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2B4 LAG3 CD160
PD-1
int
PD-1
hi
H-2
Dbg
p33
5941 58
50
7
82
93
187327
42
50
Supplemental Figure 1 Blackburn et al
PD-1
CD
44
Clone-13Day 30pi
Int
Hi
Supplemental Figure 1. Exhausted CD8+ T cell subsets have distinct inhibitory receptor co-expression profiles. (a) H-2Dbgp33+ CD8+ splenocytes from LCMV clone 13 infected mice (d30 p.i.) co-stained with anti-PD-1 and for other inhibitory receptors. The total CD8+ T cell population was used to gate on PD-1HiCD44Int and PD-1IntCD44Hi populations (top). Numbers indicate the percent of tetramer+ CD8+ T cells in the indicated quadrant (bottom). Data are representative of 3 independent experiments with 3-5 mice per experiment.
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Ly5.
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Supplemental Figure 2 Blackburn et al
64
73
87
15
99
25
85
2986
14
5
Inhibitory receptors expressed
a
b
24
Supplemental Figure 2. Concurrent expression of multiple inhibitory receptors is not dependent on TCR diversity. The simultaneous expression of multiple inhibitory receptors (PD-1, LAG-3, 2B4, and GP49) on P14 Ly5.1+ H-2Dbgp33+ CD8+ T cells from three infection groups was examined on d30 p.i. using Boolean gating analysis. (a) Individual populations were grouped according to the total number of inhibitory receptors expressed. (b) Single inhibitory receptor staining (PD-1, LAG-3, 2B4, and GP49) on P14 Ly5.1+ H-2Dbgp33+ CD8+ T cells from three infection groups examined on d30 p.i. Numbers are the percent of Ly5.1+ P14 cells expressing the indicated receptor. Data are representative of three independent experiments with 3-5 mice per experiment.
PD-1 2B4LAG-3 CD160
1
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Day 30ARM T1B Cl-13
-CD4Cl-13
Supplemental Figure 3 Blackburn et al
Inhibitoryreceptorsexpressed
Supplemental Figure 3. Pattern of co-expression of multiple inhibitory receptors. The simultaneous expression of multiple inhibitory receptors (PD-1, LAG-3, 2B4, and CD160) on H-2Dbgp33+ CD8+ splenocytes from the four infection groups was examined on d30 p.i. usingBoolean gating analysis as in Fig. 4. The outer rings represent a single inhibitory receptor and denote the population on which it is expressed. Data are representative of three independent experiments with 4-5 mice per experiment.
No Blk
-CD16
0
-PD-L1
-LAG-3
-CD48
0
10
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PD-1 int
No Blk
-CD16
0
-PD-L1
-LAG-3
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e ca
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No Tx -CD2
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No Tx -CD48
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2B4 Int 2B4 Hi 2B4 Int 2B4 Hi
a
b
c
P = 0.0263 P = 0.0161P = 0.0071 P = 0.0073
P = 0.0003
P = 0.0183
Supplemental Figure 4 Blackburn et al
Spec
ific
Lysi
s (%
)
IFN
-γ (%
)
Supplemental Figure 4. In vitro blockade of inhibitory receptors. (a) A VITAL killing assay was performed using gp33-41 pulsed targets and magnetic bead purified CD8+ splenocytes from LCMV clone 13 infected mice d30 p.i. The assay was performed in the presence or absence of anti-PD-L1 (clone 10F.9G2), anti-CD160 (clone CNX46-3), anti-LAG-3 (clone C9B7W), or anti-CD48 (clone BCM1) with an effector to target ratio of 4:1 for 18h. Representative of four independent experiments with n=3 mice per experiment (b) Splenocytes from clone 13 infected mice were incubated at 37 C for 5 h with or without anti-PD-L1, anti-CD160, or anti-LAG-3 (10ug/ml) and active caspase 3 was detected in the live cell subset by flow cytometry. Representative of two independent experiments with n=3-4 mice per experiment. (c) Splenocytes from clone 13 infected mice were stimulated with LCMV peptides (pooled) and incubated at 37 C for 5 h with or with out anti-CD48 or anti-CD2. Cytokine production was measured by ICS. Representative of two independent experiments with n=3 mice per experiment. Blockade of CD48 on functional LCMV-specific memory CD8+ T cells that are uniformly 2B4lo or 2B4int reduced cytokine production in this assay (data not shown).
