV has shown thatthe i
`mune regulator Po cell mimunodebulin maucia CEEM) 3 is up-
`revulated on exhausted CDS)
`1 cellsones etal, 2008). Tin-3
`Ka molecule orally identified as beme selectively expressed
`on IPN-ysseeretine, Tht and Tel cells (Monney et al, 2002)
`Interaction of Tim-3 wath ats
`ligand, galectin-9, triggers cell
`deathan Tuned? TP cells. Vhos. both Tims and Plt can taine
`hon as negative reguhitors oF T cell responses. In FEV: patients,
`
`2188
`
`PIM 4 and PDT mark distinet populations of exhausted cells,
`with cells positive tor borh PHS) and PIM-3 comprising the
`stitallest
`friction. Glones et al, 2008) of CDS!
`TE
`cells, Similirly,
`mother wroup bas shown that PIMe3 is up-regulated on ex-
`Hiusted Tocells tn patients with EICV (Golden Mason et al.
`009) Th Cis case cells that coesxpress PIM-3 ane PD} 1 are the
`Hostabunda faction amone PROV specie Cbs bh eells.
`Hh hot) studies, blockine: EEIM-
`4 restored Ecol proliterarion
`dl Colancecbeytokine production,
`Because tirvenbeothe PR bebe pathoaway alone dows not
`result
`iy complete rostonition oat
`Pb
`ocell fimetbon Gibhick burn
`chal, 2008) andi some cancers Gapeome the PD b-Ph
`pathway does not restore bool faneton at all (Geli etal,
`HOO) theres acneed to tdene|ty other molecules and mbibibory
`pathways that are divolved i fo celbestarston Pideed, one
`tHThis denen LAGE A as be Wp Apr sacd on ostnrusted
`Pocell. and although treamment wath ante PAG)
`3s alone chal
`not restore TP
`ocell fanctoan i LOOMinfected mince.
`it syne
`ized with PE | blockade tomprave Pocell responses and re
`duce viratload (Blick barn eral, 200). Cotortumately, Chis sandy
`did not idenuty whether LAG Sand VEY bare expressed ent adis
`tick or overlapping, populations of exhausted Pocells Cavern
`these Observanons, (appears that
`tameciig miulaple pathways
`Hay prove most eHectve ta reversing DP cell exhaustion
`We report
`in this paper the coexpression of Pim 4 and
`PDT oon oa lirge fraction of tumor inbloatiog: Lymphocytes
`
`Taeyetiog Tie re fee aie poer merely |
`
`akosho etal
`
`

`

`Tina? Ppa?
`
`
`A
`
`Ting PD
`
`hin S PD
`
`PMO
`(CD44)
`
`PMO
`(CO62L)
`
`z
`
`
`~ Ola
`
`9728
`
`18.6)
`
`Figure 2. CD44 and CDG62L expression in Tim-3- and
`PD-1-expressing TILs. Tis were harvested from CT26 tumor
`bearing mice and stained with 7AAD to exclude dead cells
`anc antibodies against CDS, CD44, CD62L, Tint
`3, and PD-1
`(A) Representative staining on CDS8* Tem-3 PD-1 , Tirn-3
`PD-1), and Tirm-3°PD-1>
`TILs is shown. PMO, controls for CD44
`and CDG2L staining are shown, Data are representativeof
`three independent analyses,
`(6) Summary data shawing [he
`frequencyof effectorimemory (CD44"CD62L") and central
`memory (CD44"CDG62L") and CD44" cells within the CD8*
`fine PDT Time 3 POAT, and Tim-3'PD-)°
`Tihs,
`*) P< O.08,
`"P< 0,01; , Pee 0.001, one-way ANOVA and Tukey's mul
`liple comparison test n=
`J. Error bars represent SEM.
`
`94.5
`
`PILs wn mice
`other cineers, we csamuined the C18!
