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Assessment of HumanRegulatory T cells (Treg) andOther Monitoring AssaysTheresa L. Whiteside, Ph.D.Slide 1

Treg subsets promote tumor escapefrom the host immune system Types of regulatory T cells :• nTreg: CD4 + CD25 high Foxp3 high•thymus derived•suppress immune responses against “self” by mechanisms involvingcontact inhibition•require exogenous IL-2 for function and expansion• Tr1 cells: CD4 + CD25 neg IL10 + TGF-β+1•induced in the periphery upon Ag presentation•suppress immune responses through IL-10 and TGF- β 1secretion• Th3 cells: are also inducible and Ag-specificsecrete TGF- β1and are IL-4 independent An increased frequency of Treg in the tumor and in the peripheral circulationof patients with cancer was previously reported by us and others: In patients with ovarian cancer, accumulations of Treg at the tumor site wereassociated with shorter survival (Curiel, Nat Med 2004)Slide 2

Methods for studies of TregIsolate CD4+CD25 high (S) and CD4+CD25 neg or CD8+CD25 neg T cells (R) fromPBMC of NC or patients with cancer bv single-cell sorting (FACS)Multicolor flow cytometry: phenotypeSuppressor function:CFSE-labeled R (+ OKT3+ IL-2) + Unlabeled S5-day co-cultureSuppression ofR proliferationLaser+ 7-AADFLOCA(Flow cytometry-basedcytotoxicity assay)Slide 3

Only CD25 high clones are strong suppressorsNormal ControlHNSCCAutologous CD4 + CD25 -RespondersEvents0%Events0%+ CD25 low R0 - cloneEvents0 30 60 90 CFSE1201 3 %*Events0 30 60 90 CFSE12029 %*Weaksuppressorclones+ CD25 interm. R0 - cloneEvents0 30 60 90CFSE1202 6 %*Events0 30 60CFSE90 1206 9 %*Strongsuppressor0 30 60 90 120CFSE0 30 60 90 120CFSEclones+ CD25 high. R0 - clone+ CD25 high R1 cloneEventsEvents36 %*0 30 60 90CFSE12079 %*EventsEvents8 7 %*0 30 60CFSE90 1209 7 %*CD25 highCTLA4+Foxp3+CD62L+0 30 60 90 1200 30 60 90 120CFSECFSE*% suppression relative to controlSlide 4

CD25 high nTreg are expandedin HNSCC patients vs. NC1.7%NC5%HNSCC3.53.0gated on CD4 + CD25 + T cellsCD25 high T cellsCD250.3%CD25 high2%% positive cellsCD25 high

Treg in TIL are stronger suppressors compared to Treg inPBMC (AD) in HNSCC patients10090***◊NC (n= 5)TIL (n= 10)80PBMC (n= 10)% Suppression70605040*p≤ 0.001◊p≤ 0.04430◊20100R alone(Aut. CD4 + CD25 - )1S:2R Ratio1S:5R RatioSlide 6

Phenotypic analysis of the CD4 + CD25 high T- cell fraction in PBMC ofHNSCC patients vs. NC% positive cells for Treg marker1009080706050403020Gated on CD4 + CD25 highNC (n=15)HNSCC (n=35)**** *** p≤ 0.0001****100CD62LGITRCD45ROFasCTLA-4Foxp3CD122CD132HLA-DRCCR4CCR7Foxp3,Fas,CTLA-4,CCR-4 and CCR-7 were significantly upregulatedon CD4 + CD25 high T cells in patients with cancer. GITRwas exclusively up-regulated on CD4 + CD25 high T cells in NC.Slide 7

uppressor function of CD4 + CD25 high T cells isolated from patients’ PBMC vs. NC10090NC (n=10)HNSCC (n=10)P≤ 0.00018070% suppression6050403020100NCHNSCCCD4 + CD25 highT cells isolated from HNSCC co-cultured cultured with autologousCD4 + CD25 - responder T cells almost completely inhibited their proliferation. on. Incontrast, Treg isolated from NC demonstrated weak suppressor function.Slide 8

Phenotypic characteristics of CD25 high nTregin different compartments (HNSCC patients)PBMCgated on CD3 + CD4 + 70%31%60%CD4+CD25highCD25highFoxp3+CD62L+CCR7+IL-10Foxp3TGF-β 1TIL72%93%96%CD4+CD25highCD25highFoxp3+GITR+IL-10+TGF10+TGF-β1+ 1+Slide 9