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e ca
spas
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IgG
2b-PD
-L1-LA
G- 3
IFN-
TNF
3.34.0
3.73.0
Sup
plem
ental Fig
ur e 5 B
lackbu
rn et al
α-LA
G3 +
α-PD
-L1
Sup
plem
ental Fig
ure 5
. IFN-γ an
d TN
F co-p
rod
uctio
n fo
llow
ing
in vivo
PD
-L1, LA
G-3
or P
D-L1
plu
sLA
G-3
blo
ckade. D
ual IFN
-α an
d TN
F co-p
rod
uctio
n w
as assessed b
y ICS fo
llow
ing
2 weeks o
f in vivo
in
hib
itory recep
tor b
lockad
e. Stainin
g w
as perfo
rmed
on
the sam
e po
pu
lation
s as sho
wn
in Fig
. 6B
. D
ata are represen
tative of th
ree ind
epen
den
t experim
ents w
ith 5-7 m
ice per exp
erimen
t.
0 102 103 104 105
-LAG3
Isotype
PD-1(gated on DbGP276 tetramer+ CD8+)
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2750P = 0.0039
P = 0.0403
P = 0.1636
Isotype -PD-L160708090
100110120130140150160170
a
b c
Supplemental Figure 6 Blackburn et al
{In vivoTreatment
Iso
typ
e
PD-1
(MF)
LAG
3 (M
F)
α-P
D-L
1
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AG
-3
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-3
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Supplemental Figure 6. Change in inhibitory receptor expression following in vivo inhibitory receptor blockade. (a) PD-1 expression on H-2Dbgp276+ CD8+ T cells from mice treated with anti-LAG-3 or an isotype control. (b) PD-1 expression (expressed as mean fluorescence intensity) on H-2Dbgp276+ CD8+ T cells from mice treated with anti-LAG-3, anti-PD-L1, anti-LAG-3 plus anti-PD-L1, or an isotype control. (c) LAG-3 expression (expressed as mean fluorescence intensity) on H-2Dbgp276+ CD8+ T cells from mice treated with anti-PD-L1 or an isotype control. Expression of LAG-3 could not be assessed following LAG-3 blockade since the blocking and detecting antibody are the same. Data are representative of three independent experiments with 5-7 mice per experiment.
Rank GENE NAME DESCRIPTION Score0 PDCD1 PDCD1:programmed cell death 1 01 CCL3 424800.03 dnagil )fitom C-C( enikomehc:3LCC2 PTPN13 676720.0 31 epyt rotpecer-non ,esatahpsohp enisoryt nietorp:31NPTP3 CASP3 293820.0esaetorp enietsyc detaler-sisotpopa ,3 esapsac:3PSAC4 CD244 417930.04B2 rotpecer llec rellik larutan 442DC:442DC5 GP49A na 0.041366 NR4A2 387140.02 rebmem ,A puorg ,4 ylimafbus rotpecer raelcun:2A4RN7 TRG@ 352940.0sucol ammag rotpecer llec T:@GRT8 EEA1 986940.0Dk261 ,1 negitna emosodne ylrae:1AEE9 GPD2 640050.0)lairdnohcotim( 2 esanegordyhed etahpsohp-3-lorecylg:2DPG
