Immudex ApS
 
작성일 : 13-04-21
[Immudex ApS] MHC Dextramer® reagents
The ultimate reagent for detection of antigen-specific T cells in blood and solid tissue
 
MHC Dextramer® reagents
High avidity, brightness and excellent resolution make MHC Dextramer® reagents the superior choice for detection of
antigen-specific T-cells in flow cytometry and immunohistochemistry
 
 
T cell receptor(TCR)는, 특정 MHC 분자에서 표출되는 항원 특이적인 peptide를 인식합니다. 이를 이용해 같은 특이성의
TCR을 갖는 별도의 T 세포 집단을 검출 및 분리 할 수 있는 기술 개발 되었으나, MHC 복합체와 TCR에서는 매우 affinity가 
낮고, 충분한 결합 강도와 검출을 얻는 것은 쉽지 않았습니다. 형광표지 한 multimer의 MHC 시약이 개발되어 항원 특이적
T 세포의 검출과 정량이 가능하게 되었습니다.
MHC Dextramer®는 최적 분자수의 MHC와 형광색소를 갖는 dextran 골격으로 구성됩니다. MHC 분자는 dextran 골격에 끈으로 연결된 목걸이처럼 정렬되어 있습니다. 또한 MHC Dextramer®는 monomer MHC 분자에 비해 높은 TCR 결합 affinity를
나타내는 multimer의 시약입니다. 이 높은 결합 affinity로 결합 활성이 강화됩니다.
MHC Dextramer®는 감염증이나 종양, 백신접종 등 세포성 면역반응에 있어서 항원 특이적 T 세포의 Identification,
enumeration 및 tracing에 적합합니다.
 
  Schematic drawing of the MHC Dextramer
 
 
Performance of MHC Dextramer® reagents
 
MHC Dextramer® reagents are used in the study of antigen-specific T cells. High avidity, brightness and excellent
resolution make MHC Dextramer® reagents the superior choice for detection of antigen-specific T-cells in flow cytometry and immunohistochemistry.
 
Examples of Dextramer performance are listed below.
 
The MHC Dextramer™ reagents come with any of three different flurochromes (PE, APC or FITC). When using flow
cytometry they may be used to accurately monitor CD8+ T-cell responses in blood, CSF or other fluid cell samples.
 
Below the benefits of using Dextramer™ reagents compared to conventional MHC multimers are shown.
  
 
MHC Dextramers can detect low-affinity antigen-specific T cells, Tetramers cannot
 
 
A, B, and C: Flow cytometry analysis of a cell sample constituting 99% "negative" HPBMC cells spiked with 1% cells of a
T- cell line specific for the multimer reagents. Cells were stained with either Tetramers or Dextramers carrying pMHC with A)
low affinity for TCR, B) medium affinity for TCR, or C) no affinity for TCR (negative control). Gated cells; live singlets,
CD3+, CD14- and CD19-. D) Signal intensity of the T-cell line when stained with the respective MHC multimers. Cell count
and multimer signal is shown for each of the three multimer specificities. Given KD-values are for monomeric pMHC-TCR
interaction as measured by SPR.
(Preliminary data kindly provided by independent UK lab)
 
 
Dextramers have stronger signals, less background staining
 
MHC Dextramer reagents come with either FITC, PE or APC label. Each of them gives rise to a strong signal with low
background staining:
 
 
Ficoll-purified human peripheral blood mononuclear cells (HPBMC) were stained with Dextramer™, Tetramer and
Pentamer according to each product's recommended procedures. Gating strategy: CD4-, CD14- and CD3+. As may be
seen, MHC Dextramer™ reagents provide the highest resolution yet has the lowest background staining.
 
 
Dextramers stain 10x stronger
 
 
An approximately 10-fold brighter staining of a human T cell clone specific for the HLA-A2 restricted HY derived epitope
FIDSYICQV is observed when stained with HLA-A2(FIDSYICQV)/PE Dextramer™ than with HLA-A2(FIDSYICQV)/PE
Tetramer.
(The data was kindly provided by David Lissauer, Karen Piper, Professor Mark Kilby and Professor Paul Moss,
  Birmingham University, Birmingham, UK)
 
  
Dextramers detect antigen-specific T cells in situ
 
Due to their high avidity, flexibility and brightness MHC Dextramers are particularly well suited for in situ detection of
antigen-specific T-cells using immunohistochemistry (IHC), e.g. in tissue sections from solid tumors as shown below.
 
 
Survivin-specific cytotoxic T cells were detected in biopsies of metastatic lesions from a melanoma patient. Cryopreserved
sections were dried, fixed in acetone, incubated with TRITC-labeled anti-CD8 antibody followed by incubation with FITC-
labeled HLA-A2(survivin), Dextramer, and finally nuclei counter-stained with DAPI. A), B) and C) shows stained nuclei,
stained CD8+ cells and stained antigen-specific T cells, respectively. D) is an overlay of A, B and C.
 (The data was kindly provided by Professor Jürgen Becker, University of Würzburg, Würzburg, Germany)
 
 
Monitoring Cancer-specific T cells
 
Tumor-specific T-cell cells often display T cell receptors with low-affinity for their target MHC-peptide complex. MHC
Dextramer® reagents are particular superior for detection of low-affinity T cells. Below an example of Dextramer staining
of Mart-1-specific T cells.
 
 
Mart-1(A*0201 ELAGIGILTV)- specific T cells were detected with tetramers and Dextramers.  Tumor-Infiltrating
Lymphocytes were stained with Mart-1 multimer, anti-CD3, anti-CD4 (dump) and anti-CD8. Using Dextramers the specific
cells were detected with median fluorescence and resolution 3 times higher than for tetramers.
 
 
Analyzing multiple specificities in same sample
 
With MHC Dextramers it is possible to stain with high numbers of Dextramers in the same tube. Pools of Dextramers
covering numerous alleles and multiple epitopes for each allele can be applied for staining in a single tube. An example
with 8 Dextramers in a pool is given below. The number of Dextramers per pool may be increased even further.
A positive population may be identified in single color pools with up to 15 Dextramer reagents in the same tube.
 
 
HPBMCs from a positive donor were stained with the cognate Dextramer, either as a single Dextramer staining or in a
pool of 8 Dextramers (comprising the cognate Dextramer and 7 Dextramers that were negative on the donor). In both
the single and in the pool Dextramer staining reaction a positive population of 0,4% of the CD3+ cells was detected.
 
The number of Dextramers per pool can be increased even further by combining Dextramers with different labels.
Background staining from Dextramers in one color does not interfere with Dextramer staining in another color.
 
 
Applications
 
 항원 특이적 T cell response monitoring
 
   - 암 백신 개발
 
   - 암 면역요법 연구
 
   - 임상실험
 
 항원 특이적 면역상황 결정
 
   - 이식 환자
 
   - 면역 결핍 환자 (예를 들어 HIV 환자)
 
   - 면역 억제제를 투여하는 환자
 
 감염증, 암, 자가면역질환이나 악성종양의 T 세포 반응 profiling
 
 면역연구에 있어서 항원 특이적 T 세포 반응의 검출
 
 T 세포 clone의 특이성 확인
 
 T 세포 면역요법의 validation
 
 
 
 
Ordering information:  
 
 
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