`bearing other solid tumors: FEE mammary adeno
`carcinoma dnd BLOF LO melinoma, Consistent with
`our observations m mice bearme C126, cells chat
`coexpress Time ind PDT also comprise 50%ol
`the CDS!) Tihs in mice bearing 4b tumor, wath
`cells expressing PDE alone or neither Pim-3 nor
`PID-1 abo comprising smaller populations (25 and
`15"respectively; Fig. 1B). hninice bearing BLGOE TO
`melinoma,
`all
`three populations of CIS)
`ET»
`(Vined PILL. Vime3d PD-L and Pines PD)
`are present at roughly equal frequency, Interest
`ingly. in all three of the tumor models examined we
`did nor observe any Tim-3'PD-1
`Pky (Pig. 1A
`and not depicted). We also exammned CDA)
`TTEs:
`however. these are less abundant, and among these
`we found that
`the majority were Fined PI-4
`with the Tim-3'PD-
`1! and Vim-3 PD-1*
`popula
`noms being roughly equivalent (lig.
`| A cand not
`depicted),
`( ‘ollectively,
`these date
`indicate that
`Vines and PD) coexpressing, C18"
`Ibs com
`prise a major population of
`T cell present m THs
`iifilrrating: chtferent solid’ Gamors,
`We also examined ‘Timed and PD-1 expression
`in the spleens of
`tumor-bearmg mice, There, we
`observed a trend toward inereased trequeney ol
`CDS'Tin-3° cells compared with naive mice; however, the es
`rent of this increase was varnble among mice bearing, ditlerent
`solid tumors (Pig.
`| CC). In contrast 6 the CDS)
`FILS, we found
`little af any evidence tor coexpression of PI) Lowith Dies
`amongsplenic CDS8*
`TT cells in tumor-bearing mice (Fig. St),
`suggesting that up-regulation of PIT on CHS! ‘Pime3)
`cells
`may happen in the tumor environment in response to environ
`imental cues. Llowever, we could cistingtish (we distinct popu.
`Dh
`Jin the peripheral
`lations of Pim-3° cells, Pine 3e! and Pune 3
`Ivniphoid tisstic oF tumor-bearing mice, Similarly. among
`splenic CIA) DT cells i tumor-bearing immer, we observed
`Ping 3!!! and Pime3!population, Interestingly, the Tin a"
`population was characterized by coexpression of PI-1, stigeest
`ing: that these cells may be the precursors of Tim) PDT" TEs
`and that they umayrepresent
`FE cells that are nia difterent fine
`tonal state frome Pied cells (Pig, S12).
`
`
`Central Memory
`Elfector/Memory
`cbsa"cpeel!
`cDaa"Cpea™
`)
`we
`10 2 is 100
`he
`i” i
`oO
`o 4A
`3
`Fe
`§
`a
`oO 4
`2
`ws
`0
`
`B
`
`3 7h
`oO
`B
`a 50
`a
`or
`2:
`uw
`i
`
`
`
`SE LS SL
`
`cD4a"
`_*
`
`
`
`co fy
`gS”
`3
`5 0
`=
`oT
`ay
`oP
`ic 25
`0
`RX Rh Kk
`OO a@
`.& 2
`& wo
`s
`a
`<”
`>
`
`CLES) ai tnice bearing solid tumors. PTLs that coexpress Pini
`and PD-T predominate among CDs" bs and exhibit
`the
`most profound defects in T cell etfector function. We further
`show that combined targeting of the Tinted and PD-1 path
`
`
`wavs is biehly efeerive in controlling tamer growth,
`
`RESULTS
`Tim-3 and PD-1 co expression on T cells in cancer
`Lo examine a potential role for Pind in 7 ell exhaustion mn
`Caneer, We first examined the expression of Pineas well as
`Ph dan DP cells (rom mice bearing: the solid turner CoP 26 4 olon
`circmoni. We observed thar among CDS!