10 .SEPT4 SEPT4:septin 4 0.05075411 FASLG 194260.0)6 rebmem ,ylimafrepus FNT( dnagil saF:GLSAF12 CD160 CD160:CD160 antigen 0.06257813 IFIH1 230360.01 niamod C esacileh htiw decudni norefretni:1HIFI14 TANK 90360.0rotavitca BKFN detaicossa-rebmem ylimaf FART:KNAT15 MDFIC 681360.0gniniatnoc niamod rotibihni ylimaf DoyM:CIFDM16 WBP5 118460.05 nietorp gnidnib niamod WW:5PBW17 PTGER4 59560.0)4PE epytbus( 4 rotpecer E nidnalgatsorp:4REGTP18 SH2D2A SH2D2A:SH2 domain protein 2A 0.06601719 4631408O11RIK na 0.07430820 NRP1 NRP1:Neuropilin 1 0.07434221 ISG20 806670.0aDk02 eneg detalumits norefretni:02GSI22 GPR56 368770.065 rotpecer delpuoc-nietorp G:65RPG23 CD7 CD7:CD7 antigen (p41) 0.07964424 1110067D22RIK na 0.08045325 CCL4 297280.04 dnagil )fitom C-C( enikomehc:4LCC26 GAS2 GAS2:growth arrest-specific 2 0.0870627 ENTPD1 285880.01 esalordyhohpsohpid etahpsohpirt edisoelcunotce:1DPTNE28 NR1I4 932290.04 rebmem ,I puorg ,1 ylimafbus rotpecer raelcun:4I1RN29 NFATC1 NFATC1:nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 0.09227730 RSAD2 622490.02 gniniatnoc niamod eninoihtem lysoneda-S lacidar:2DASR31 FGL2 FGL2:fibrinogen-like 2 0.09447332 COCH 739790.0)sumehpylop sulumiL( nilhcoc ,golomoh C rotcaf noitalugaoc:HCOC33 PBX3 943890.03 rotcaf noitpircsnart aimekuel llec-B-erp:3XBP34 GPR65 336890.056 rotpecer delpuoc-nietorp G:56RPG35 EOMES 517401.0)siveal suponeX( golomoh nimredosemoe:SEMOE36 LITAF 409801.0rotcaf FNT decudni-edirahccasylopopil:FATIL37 SERPINA3G na 0.11069638 CCRL2 283211.02 ekil-rotpecer )fitom C-C( enikomehc:2LRCC39 PTGER2 697411.0aDk35 ,)2PE epytbus( 2 rotpecer E nidnalgatsorp:2REGTP40 FYN 470511.0SEY ,RGF ,CRS ot detaler enegocno NYF:NYF41 LAG3 640611.03 eneg noitavitca-etycohpmyl:3GAL42 PERP 721021.0rotceffe sisotpopa 35PT ,PREP:PREP43 SPP1 345121.0)I nietorpolais enob ,nitnopoetso( 1 nietorpohpsohp deterces:1PPS44 TNFRSF1B 164221.0B1 rebmem ,ylimafrepus rotpecer rotcaf sisorcen romut:B1FSRFNT45 1300007C21RIK /// LO na 0.12272946 SFRS2IP 333521.0nietorp gnitcaretni ,2 hcir-enires/eninigra ,rotcaf gnicilps:PI2SRFS47 CD200 CD200:CD200 antigen 0.12637348 CXCL10 871821.001 dnagil )fitom C-X-C( enikomehc:01LCXC49 H2-T23 na 0.12819450 1810035L17RIK na 0.12917751 PENK1 na 0.13064252 IIGP1 na 0.13172453 AHR AHR:aryl hydrocarbon receptor 0.13281854 2010100O12RIK na 0.13282555 D17H6S56E-5 na 0.13301156 RNF11 RNF11:ring finger protein 11 0.13325157 LYCAT 957531.0esarefsnartlyca nipiloidracosyL:TACYL58 ADAM19 588731.0)ateb nirtlem( 91 niamod esanietorpollatem dna nirgetnisid a:91MADA59 CD9 CD9:CD9 antigen (p24) 0.14089360 ART3 ART3:ADP-ribosyltransferase 3 0.14247361 PGLYRP1 87241.01 nietorp noitingocer nacylgoditpep:1PRYLGP62 GM1066 na 0.14315763 IFIT1 552341.01 staeper editpepocirtartet htiw nietorp decudni-norefretni:1TIFI64 TCRB-J /// LOC381765 na 0.1462765 BC039093 na 0.14687266 TNFRSF7 880741.07 rebmem ,ylimafrepus rotpecer rotcaf sisorcen romut:7FSRFNT67 GZMK 20841.0)II esatpyrt ;3 emyznarg ,esaetorp enires( K emyznarg:KMZG68 MLLT3 