Supplementalgfigureg1gSchematicgdiagramgofgtumor1modellingg

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1 SChinjectionh F:LuchLCLsh IVhinjectionh T:cellsh Monitorhforhtumorh growthhandhxeno: reactivehgvhd GVLgexperimentg kcbgvsgpbgt1cellse Xeno1reactiveg experimentg kcbgvsgpbgt1cellse IVhinjectionh 5xh,N^6 T:cells Monitorhforh xenoreactivityh Correlationgbetweeng GVLgandgxeno1 reactivitygfollowingg PBgT1cellginfusion SChinjectionh p85hxh,n^6 F:LuchLCLs IVhinjection 5xh,N^6 PBhT:cells Monitorhforhtumorh growthhandhxeno: reactivity Allo1reactivegvsg anti1viralg experiment SChinjectionh F:LuchLCLs IVhinjection 5hxh,N^6 allogeneichvsh autologoushcbht:cellsh Monitorhforhtumorh growth Quantitygandg phenotypegofgtilsg aftergcbgandgpbgt1 cellginjection Differentialgkineticsg ofgtilsgaftergcbgandg PBgT1cellginjection Effectorgfunctionsg gainedgbygcbgandg PBgTILsgduringg tumorgregression SChinjectionh F:LuchLCLs IVhinjectionh T:cellsh Histologyhofhtumorh sectionshandhtilshforh CD4:CD8hratiohandh memory/effectorh differentiation SChinjectionh F:LuchLCLs SChinjectionh F:LuchLCLs IVhinjectionh T:cellsh IVhinjectionh T:cellsh CD4:CD8handh CD8:T:reghratio TILshstudiedhforhgainh ofheffectorhfunctionh afterhonsethofhtumorh regression:hifn:y4h TNF:a4hperforinhand Th,/Thphbalanceh Dayg12 DaygS Day01S Day015 DaygS Day6 Supplementalgfigureg1gSchematicgdiagramgofgtumor1modellingg Day06S CD4:CD8handh CD8:T:reghratio

2 RGB^colour^image^^^^^^^^^^^^^^^^^^^^^^^Black^and^white^mask^image^^^^^^^^^^^^^^^^^^^^Out-line^image^^^^^^^^^^^^^^^^^^^^^^^ Out-lined^area^=^ ^sq.^microns T-cell^area^=^54.665^sq.^microns 172^cells/ ^sq.microns 1817^cells/mm^2 Out-lined^area^=^ ^sq.^microns T-cell^area^=^54.665^sq.^microns 329^cells/ ^sq.microns 3476^cells/mm^2^ Supplementalbfigureb2.bTheb10bRGBbimagesbofb40xbmagnificationbforbeachbtumorbslidebwerebconvertedbtobblackbandb whitebmaskboutputbusingbabcalibratedbthresholdbforbeachbstackbofbimages.bthebblackboutputbareasbcorrespondingbtob thebcd3bimmuno-histochemicalbstainingbdefinedbinbthebmaskbwereboutlinedbandbmeasuredbinbsquarebmicrons.b RepresentativebRGBybmaskbandboutlinebimagesbofbPBbandbCBbgroupbarebshownbinbthisbfigure.bThebnumberbofb infiltratingbt-cellsbinbeachb40xbimagebofb94901bsquarebmicronsbwerebcalculatedbasb=btotal out-lined area/area of Tcell. ThebnumberbofbinfiltratingbT-cellsbperbmm^2bwasbderivedbforbeachbimagebtobdeterminebthebaveragebdensitybofbTcellsbperbmm^2bofbtumor.bb

3 Supplementalqfigureq3qshowsqcumulativeqtumorqvolumesq(meanqandqstandardqerrorqofqmean)qfromqadditionalqfunctionalqexperiments.qTheq tumorqvolumesqwereqsignificanltyqlowerqinqtheqmiceqinjectedqwithqcbqtqcellsqcomparedqwithqthoseqwhoqreceivedqequivalentqnumbersqofqpbqtq cells.q

4 4 (A) 4 (B) Supplemental-figure.-4-CA6-shows-onset-of-weight-loss-three-weeks-after-PB-T-cell-injections y-weight-loss-occurred-by-four-weeks-in-the-PB-group-andhence-these-mice-were-sacrificed.-No-weight-loss-was-observed-in-mice-receiving-CB-T-cells-up-to-6-weeks-after-the-injection-of-T-cells.-CB6-Haematoxylinand-eosin-sections-of-liver-and-skin-of-mice-receiving-PB-T-cells-show-infiltrating-lymphocytes-around-the-bile-duct-and-in-the-intra-dermal-region.Cleaved-caspase-3-staining-was-apparent-at-the-site-of-lymphocytic-infiltration-confirming-apoptosis-of-bile-duct-epithelium-and-keratinocytespresent-in-epidermis-and-hair-follicle.-CB-T-cells-were-not-observed-to-infiltrate-either-liver-or-skin.--

