Dextramer teachology slides (PDF)Dextramer技术的全面介绍 (20页 ppt)

MHC Dextramer TM technology

Improving immune monitoring

Liselotte Brix, PhD

COO, Immudex

Agenda

Dextramer structure and function

Detection of low-affinity interactions Stability of MHC Dextramers Detection of CD4 + antigen-specific T cells Detecting antigen-specific T cells in situ

Traditional MHC multimers

?Detection of low-affinity TCR-MHCp interactions:–

Cancer-specific CD8+ T cells –Antigen-specific CD4+ T cells

?

Low reproducibility ?

Reagent-stability Shortcomings

Detection of antigen-specific T cells ?Isolated MHC Multimer

specific population

?Easy to enumerate ?No clear MHC Multimer

specific population

?Difficult to enumerate MHC multimer

A n t i -C D 8

More MHCs, stronger cooperative binding

Benefits

?Dextramers have 10-100 fold higher

avidity than Tetramers ?The Dextramer is a flexible ”ball of yarn” that can adapt (unfold-refold) around its target, to position the MHC monomers for optimal interaction with TCRs, and lock the structure

CTL TCR

MHC

Fluorochrome

Dextramer

T? >> 10 min

(hours?)MHC monomer

T? ~ 10 sec MHC dimer

Tetramer

T? ~ 10 min

Pentamer

Detection of Tumor Infiltrating Lymphocytes

C D 8 /P a c i f i c B l u e Neg.control /RPE A*0201 ELAGIGILTV /RPE Dextramer

Tetramer Resolution: 373Median fl.: 1104Resolution: 131

Median fl.: 301

Resolution using Dextramers is 3 times as high as for Tetramers.

Median fluorescence using Dextramers is three

times higher compared to tetramers

Staining Mart-1 -specific T cells

Dextramers stain 10 times stronger than Tetramers

Tetramer vs. Dextramer

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 product’s

protocol

?

Dextramer stains antigen-specific T

cells 10x stronger ?Data kindly provided by David Lissauer, Professor

Paul Moss’ lab, Birmingham University, UK

Tetramer-PE Dextramer-PE Tetramer Dextramer Dextramers are particularly superior

reagents for detection of T cells that have

low signal intensity C D 8

What is the lowest affinity of the MHC-TCR interaction that

allows detection of Ag-specific T cells?

Multimers with peptide variants A: Kd ≈ 4 μM wt: Kd ≈40 μM B: Kd ≈100 μM C: Kd ≈250 μM D: Kd > 500 μM Fl Fl Fl

Fl Fl

Experimental 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 according to recommended

protocols

?Kd refers to the interaction of theTCR with

MHC monomer Fl

Dextramers can stain T cells of very low affinity

(MHC-TCR interaction: Kd = 500 μM)

Data

Comments Dextramer-PE Tetramer-PE Tetramers

?Significant separation for affinities

higher than 100 μM

?Detectable staining down to 250 μM

Dextramers ?Significant separation for affinities higher than 250 μM ?Detectable staining down to 500 μM

A (4 μM)

wt (40 μM)

B (100 μM)

C (250 μM)

D (> 500 μM)

Neg. control

Dextramers detect T cells that Tetramers cannot

99% neg HPBMC + 1% pos CTL clone100 % CTL clone

A

Low affinity

MHC/TCR

interaction

K D~250 μM

Tetramer Dextramer

C

D

8

B

Medium affinity

MHC/TCR

interaction

K D~40 μM

Tetramer-PE Dextramer-PE

wt (40 μM)

C (250 μM)

Neg. control

?Low affinity: T cells are only

detected by Dextramers

?Medium affinity: T cells are also

detected by Tetramers but with

lower resolution

Dextramers are particularly superior

reagents when working with low affinity

TCR-MHC interactions

Tetramer Dextramer

C

Negative control

Dextramer stability at 4°C

Test and conclusion

Data ?Incubate CMV-specific Dextramers

(APC, PE or FITC label) at 4°C for 12 months

?Test by flow cytometry (resolution, percent positives).

?

Dextramers (APC, PE, FITC) are fully functional after 12 months at 4°C ?Different Dextramers have different stability depending on MHC-peptide dispayed T = 0T = 12 mths

% Positive 0.49%0,43 %

Resolution 772

707Month T = 0 T = 12APC log APC log

Some Dextramers withstand repeated freeze-thaw cycles Test and conclusion Data

?CMV-specific Dextramers(APC, PE, FITC)

?10 Freeze/thaw cycles: -20°C <> 20°C

?Test by flow cytometry(resolution, percent

positives)

?APC-and FITC Dextramers:

Fully functional after4 freeze-thaw cycles

?PE Dextramer:

Functional but lower resolution, more

background

?Storage at 2-8°C is recommended

Before and after repeated freeze-thaw

FITC

PE

Conjugation to dextran stabilizes protein

conformations dramatically

Products under development at Immudex

Products 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 have 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 on CD1d with low affinity ?Dextran is likely to stabilize class II MHC/peptide complexes ?High avidity circumvents the problem relating to

the low-affinity MHC-TCR interaction ?Dextramer flexibility and many MHC molecules enable detection of multiple TCR’s despite:–Missing TCR patch formation i fixed tissue –Many denaturated TCRs/ cell

?More fluorochromes increase signal

?High avidity circumvents the problem relating to the low-affinity MHC-TCR interaction

Mouse class II Dextramers stain better than Tetramers US collaborator -prel. data Dextramer –Tetramer comparison

Dextramers are superior class II reagents C

D

4

Tetramer class II

Dextramer class II

C

D

4

Positive control Negative control

?Class II Tetramer-experienced lab

?Lymph node cells from immunized mice

?Dextramers have less background ?Dextramers have higher staining intensity

?Dextramers detect more positives Tetramer Dextramer

Human class II Dextramers detect T cells that Tetramers

(almost) cannot

UK collaborator -prel. data

Dextramer –Tetramer comparison Dextramers 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 distinguish positives from negatives –Dextramers: Positives would be well

separated from negatives Unstained Neg. control HA Tet. Unstained Neg. control HA Dex Tetramer/Dextramer-PE

C D 4-A P C No Tet/Dex +MAGE Tet

+MAGE Dex +HA Tet +HA Dex 1611557491611603810% o f M a x .

Tetramer-PE Dextramer-PE

A B 3883091

In situ

staining –MHC Dextramers gives bright staining Context and principle

Data Prof. Jürgen Becker, University of Würzburg:

?

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 microscopy

Nuclei CD8+Survivin-

specific T cells Overlay MHC Dextramers with more MHC and more fluorochromes are being tested!!

Summary

Improved immune monitoring can be obtained using MHC Dextramers –MHC I Dextramers detect low-affinity T

cells previously undetectable by traditional

multimer technologies

–MHC II Dextramers detect T cells that

Tetramers cannot (preliminary

data)

–Dextramers are strong reagents for in situ

staining

–Dextramers have high stability

–High reagent consistency and assay

reproducibility Dextramers are particularly advantageous in the following fields:?Enumeration and sorting of low-affinity Ag-specific CD8+ cells ( e.g. cancer)?Immune monitoring of cancer vaccine clinical trials ?Monitoring CMV-specific immune status ?Enumeration and sorting of Ag-specific CD4+ cells

?

Detection of T cells in situ

Dextramer Publications

1. Goodyear OC, Dennis M, Jilani NY, Loke J, Siddique S, Ryan G, Nunnick J, Khanum R,

Raghavan M, 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

2. Uzana R, Eisenberg G, Sagi Y, Frankenburg S, Merims S, Amariglio N, Yefenof E, Peretz T,

Machlenkin A, Lotem M (2011) Trogocytosis is a gateway to characterize functional

diversity in melanoma-specific CD8+ T cell clones J Immunol 2012;188;632-640, online Dec 7, 2011; doi:10.4049/jimmunol.1101429

3. Kim Y-H, Faaij CMJM, van Halteren AGS, Schrama E, de Jong TAM, Sch?ller 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 BBMT, online 4 Nov 2011, DOI:

10.1016/j.bbmt.2011.10.038

4. Hofmann C, H?fflin S, Hückelhoven A, Bergmann S, Harrer E, Schuler G, D?rrie 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/Blood-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, Peppa 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, Beaudoin 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 (2011) Interrogating the repertoire: broadening the

scope of peptide-MHC multimer analysis Nature Reviews Immunology 11, 551-558, Aug 2011, doi: 10.1038/nri3020

9. M?rner A, Jansson M, Bunnik EM, Sch?ller J, Vaughan R, Wang Y, Montefiori DC; Otting N,

Bontrop R, Bergmeier LA, Singh M, Wyatt RT, Schuitemaker H, Biberfeld G, Thorstensson R, Lehner T (2011) Immunization with Recombinant HLA Classes I and II, HIV-1 gp140, and SIV p27 Elicits Protection against Heterologous SHIV Infection in Rhesus Macaques J Virol, July 85(13)6442-52, doi: 10.1128/JVI.00129-11

10. Fierabracci A (2011) The potential of 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, doi: 10.4049/jimmunol.1001251

12. Yoshikawa T, Nakatsugawa M, Suzuki S, Shirakawa H, Nobuoka D, Sakemura N, Motomura Y,

Tanaka Y, Hayashi S, Nakatsura T (2011) HLA-A2-restricted glypican-3 peptide-specific CTL clones induced by peptide vaccine show high avidity and antigen-specific killing activity against tumor cells Cancer Science May(102)918-925, doi: 10.1111/j.1349-

7006.2011.01896.x

13. Yong ASM, Stephens N, Weber G, Li Y, Savani BN, Eniafe R, Keyvanfar K, Kurlander R

Rezvani K, Barrett AJ (2011) Improved outcome following allogeneic stem cell

transplantation in chronic myeloid leukemia is 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 tumor 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