`PELs, cells that
`coexpress
`Pinte and PL}
`T comprise the major popalanon
`(50%) with cells expressing, PIT alone on neither Tine3
`Hor PD b comprising smaller populations (408% and 20%,
`respectively: Pie. EAB) Pe extend these ObSerViithOns bo
`
`This material was copied
`eea ee ees
`
`2789
`
`

`

`A
`
`Tin-3 PD
`
`Timed PD"
`
`ries Ppt
`
`IWNy Tihs
`
` INFee Tihs
`(°)
`
`el
`
`Frequency{
`
`Frequency
`
`Coa
`
`Figure 3.
`Cytokine production in TiLs from C126 tumor-bearing mice. [lls were harvested (rom CT26 tumor-bearing mice and stimulated with
`PMAand hoOncrnyern before vtrad WOpTSAIe
`ey foie snacitoe,
`DAD Papress
`f
`WOE eYTORIe Tn Tin 4 PD-1 : Tim} PO-1', and Tim-3'PD-1° CD8>
`TILs
`Data shown are representative al levee pendent aoe yoes, PMO
`ycage
`cence minus une (aAt-eytoking antibody), (B) Frequency of Tima POS and
`Time PD 1
`cells armed f DE eylokine pierehar Ar Pardee Ty
`4) The honzornital bars indicate means. "P< 0.0001; Po»
`GLO26T,
`unpaired Student's p test
`
`T cell dysfunction in TiLs expressing Tim-3 and PD-1
`we
`lo further characterize the ditlerent subsers of CDR ET
`first examined their expression ob CME and © biel Wa
`found that
`the pattern of CD62) and © Dobe PPUSsTOnL Was
`quite different amene the Pim Phd bin Phebe ane
`Fam PPT) PEs (Pigs, 2A iad 1S), Adpvoonase Che tlie propor
`lavions, the Lime
`3! Pby |
`population Contam che Lircest
`poolls but
`the
`Haiction of efector memory (Ob eq Poop
`(ODAC DOZEN) vells
`lowest
`fraction of central memory.
`Indeed. the majoriny of Pam ao Pb |
`TILs were C1621!
`Phe iajority of PiLs in all three populations expressed bow
`todtertecdnite levels of C144. Although this Co) doggie’ pu pul
`Hon thay comprise some mauve cells, thas more likely that this
`
`population comprises cells that are (rinsitronmey from naive
`to effector sertus. Phe CD44" population was lowest among
`Pin-3 Poe I
`eclls. Phese data gave the firse meticatron chat
`the three populations of Tihs characterized by differential
`expression Of Fined and Pe? conrain cells i different Hane
`tonal states,
`
`In chrome viral mtection, PIT has been identified
`asd otarker of dvstuneuonal or exhausted C18 1
`cells
`
`2190
`
`(Marber etal,
`
`2006). Furthermore, has been observed that
`there as a dnerarchy at
`PT ocelleshaustion wath CoPL finetuon
`ind production of El
`home« onipronmiused Hirst, followed by
`WHY)
`| herete ye,
`lossol PNP and then TPN oy (Wherry etal
`todetormine whether any ofthe Pine and PP | CNPressinnye
`Hhs
`eshibied exteusted plenary pe, we asoahiled (Ds PTs
`ancl esamimect ther production ob Tl
`ENTE, and TEIN y ch
`4 Shab
`found) thatthe Pred Phe br
`reetly os vive, We
`the most Profound Gapaecrt to proctietron of Pb,
`Weal
`PINE and ib ry When onnpared with Fane PRE PTs ad
`lie PIT OTs i AY Surpristagly, the Pre 4 Phe I
`HDs produced the most FEN-y ong the three popularons
`OF TTLs and showed stemteanely less HpArent ia the pre
`duction of Tl
`Dame PINE than the Pine SPRL ELLs. Pliese
`dite stiverest Chat the Pine
`3 Php!
