Many thanks to all of you. Here is a long email with all of the tetramer
messages that weren't sent to the list:
******************
This assay is a peptide epitope-specific functional assay for antigen
specific T-cells. Normally this is done to detect virus-specific
(influenza, HIV, EBV ect) CD8+ T cells, but can be done for any
antigen. What one stains with a major histocompatibility complex class
I-peptide complex which forms a tetramer in solution. Normally the
domanant epitope of the antigenic peptide bound is bound to the
appropriate class of HLA. Since this mimics how the macrophage, B cell
or dendritic cell presents antigen, it can identify antigen-specific
cells. The binding affinity of the complex to the T cell is near
antibody-antigen complexes and is suitible for flow. Many different
papers for a lot of different antigens.
For a review of the technique see
Curr Opin Immunol 1998 Aug;10(4):393-6 HLA-peptide tetrameric
complexes. Ogg GS, McMichael AJ, Dunbar PR, et al.
Direct isolation, phenotyping and cloning of low-frequency
antigen-specific cytotoxic T lymphocytes from peripheral blood.
Curr Biol. 1998 Mar 26;8(7):413-6.
Murali-Krishna K, et al. Counting antigen-specific CD8 T cells: a
reevaluation of bystander activation during viral infection. Immunity.
1998 Feb;8(2):177-87.
Altman JD, et al. Phenotypic analysis of antigen-specific T
lymphocytes. Science. 1996 Oct 4;274(5284):94-6.
Harry D. Dawson, Ph.D.
Laboratory of Immunology
National Institute on Aging, NIH
5600 Nathan Shock Dr.
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The assay is not for antigen presenting cells but antigen specific
cells (mostly for CTLe). The technique is very new and you have to have
"connections" to get access to the reagents. This will change with the
opening of the NIH's tetramer faclilty. The webpage address for that is...
http://www.niaid.nih.gov/reposit/tetramer/index.html
Only one paper has been published using the technique for CD4 cells (
using MHC Class II tetramers). It was published by Mark Davis et al. in
Immunity. Many papers have been published by Andrew McMichael's group and
others using Class I tetramers. There was a review written by Andrew
McMichael and Graham Ogg about the assay in Current Opinion in Immunology
last year.
Craig Irwin
********************************************
Try these for tetramer assay:
Altman, J.D., Moss, P.A.H., Goulder, P.J.R., Barouch, D.H.,
McHeyzer-Williams, M.G., Bell, J.I., McMichael, A.J. and Davis, M.M. (1996)
Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94.
Callan, M.F.C., Tan, L., Annels, N., Ogg, G.S., Wilson, J.D.K.,
O'Callaghan, C.A., Steven, N., McMichael, A.J. and Rickinson, A.B. (1998)
Direct visualization of antigen-specific CD8+ T cells during the primary
immune response to Epstein-Barr virus in vivo. J. Exp. Med. 187, 1395.
Ogg, G.S., Jin, X., Bonhoeffer, S., Dunbar, P.R., Nowak, M.A., Monard, S.,
Segal, J.P., Cao, Y., Rowland-Jones, S.L., Cerundolo, V., Hurley, A.,
Markowitz, M., Ho, D.D., Nixon, D.F. and McMichael, A.J. (1998)
Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of
viral RNA. Science 279, 2103.
Alice Givan
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Please see ref: Science; Vol.274, 4, October 1996. This procedure is
described
by John Altman et al. The tetramers, because they and made with PE, are
actually very bright but because of the frequency you will need to collect at
least 100,000 events. I believe the NIH repository is offering samples for
analysis which we are currently using. Please see...www.niaid.nih.gov/repost/
tetramer/listing
Stephen P. Perfetto, MS.,MT. (ASCP)
Walter Reed Army Institute of Research
Department of Molecular Diagnostics and Pathogenesis
*************************************************************
This is in response to your question about the tetramer assay. The
technology was developed by John Altman at Emory Univ. The original paper
was in Science 274:94-96 (1996). Several of the investigators here at St.
Jude use the system to look for T cells specific for different viral
peptides. See papers by Peter Doherty, et. al.
Richard Cross, Ph.D
Dept of Immunology Flow Cytometry Facility
St. Jude Children's Researc Hospital
Memphis, TN
***************************************************
Science 1996 Oct 4;274(5284):94-6
Published erratum appears in Science 1998 Jun 19;280(5371):1821
Phenotypic analysis of antigen-specific T lymphocytes.
Altman JD, Moss PAH, Goulder PJR, Barouch DH, McHeyzer-Williams MG, Bell JI,
McMichael AJ, Davis MM
Department of Microbiology and Immunology, Stanford University School of
Medicine, Stanford, CA 94305-5428, USA.
Identification and characterization of antigen-specific T lymphocytes during
the course of an immune response is tedious and indirect. To address this
problem, the peptide-major histocompatability complex (MHC) ligand for a
given population of T cells was multimerized to make soluble peptide-MHC
tetramers. Tetramers of human lymphocyte antigen A2 that were complexed with
two different human immunodeficiency virus (HIV)-derived peptides or with a
peptide derived from influenza A matrix protein bound to peptide-specific
cytotoxic T cells in vitro and to T cells from the blood of HIV-infected
individuals. In general, tetramer binding correlated well with cytotoxicity
assays. This approach should be useful in the analysis of T cells specific
for infectious agents, tumors, and autoantigens.
Paul Fallon
********************************************************
Basically, one prepares a recombinant MHC class I chain (your choice
of allele), bind it with microglobulin and an epitope peptide. A
signal sequence added to the recombinant allows this to be
biotinylated selectively.
One the class I chain/peptide/microglobuline construct is ready, one
mixes it with fluorescinated straptavidin, thus forming a tetramer of
peptide-loaded MHC class I molecules (since strep has 4 biotine
binding sites).
One can then use this reagent (the complex is supposed to be very
stable) to stain T-cells with it as if it were a flow cytometry
anti-CD monoclonal, for instance. The low shifts in fluorescence
typically seen are attributed to antigen-reactive class I -restricted
T-cells present in a stained polyclonal population, eg PBMC, since
these are the only ones thought to interact with a given class
I/epitope complex.
There seem to be more problems with class II constructs.
Guy Hermans, PhD
Dept. of Neurology and Neurological Sciences
Beckman Institute
Stanford University, CA 94305-5429
****************************************************************************
Elaine Kunze
Flow Cytometry.....Image Analysis...
The Biotechnology Institute for Research and Education
Life Sciences Consortium
8B Althouse Laboratory (814-863-2762)
Penn State University
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