5 5h%A/ 5h%B/ Supplementalhfigureh5hillustrateshGVLheffecthmediatedhbyhPBhThcellsh %5hmillion/hagainsthahlowerhburdenhofhBXcellhlymphomah%8:5hmillionh tumorhcells/handhahcorrelationhbetweenhxenoreactivityhandhtumorh regressionhfollowinghpbhtxcellhinjection:h%a/hshowshserialhweeklyh weighthmeasurementshofhmicehplottedhashjhofhbaselinehweighth%meanh andhstandardherrorhofhmean/honhrighthyxaxishandhbioluminescencehofh tumorhmeasuredhashphotons0sec0cm^80srh%meanhandhstandardherrorh ofhmean/hplottedhonhlefthyxaxis:h Inhthishexperiementhwithhlowerhburdenhofhtumorhcells,htumorh regressionhcorrelatedhwithhthehonsethofhhweighthloss,hanhobjectiveh measurehofhxenoreactivity:hthishexperimenthsuggestshthathpbhthcellsh alsohmediatehtumorhregressionhbuththisheffecthmayhbehattenuatedhandh typicallyhoccurshwithhthehonsethofhxenoreactivity:h%b/hshowshtumorh bioluminescencehmeasuredhashphotons0sec0cm^80srhusinghxenogenx IVIS:hAllhimagesharehshownhonhonehscaleh%range:h8:66e6htoh1:66e8h photons0sec0cm^80sec/:

6 SupplementalPfigureP6PshowsP representativepcolorp3dpinspectorpimagesp ofp10xpmagnifiedptumorpsectionspstainedp withpfoxp3pimmuno-histochemicalpstain.p ThesePimagesPshowPFoxp3+PcellsPinPtheP PBPPandPCBPgroupPonPdayP+10.P However,PonPdayP+20PthesePFoxp3+P cellsponlyppersistedpinptheppbpgrouppandp nopobviouspinfiltrationpofpfoxp3+pcellsp waspdetectedpinpthepcbpgroup.

7 7L.A2 7L.B2 7L.D2 7L.E2 SupplementalLfigureL7L.A21L.B2LandL.C2LshowLrepresentativeLflow/ cytometryldotlplotsloflil/41lifn/yl andltnf/alsecretinglcblandlpbl CD4ELTILsLrespectively6L.D2LshowsLsignificantlyLhigherL percentageloflcd4elpbltilslwerel IL/4LsecretingL.plottedLasLmeanL andlstandardlerrorloflmean2landl hencelth2lbiasedlcomparedltol CD4ELCBLTILs6L.E2LandL.F2LshowLhigherLTh1/Th2L ratiol.plottedlaslmeanlandl standardlerrorloflmean2linlthelcbl TILsLcomparedLtoLPBLTILs6L.Th1/Th2LratioLwasLcalculatedLasL thelratioloflifn/y/il/4landltnf/a/ IL/426 7L.C2 6L.F2

8 Supplemental methods 1) IFN-γ, TNF-α, IL-4 responses TILs were separated from lymphoma cells using CD20 microbeads (Milteyni Biotec). Isolated TILs were then re-stimulated at 37 C for 6 h with lymphoma cells from the same donor at the effector:target ratio of (1:10) in the presence of 10 µl of Brefeldin A (1 mg/ml) in 1 ml of RF-10. TILs were then surface stained with mouse anti-human antibodies, including APC-Cy7-conjugated CD3, BV605-conjugated CD4, and BV421-conjugated CD8. Following fixation and intracellular permeabilization with the ebioscience buffer set ( ), TILs were stained for PE-conjugated INF-γ, FITC-conjugated TNF-α, and APCconjugated IL-4. 2) Perforin expression Unstimulated TILs were surface stained with mouse-antihuman antibodies, including APC- Cy7-conjugated CD3, BV605-conjugated CD4, and BV421-conjugated CD8 for 20 mins. For intracellular perforin staining, TILs were fixed with 1% PFA in PBS for 20 min at room temperature and incubated 10 min with BD perm/wash buffer 1X (BD Biosciences). Cells were washed twice with BD perm/wash buffer 1X and then stained with PE-conjugated mouse anti-human perforin mab (δg9, IgG2b; BD Biosciences) in BD perm/wash buffer 1X for 30 min at room temperature. Unbound antibodies were removed by washing with perm/wash buffer 1X and fixed again with 1% PFA. 3) Naïve-memory-effector differentiation Unstimulated TILs were surface-stained with mouse anti-human antibodies, including APC- Cy7-conjugated CD3, BV605-conjugated CD4, BV421-conjugated CD8, PerCP-Cy5.5- conjugated CD45RO, and APC-conjugated CCR7.

9 4) FoxP3 staining Unstimulated TILs were surface stained for mouse anti-human antibodies, including FITC-conjugated CD4, PE-conjugated CD8, and BV421-conjugated CD25. Following fixation and intra-cellular permeabilization with the ebioscience FoxP3 buffer set ( ), TILs were stained for APC-conjugated FoxP3. T-regulatory cells were identified as CD4+CD25+FoxP3+ cells using a gating strategy based on CD4+ CD25- non-tregs. 1 5) Th1/Th2 TILs CD4+ TILs were identified as IFN-γ or TNF-α secreting Th1 cells and IL-4 secreting Th2 cells. The Th1/Th2 balance of CD4+ TILs was quantified using IFN-γ/IL-4 and TNFα/IL-4 ratios. References 1) Law JP, Hirschkorn DF, Owen RE, et al. The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cells. Cytometry A Dec;75(12):

10 Supplemental table 1: CB T cells vs LCLs and PB T cells vs LCLs show a similar degree of HLA-mismatch in the two experiments. All 10 HLA antigens and 7 of 10 HLA antigens were mismatched in experiment 1 and 2 respectively. HLA A HLA B HLA C HLA DRB1 HLA DQB1 CB donor PB donor LCL donor CB donor PB donor LCL donor Supplemental table 2: The representative CD3+ population obtained after negative selection is shown as APC-Cy7 flourescence. Cell debris were excluded from the analysis based on scatter signals. The purity of T cells obtained after negative selection was > 94% in the two primary experiments. CB PB Expt % 94.6% Expt % 94.5%