MLLT3:myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila) 0.14909669 RGS16 300151.061 gnillangis nietorp-G fo rotaluger:61SGR70 IFIT3 681151.03 staeper editpepocirtartet htiw nietorp decudni-norefretni:3TIFI71 CBX6 CBX6:chromobox homolog 6 0.15298972 ITM2C 953451.0C2 nietorp enarbmem largetni:C2MTI73 ALCAM 874551.0elucelom noisehda llec etycokuel detavitca:MACLA74 BCL2A1A /// BCL2A1B na 0.155675 C330007P06RIK na 0.15737476 IRF8 119851.08 rotcaf yrotaluger norefretni /// 8 rotcaf yrotaluger norefretni:8FRI77 GP49A /// LILRB4 na 0.16075478 TRIM17 /// HIST3H2A na 0.16102779 IFI204 na 0.16111680 RBL2 RBL2:retinoblastoma-like 2 (p130) 0.1614181 ITGAV ITGAV:integrin, alpha V (vitronectin receptor, alpha polypeptide, antigen CD51) 0.16188882 CASP1 CASP1:caspase 1, apoptosis-related cysteine protease (interleukin 1, beta, convertase) 0.16206283 CTLA4 463261.04 nietorp detaicossa-etycohpmyl-T cixototyc:4ALTC84 EIF3S1 179261.0aDk53 ,ahpla 1 tinubus ,3 rotcaf noitaitini noitalsnart citoyrakue:1S3FIE85 SPG21 182461.0)emordnys tsaM ,evissecer lamosotua( 12 aigelparap citsaps:12GPS86 CAPZB 393661.0ateb ,enil-Z elcsum )tnemalif nitca( nietorp gnippac:BZPAC87 ACADL 386661.0niahc gnol ,esanegordyhed A emyzneoC-lyca:LDACA88 MX1 MX1:myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) 0.16804589 NDUFA5 795961.0aDk31 ,5 ,xelpmocbus ahpla 1 )enoniuqibu( esanegordyhed HDAN:5AFUDN90 RASA1 779071.01 )nietorp gnitavitca esaPTG( rotavitca nietorp 12p SAR:1ASAR91 SERPINB6A na 0.17275192 RCN1 884571.0niamod gnidnib muiclac dnah-FE ,1 niblacoluciter:1NCR93 TNFSF11 756571.011 rebmem ,ylimafrepus )dnagil( rotcaf sisorcen romut:11FSFNT94 HCCS 534671.0)esayl-emeh c emorhcotyc( esahtnys c emorhcotycoloh:SCCH95 CCR5 275771.05 rotpecer )fitom C-C( enikomehc:5RCC96 2310015N07RIK na 0.17758297 5830471E12RIK na 0.1796498 CPEB2 498971.02 nietorp gnidnib tnemele noitalynedaylop cimsalpotyc:2BEPC99 NUCB1 NUCB1:nucleobindin 1 0.179917
Supplementary Table 1 Blackburn et al
Supplementary Table 1 Nearest neighbor analysis
Supplemental Table 2 Blackburn et al
Supplemental Table 2 Flow Cytometry Antibodies Used
Molecule CloneCD8 53.6-7CD44 IM7PD-1 RMP1-30LAG-3 C9B7WCD160 CNX46-3CD244/2B4 2B4 and ebio244F4CTLA-4 Uc10-4F10-11 and UC10-4B9PIR-B 6C1GP49 H1.1KLRG1 2F1NKG2A 16a11NKG2D CX5BTLA 6F7CD62L Mel-14CD127 A7R34TNF MP6-XT22IFN-g XMG1.2CD107a 1D4BKi67 B56CD48 BCM1
Supplemental Table 3. Blackburn et.al.
LCMV H2b CD8 peptides Included in peptide pool GP33-41 √GP44-51 √GP70-77 √GP92-101 √GP118-125 √GP166-173GP221-228 √GP276-286 √GP365-372L156-163 √L313-320L338-346 √L349-357 √L455-463 √L663-671L689-697 √L743-751L775-782 √L1189-1196L1302-1310L1369-1377L1428-1435 √L1878-1885 √L2062-2069 √NP165-175 √NP205-212 √NP238-248 √NP396-404 √