Treg detection in tissues: ImmunohistochemistryTumorFoxP3 +Slide 10

Percentages of CD4+CD25 high Treg in freshly harvestedTILor PBMC in HNSCC patients or controls and in tissueCD25 high expression (% positive cells)Gated on CD3 + CD4 + T cells16 Normal Donors/PBMC(n=10)P< 0.001141210P< 0.018*642*HNSCC/PBMC (n=25)HNSCC/TIL (n=15)1C.0ImmunofluorescenceSlide 11

Other monitoring assaysSlide 13

Lymphoid DC predominate in peritoneal LNof patients with ovarian cancerLSScControl PBMCCD3,14,19HLA-DRCD4AscitesFL1 FL2 FL3 FL4 FL5FITC PE ECD PC5 PC7CD11c CD123 CD3,14,19 HLA-DR CD4Multiparameter flowcytometryLNCD123CD123 CD12324%Slide 1468%CD11c13%75%CD11c65%28%CD11cSScSScSScFScFScFSc

Apoptotic CD8+ T cells in the nest oflymphocytes at the tumor site(TUNEL ASSAY)TumorRed = alive CD8+ T cellsBlue = dying CD8+ T cellsSlide 15

HLA class I peptide tetramers:Multiparameter flow cytometryStreptavidin-PE tetramer instead of APCTCRMHC/peptideX 4T cellSlide 18

ELISPOT: enumeration of cytokine-secretingsingle T cellsPBMC, CD4 + orCD8 + T cells + DC+ Ag placed on Ab1-coated well insertOver next 24-48hAg-specific T cellssecrete cytokineCaptured cytokinedeveloped with Ab2,enzyme, AEC“Footprints”Ab1 = capture AbAb2 = detection AbEnumerateSpotsA monolayer of singlecells on a NC membraneSlide 21(SFC + Antigen) – Control SFC= SFC /cells plated/ well

Multi-color flow cytometry for phosphorylatedSTAT1 levels in activated immune cells Sensitive, quantitative, fast, usesfew cells; measures early events1. Surface staining with anti-CD3 Ab2. Cell permeabilization3. Intracytoplasmic staining with Abto phosphorylated STAT1ActivationsignalLaserPSTAT1T cellSTAT1CD3LaserSlide 22

The CD107 de-granulation assayGranulecore withenzymesGranule membraneexpressing CD107a,CD107b and CD63Cell surface exposureof CD107a, CD107band CD63CytotoxicgranuleCellmembraneGranule is transportedto the cell membraneDe-granulation andrelease of contentsSlide 23

M BNKTMedium24hIn vivo activationSpontaneous cytokinesecretionSupernatants testedfor cytokines by LuminexMTBNKMedium+Ag24hAg-induced cytokinesecretionAbility to respond“Cytokine screen” with PBMCSlide 24

How The Bio-Plexprotein array system worksUp to 100 microspheresare in a bead set. Eachis color-coded andconjugated with a MAbspecific for a uniqueprotein analyteA flow-based instrumentwith 2 lasers andassociated opticsmeasures biochemicalreactions that occur onthe surface of thecolored microspheresA high-speed digitalsignal processorefficiently manages thefluorescent output.Slide 25LasersFluorescence Intensity (FI)30000.0020000.0010000. (21)8.465.260.0110.00 100.00 1000.00Concentration (pg/ ml)2.512.55reporterclassifier

Cytokines Chemokines & More Human– TNFα, IFNγ, TGF-β1, IL-1 Ra, IL-2 sR– IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7,IL-8, IL-10, IL-11 ,IL-12, IL-13, IL-15, IL-18– G-CSF, M-CSF,GM-CSF, EGF, FGF-7, SCF, MIG, VEGF, HGF– FLT-3, MCP-1, MIP-1α, MIP-1β, Rantes, IP-10, LIFInflammationTh-1/ Th-2AutoimmuneHematopoeisisG-CSFIFN-γIL-4IL-1bG-CSFIL-6IL-5TNF-αIL-6IFN-γIL-8IL-2IL-7TNF-αIL-1βTNF-αIL-12GM-CSFIL-12IL-6IL-13IL-18GM-CSFSlide 26

QC/QA issuesCellular assays are difficult to standardize:the inter-assay CV is usually >20%Thus, “batching” of serial samples todecrease in between-assay variabilityYou can only batch if cryopreserved/thawedcell = fresh cell in your own laboratorySOP and an assay, which is robust andvalidated for specificity, sensitivity, accuracy,precisionDocumentation that assay is in control forlarge-scale testingSlide 27

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