1、Improving immune monitoringLiselotte Brix, PhDCOO, ImmudexMHC DextramerTM technologyAgendaDextramer structure and functionDetection of low-affinity interactionsStability of MHC DextramersDetection of CD4 + antigen-specific T cellsDetecting antigen-specific T cells in situDextramers a new generation
2、of MHC multimers with more MHCsDextramer structure Flow cytometer principleDextramers bind and label antigen-specific T-cellsPeptide antigenMHC proteinFl FluorophoreDextranLaser beam fluorescenceFlFlde xFlFlde xFlFlde xFlFlde xFlFlde xFlFlde xFlFlde xFlFlde xCells and ReagentsflowFlow CytometerCells
3、 pass by a laser beam, one at a time, allowing detection of fluorescence-labelled cellsDextramer Tetramer PentamerFl FlFlFlFl FlFlTraditional MHC multimers Detection of low-affinity TCR-MHCpinteractions: Cancer-specific CD8+ T cells Antigen-specific CD4+ T cells Low reproducibility Reagent-stability
4、ShortcomingsDetection of antigen-specific T cellsIsolated MHC Multimerspecific populationEasy to enumerateNo clear MHC Multimerspecific populationDifficult to enumerateMHC multimerA nt i -C D8More MHCs, stronger cooperative binding Benefits Dextramers have 10-100 fold higheravidity than Tetramers Th
5、e Dextramer is a flexible ”ball of yarn” that can adapt (unfold-refold) around its target, to position the MHC monomers for optimal interaction withTCRs, and lock the structure CTLTCRMHCFluorochromeDextramerT 10 min(hours?)MHC monomerT 10 secMHC dimerTetramerT 10 minPentamerDextramers have higher re
6、solution and less background than conventional multimersComparison Experiment and conclusion CMV-specific detected in HPBMC Each multimer used according to itsrecommended protocol Gating: Live lymphocytes, CD3+, CD4-, CD14- Dextramers have less background and better resolutionDextramers allow more a
7、ccurateenumeration of antigen-specific T cells. Dextramers Highresolution LowbackgroundTetramers Lowresolution Medium backgroundPentamers Medium resolution High backgroundDetection of Tumor Infiltrating LymphocytesC D8 /P ac i fi c B lu eNeg.control /RPE A*0201 ELAGIGILTV /RPEDextramerTetramerResolu
8、tion: 373Median fl.: 1104Resolution: 131Median fl.: 301Resolution using Dextramers is 3 times as highas for Tetramers.Median fluorescence using Dextramers is threetimes higher compared to tetramersStaining Mart-1 -specific T cellsDextramers stain 10 times stronger than TetramersTetramer vs. Dextrame
9、r Experiment by David Lissauer T cell line specific for HY-derived epitope (mHAg) Presumed low affinity or low abundance of TCRs Staining according to each products protocol Dextramer stains antigen-specific T cells 10x stronger Data kindly provided by David Lissauer, Professor Paul Moss lab, Birmin
10、gham University, UKTetramer-PE Dextramer-PETetramer DextramerDextramers are particularly superior reagents for detection of T cells that have low signal intensityC D8What is the lowest affinity of the MHC-TCR interaction that allows detection of Ag-specific T cells?Multimers with peptide variantsA:
11、Kd 4 Mwt: Kd 40 MB: Kd 100 MC: Kd 250 MD: Kd 500 MFlFlFl FlFlExperimental set-up T cell line recognises the MHC/wt-peptide complex ”wt” is a tumor-derived peptide Different variants of wt peptide give different MHC-TCR affinity Stain with Tetramers and Dextramers carrying MHC-peptide variants Stain
12、according to recommended protocols Kd refers to the interaction of theTCR with MHC monomer FlDextramers can stain T cells of very low affinity(MHC-TCR interaction: Kd = 500 M)Data CommentsDextramer-PETetramer-PE Tetramers Significant separation for affinities higher than 100 M Detectable staining do
13、wn to 250 M Dextramers Significant separation for affinities higher than 250 M Detectable staining down to 500 M A (4 M)wt (40 M)B (100 M)C (250 M)D ( 500 M)Neg. controlDextramers detect T cells that Tetramers cannot99% neg HPBMC + 1% pos CTL clone 100 % CTL cloneALow affinity MHC/TCR interaction KD