`Ilhs represen the THOst os
`hausted TILs andthat bined PDs bob bs maay contun a mistire
`
`obeshausted 1 cel
`said etfector PF
`ecells. Fo tunther Combine hese
`observations, we determined the abundance of Pun 3oPbe |
`cells ancl Time
`3 PP |
`cells within the evtokine prodtienna
`wand
`-nonproducing Fibs (bie 4B) We found thar bia 3) Phy
`cells are the most abundance (35 60%) populithon anions
`
`aeserbiniry Lire
`
`He PE) fe citeeloimen iereenite:
`
`|
`
`sakiped
`
`al
`
`

`

` 1O-PRO-3
`
`exnnmed the abrhity et Tihs to proliferate direc thy es vive by
`determine expression ob i-G7, a nue lear protem expressed
`by cells whielr have entered mete cell cycle, However,
`it tis
`been nered thar im aidividials clromieally infected wach ELEY,
`cells that are arrested in Gil can UXPress Ki-67)
`(Cloambadiere
`etal, 2000). We therefore also examined LINA content by si
`multaneously staining with POLPR OR iodide. By dome so.
`we can discern cells arrested in Gil
`from cells that have pro
`eressed to S, G2. and M phase. We isolated Tihs and stan
`lated them: directly cx vivo before examination ob KreG7
`expression and DNA content. We then determined the abun
`dance of Timea PI) and Pind Plt)
`cells ta Gu, Gil, and
`Proliferation and cell cycle entry in TiLs from CT26
`Figure 4.
`S-Mphases of cell eyele (Pi, +A), We found that Tim Ph |
`tumor-bearing mice. [|Ls were Harvested from C126 tumor-bearing mice
`cells are the miost abundance population that
`is stue kon Gi),
`
`
`and stimulated wi
`pg/ml
`of ant-CD3
`re SLA With antibodies
`outnumbenny Pin Pet) cells by 5 te | (hig. 414). Inter:
`and TO-PRO-:
`ynst CDS, Tim-3, PD-1, and Ki-G7
`rodide.
`(A) Expr
`
`
`estingly, when we examined cells that have progressed to the
`Tiks showing the diffe
`af i-67 and TO-PRO-3 staining i
`Cl and S-Mphases, we found that Pint 3 PIT!
`cells steactily
`»M. Data shown are represe
`of the cell eyele: GO, GV, and 5S
`Ive
`decrease in number, whereas Timed PIT?
`cells steadily im
`ThLs
`(B) Ratio ul Tim-3°PO-1°
`to Tine} PO
`ndependent analyses.
`crease with progression through cell evele. Collectively, out
`
`
`lo@Glin different phases. of cell le, *, P 0.05, one-way ANC
`
`
`
`
`data strongly support
`that coexpression of ‘Tuned and PD]
`a=
`6 Error bars represent
`SEM
`lukey's multiple comparison test
`marks the mast exhausted populigon ol Fibs, whieh tual
`te
`proliferate and produce [IL-2. TNE, and TPN-y
`
`Ce Ons th tions
`J WIS:
`
`Effect of targeting the Tim-3 and PD-1 signaling
`pathways in cancer
`Qur observations, alone with the previous
`that Mockade of either the PIT or Fined Qloanes et Al:
`CGolden-Muson et ale, 2009) signaling pathways can maiprove
`Pecell limetion in the contest at chromic infeetions, ramed the
`possibility that combined targeting of
`these two pathways
`may prove to be the most eficicious nicais fo restore ain
`Cuimtor immune) in viwe. Before commencing in vive: (reat
`ments,
`awe
`first contirmicd the expression of
`the PI-1 and
`Pim-3 ligands (PIO-LT ane galectin-), respec tively) on CP 26
`tinmar (Pie. S23). We then treated CoP 26 tunes beanie mice
`with an ante Pine} anubody, which was previ nisly described
`
`Day 23/24
`
`B 409
`
`Day 20/21
`
`
`n
`
`Wo
`
`00
`
`200
`
`ron
`
`*
`
`*
`
`Ch
`
`0
`
`nti-PD- |
`
`2191
`
`cytokine-nonproducme ELLs, outmuinbering Pines Pb!
