PMID- 20558172
OWN - NLM
STAT- MEDLINE
DCOM- 20101110
LR  - 20210103
IS  - 1872-7905 (Electronic)
IS  - 0022-1759 (Linking)
VI  - 360
IP  - 1-2
DP  - 2010 Aug 31
TI  - A flow cytometry-based assay to assess minute frequencies of CD8+ T cells by 
      their cytolytic function.
PG  - 56-65
LID - 10.1016/j.jim.2010.06.005 [doi]
AB  - Limited sample size and low sensitivity of currently used functional assays 
      challenge direct analysis of cytotoxic CD8+ T lymphocyte activity to quantify 
      antigen-specific immunity after infection or vaccination. Our flow 
      cytometry-based assay reproducibly detects at least three epitope-specific CD8+ T 
      lymphocytes by their cytolytic function. As exemplified for viral epitopes 
      restricted to the human leukocyte antigen (HLA)-A2, the HLA-A2+ human somatic 
      cell hybrid T2 provided an about 10-fold more sensitive readout as compared to 
      autologous B-lymphoblastoid cells or the human erythroleukemia cell line K562 
      transfected to express HLA-A2 when used as target cells. We named our assay 
      VITAL-FR assay, referring to Hermans et al. (2004) and indicating the 
      modification of using Far Red (FR) dye instead of CMTMR. Under optimal conditions 
      the VITAL-FR assay proved 30 times more sensitive than the 51chromium-release 
      assay to assess epitope-specific target cell lysis. The high overall sensitivity 
      of the VITAL-FR assay basically depended on the negligible spectral overlap of 
      the emission of a stable Far Red fluorescent reporter with the green tracer for 
      target cell labelling. It also profited from long co-incubation of effector and 
      target cells of up to 72, from prior in-vitro culture increasing the frequency of 
      epitope-specific CD8+ T cells and from generic, easily accessible standardized 
      target cells that were used with only 10(3) specific and 10(3) control target 
      cells per individual experimental reaction. Our functional approach with the 
      VITAL-FR assay therefore ideally suits for monitoring CD8+ T cell-mediated 
      cytotoxicity in e.g. vaccination studies with known MHC-restricted immunogenic 
      peptides in scientific and diagnostic applications.
CI  - 2010 Elsevier B.V. All rights reserved.
FAU - Stanke, Jonas
AU  - Stanke J
AD  - Gynecology, Gynecologic Tumor Immunology, Campus Benjamin Franklin and Mitte, 
      Charite-Universitatsmedizin, Berlin, Germany.
FAU - Hoffmann, Corinna
AU  - Hoffmann C
FAU - Erben, Ulrike
AU  - Erben U
FAU - von Keyserling, Helmut
AU  - von Keyserling H
FAU - Stevanovic, Stefan
AU  - Stevanovic S
FAU - Cichon, Guenter
AU  - Cichon G
FAU - Schneider, Achim
AU  - Schneider A
FAU - Kaufmann, Andreas M
AU  - Kaufmann AM
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20100615
PL  - Netherlands
TA  - J Immunol Methods
JT  - Journal of immunological methods
JID - 1305440
RN  - 0 (Fluorescent Dyes)
RN  - 0 (HLA-A2 Antigen)
SB  - IM
MH  - CD8-Positive T-Lymphocytes/immunology/*metabolism/pathology
MH  - *Cell Count
MH  - *Cytotoxicity Tests, Immunologic
MH  - Cytotoxicity, Immunologic
MH  - Flow Cytometry/methods
MH  - Fluorescent Dyes/metabolism
MH  - HLA-A2 Antigen/genetics/immunology/metabolism
MH  - Humans
MH  - K562 Cells
MH  - Lymphocyte Activation
MH  - Monitoring, Immunologic/methods
MH  - Reproducibility of Results
MH  - Sample Size
MH  - Sensitivity and Specificity
MH  - Transgenes/genetics
EDAT- 2010/06/19 06:00
MHDA- 2010/11/11 06:00
CRDT- 2010/06/19 06:00
PHST- 2010/01/21 00:00 [received]
PHST- 2010/05/20 00:00 [revised]
PHST- 2010/06/07 00:00 [accepted]
PHST- 2010/06/19 06:00 [entrez]
PHST- 2010/06/19 06:00 [pubmed]
PHST- 2010/11/11 06:00 [medline]
AID - S0022-1759(10)00170-5 [pii]
AID - 10.1016/j.jim.2010.06.005 [doi]
PST - ppublish
SO  - J Immunol Methods. 2010 Aug 31;360(1-2):56-65. doi: 10.1016/j.jim.2010.06.005. 
      Epub 2010 Jun 15.