14、250 M Tetramer Dextramer C D8BMedium affinityMHC/TCR interaction KD40 MTetramer-PE Dextramer-PEwt (40 M)C (250 M)Neg. control Low affinity: T cells are only detected by Dextramers Medium affinity: T cells are also detected by Tetramers but with lower resolutionDextramers are particularly superior re
15、agents when working with low affinity TCR-MHC interactionsTetramer Dextramer CNegative controlDextramer stability at 4CTest and conclusion Data Incubate CMV-specific Dextramers (APC, PE or FITC label) at 4C for 12 months Test by flow cytometry (resolution, percent positives). Dextramers (APC, PE, FI
16、TC) are fully functional after 12 months at 4C Different Dextramers have differentstability depending on MHC-peptidedispayedT = 0 T = 12 mths% Positive 0.49% 0,43 %Resolution 772 707MonthT = 0 T = 12APC log APC logSome Dextramers withstand repeated freeze-thaw cyclesTest and conclusion Data CMV-spec
17、ific Dextramers (APC, PE, FITC) 10 Freeze/thaw cycles: -20C 20C Test by flow cytometry (resolution, percentpositives) APC- and FITC Dextramers:Fully functional after 4 freeze-thaw cycles PE Dextramer: Functional but lower resolution, more background Storage at 2-8C is recommendedBefore and after rep
18、eated freeze-thaw FITCPEConjugation to dextran stabilizes protein conformations dramaticallyProducts under development at ImmudexProducts in development Why may new products be even better? MHC II Dextramers Most Class II MHC/peptide complexes are unstable Typically, Class II MHC/peptide complexes h
19、ave low affinity for TCR Dextramers optimized for in situ staining Stronger signals are desirable Detection in paraffin-embedded tissue sections CD1d Dextramers NK-TCR binds some glycolipids displayed onCD1d with low affinity Dextran is likely to stabilize class II MHC/peptide complexes High avidity
20、 circumvents the problem relating to the low-affinity MHC-TCR interaction Dextramer flexibility and many MHC moleculesenable detection of multiple TCRs despite: Missing TCR patch formation i fixed tissue Many denaturated TCRs/ cell More fluorochromes increase signal High avidity circumvents the prob
21、lem relating to the low-affinity MHC-TCR interactionMouse class II Dextramers stain better than TetramersUS collaborator - prel. data Dextramer Tetramer comparisonDextramers are superior class II reagentsC D4Tetramer class IIDextramer class IIC D4Positive control Negative control Class II Tetramer-e
22、xperienced lab Lymph node cells from immunized mice Dextramers have less background Dextramers have higher staining intensity Dextramers detect more positivesTetramerDextramerHuman class II Dextramers detect T cells that Tetramers(almost) cannotUK collaborator - prel. data Dextramer Tetramer compari
23、sonDextramers are superior class II reagents A: T cell line stained with class II MHC multimers Dextramers have high resolution Positive control Tetramer signal overlap with unstained control B: HPBMC spiked with T cell line Tetramers: Would be hard to distinguishpositives from negatives Dextramers:
24、 Positives would be wellseparated from negativesUnstained Neg. control HA Tet. Unstained Neg. control HA DexTetramer/Dextramer-PEC D4 - AP CNo Tet/Dex +MAGE Tet +MAGE Dex+HA Tet +HA Dex161 155 749 161 160 3810% o fMa x.Tetramer-PE Dextramer-PEAB388 3091In situ staining MHC Dextramers gives bright st
25、ainingContext and principle DataProf. Jrgen Becker, University of Wrzburg: Biopsy from lesion in a melanoma patient Frozen tissue sections Staining: HLA-A2/(Survivin)/Fitc Dextramer Anti-CD8 /TRITC DAPI (snuclei stain) Confocal laser fluorescence microscopyNuclei CD8+Survivin-specific T cells Overla
26、yMHC Dextramers with more MHC and more fluorochromes are being tested!SummaryImproved immune monitoring can be obtained using MHC Dextramers MHC I Dextramers detect low-affinity T cells previously undetectable by traditionalmultimer technologies MHC II Dextramers detect T cells thatTetramers cannot