`
`cells by three to fourfold. Examination of evrokine producing
`cells are less abundant
`than
`Plbs revealed that Pines Pl
`lun Pb |
`mone [L-2—producing PELs. A similar
`trend
`
`wis Observed wath I Ni, although thos did noe reach statisceal
`sremiiioinee, Both populimons were equally represented
`won TeNeyeproducing Pibs. Tnterestingty,
`this. stepwise
`Joss
`to abundance of Pines Plt)
`cells among evtokime
`producimg PTLs seems to follow the Inerarchy of
`P cell ex
`a dyna
`
`haustion, sumeesting that exhaustion as
`likely
`process im vive and that Pam-3° PD}
`To
`cells may be the tise
`(ii develop exhausted phenotype.
`loss of the abiliey to
`As stated in the previous paravraph,
`profiferre in response to PCR stumulation is among the frst
`effector Himenons Tost mr exhausted To cells. We theretore
`
`
`A, 4004-2 Control
`aae Antifinng Ar
`e- AnheD
`
`An fir T
`
`ime (a)
`
`Time (dj
`
`Effect of targeting the Tim-3 and PD-1 signaling pathways on tumor
`Figure 5,
`Mie® wer
`Alfie mice
`qowithl
`a
`m-3, anti-PD-L1, anti-Tim
`eres
`NM Tw
`ndependent experiments
`are shown, Left, contral (
`
`5) Right, contol
`ir
`Hooeti
`Time
`fe 5],
`ante PDL (re
`4]
`ant
`Orne
`ent
`SEM.
`led
`necontro, Or
`
`
`
`
`|
`
`\
`
`|
`
`
`
`

`

`IFN y
`
`a Anti-Tim-3 Ab
`CO Anie POL Ab
`CO Anti Tien-3 Ab + Ant-PD-L1 Ab
`
`
`
`1750
`
`
`
`imi) 1o00
`tokine(p 2
`\Cy 500
`
`1500 1250
`
`TILS#Y
`
`TILs#2
`
`Tksw3
`
`Figure 6. Blockade of the Tim-3 and PD-1 signaling pathways
`restores IFN-y production. [Ils were harvested from CT26 tumor
`bearing mice and cultured in vitro in the presence of soluble ariti-CD3
`and anti-Tim-3, anti-PD-L1, anti-Tim-3 plus anti-PD-L1, of cantral im
`munoglobulins. After 96 h, culture supernatant was collected and IFN-y
`measured by cytometric bead array. Data are expressed as the difference
`mncytokine production over that observed in cultures with control immuno
`globulins. Data shown are from three independent TILs samples tram two
`independent experiments.
`
`to have blocking function in vive (Monney etal. 2002), anei
`Pl-LE antibody, anti-Fim-3 plus ant-PD-L 1 antibodies, or
`control nmamunoglobulins, We found that
`treatmenr with
`ano—Tin-3 alone had littl or no ctleet waned treatment wath
`antePD-LI alone showed a
`trend toward delived tumor
`crowth, but
`this varied between experiments and did) not
`reach stuustuical sinibreanee (Pie.
`Oo).
`Plowever. combined
`treamnene wath ante Dine
`4 dane Ph) DL resulted! ana dea
`mate reduction to tumor erowrh. warke
`SO! of the
`ieee es
`hibiting complete tumor resression Eideed,
`the mice trom
`the combed ane Pine
`4 pins Aa ee es | vroup thar
`esx
`hibited complete POLTESSTOT reTHed CHor Pree exer) ater
`rechallenge (anpublished clitajy Because
`(126 tumor
`e
`presses PIE E butnot Pin 4ig S52), we conmtrathoat tor ihe
`possibility that PIE) nobody could) howe dineen a
`hibitory etleets on tumor vrowelh We Cultured € 16 rune
`W the Presenue ofan PIE) or conrad Tminowhobwaliy
`vnd found that tamer protitenition was notattected (ip S34)
`We have also tested the etlect of ane
`dana
`4 plus aan Phd |
`trreatnent i nice being BLO tehmorie and found that nine
`POCCIVTNEE the combined treatment exhibit enhanced surviwsal
`relative tocontrolmmunovlobulin.nte bin a aranu Pht
`treated nice