27、(preliminary data) Dextramers are strong reagents for in situstaining Dextramers have high stability High reagent consistency and assayreproducibilityDextramers are particularly advantageous in the following fields: Enumeration and sorting of low-affinityAg-specific CD8+ cells ( e.g. cancer) Immune
28、monitoring of cancer vaccine clinical trials Monitoring CMV-specific immune status Enumeration and sorting of Ag-specific CD4+ cells Detection of T cells in situ1 WWW.IMMUDEX.COM Dextramer Publications 1. Goodyear OC, Dennis M, Jilani NY, Loke J, Siddique S, Ryan G, Nunnick J, Khanum R, Raghavan M,
29、Cook M, Snowden JA, Griffiths M, Russell N, Yin J, Crawley C, Cook G, Vyas P, Moss P, Malladi R, Craddock CF (2012) Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia. Blood, Jan 10, doi:101182/blood-2011-09-377044
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31、 3. Kim Y-H, Faaij CMJM, van Halteren AGS, Schrama E, de Jong TAM, Schller J, Egeler RM, Pavel S, Vyth-Dreese FA, van Tol MJD, Goulmy E, Spierings E (2011) In situ detection of HY-specific T cells in acute graft-versus-host disease-affected male skin after sex-mismatched stem-cell transplantation BB
32、MT, online 4 Nov 2011, DOI: 10.1016/j.bbmt.2011.10.038 4. Hofmann C, Hfflin S, Hckelhoven A, Bergmann S, Harrer E, Schuler G, Drrie J, Schaft N, Harrer T (2011) Human T cells expressing two additional receptors (TETARs) specific for HIV-1 recognize both epitopes Blood, Online Sep 16, DOI: 10.1182/Bl
33、ood-2011-04-347005 5. Massilamany C, Upadhyaya B, Gangaplara A, Kuszynski C, Reddy J (2011) Detection of autoreactive CD4 T cells using major histocompatibility complex class II dextramers BMC Immunol 12(1):40, online Jul 18, doi: 10.1186/1471-2172-12-40. 6. Schurich A, Khanna P, Lopes AR, Han KJ, P
34、eppa D, Micco L, Nebbia G, Kennedy PTF, Geretti A-M, Dusheiko G, Maini MK (2011) Role of coinhibitory receptor cytotoxic T lymphocyte antigen-4 on apoptosis-prone CD8 T cells in persistent Hepatitis B virus infection Hepatology 53(5)1494-1503, May 2011, doi: 10.1002/hep.24249 7. Diana J, Brezar V, B
35、eaudoin L, Dalod M, Mellor A, Tafuri A, von Herrath M, Boitard C, Mallone R, Lehuen A (2011) Viral infection prevents diabetes by inducing regulatory T cells through NKT cell-plasmacytoid dendritic cell interplay J.Exp.Med., 11 Apr 2011, doi: 10.1084/jem.20101692 8. Davis MM, Altman JD, Newell EW (2
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38、 multimer technologies in type 1 diabetes prediction strategies Diabetes Metab Res Rev. Mar;27(3):216-29, doi: 10.1002/dmrr.1165 11. Holst PJ, Christensen JP, Thomsen AR (2011) Vaccination against Lymphocytic Choriomeningitis Virus Infection in MHC Class II-Deficient Mice J.Immunol (186)3994-4007, d
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41、associated with higher expression of BMI-1 and immune responses to BMI-1 protein Leukemia Apr;25(4):629-37, doi: 10.1038/leu.2010.325 14. Kaida M, Morita-Hoshi Y, Soeda A, Wakeda T, Yamaki Y, Kojima Y, Ueno H, Kondo S, Morizane C, Ikeda M, Okusaka T, Takaue Y, Heike Y (2011) Phase 1 trial of Wilms t
42、umor 1 (WT1) peptide vaccine and gemcitabine combination therapy in patients with advanced pancreatic or biliary tract cancer J.Immunother Jan;34(1)92-9, DOI: 10.1097/CJI.0b013e3181fb65b9 2 WWW.IMMUDEX.COM 15. Bannard O, Kraman M, Fearon DT (2010) Cutting edge: Virus-specific CD8+ T cell clones and
43、the maintenance of replicative function during a persistent viral infection J.Immunol (185)7141-7145 16. Baba T, Sato-Matsushita M, Kanamoto A, Itoh A, Oyaizu N, Inoue Y, Kawakami Y, Tahara H (2010) Phase I clinical trial of the vaccination for the patients with metastatic melanoma using gp100-deriv
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