Summary Document
This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.
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Dear Flowers,
as always on this mailing list - a lot of good suggestions. This is a first summary that
I will update if I get more suggestions.
Thanks to everybody helping me out.
Ruedi
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Original question about:
Frequency of antigen specific T cells
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Responses:
See
"Multiparameter precursor analysis of T-cell responses to antigen"
Nadège Bercovici, Alice L. Givan,*, Mary G. Waugh, Jan L. Fisher, Frédérique
Vernel-Pauillac, Marc S. Ernstoff, Jean-Pierre Abastado, Paul K. Wallace
Journal of Immunological Methods 276 (2003) 5 - 17.
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One method to assess frequency, precursor frequency, activation and proliferation of
allo- or antigen-specific T cells:
The Journal of Immunology, 2001, 166: 973-981.
Can also be combined with ICS to assess function
The Journal of Immunology, 1999, 162: 5212-5223.
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Try:
Novel application of a whole blood intracellular cytokine detection assay to quantitate
specific T-cell frequency in field studies:
Hanekom WA et al. 2004. J. Immunol. Methods 291: 185-95. We have found it very useful.
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Hello from a former Loyola faculty member, now at BD Biosciences in San Jose, CA. I saw
your post on the Purdue Cytometry Board, and wanted to make you aware of our lab's
website which has protocols, publications, etc., on antigen-specific intracellular
cytokine staining (ICS):
http://maeckerlab.typepad.com/
While there are multiple ways to measure antigen-specific T cells by flow, ICS is one of
the most versatile and standard methods. Feel free to write me if you have any questions
on this or related methodologies.
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Use a tetramer. Or a pentamer. These can be custom made to uncommon antigens. One
source is Beckman Coulter immunomics
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Depends on exactly what you want. I can give you a brief account of several methods.
Multimers-you need to know the antigen and MHC alleles associated.
Beckman coulter makes tetramers and Proimmune makes pentamers, those are the two most
well known. Much easier for class I than class II. Should stain all specific T cells
ICC-Measures cytokine production. Several things to watch for, main thing is that many
people have found that in most cases many of the antigen specific T cells are not
functional, at least in these types of in vitro assay. When using tetramer vs ICC you can
sometimes find 90% of the tetramer + cells will not secrete cytokine in response to
stimulus.
These can be done however, when multimers often times cannot. All you need is the antigen
to stimulate the cells and decide on a cytokine to measure (often it is IFNg)
Proliferation assays-The old standard. Nowadays CFSE (membrane dye that loses intensity
as the cell divides) is the more common dye but PKH dyes are similar. This is a less
specific measure since you are actually measuring proliferation, what you hope is a by
product of activation induced by a specific antigen.
That is what comes to mind and I think it covers most of the major flow based assays used
to measure antigen specific T cells. If you want to dig into the literature do a search
for papers from Holden Maecker or here is his website.
http://maeckerlab.typepad.com/maeckerlab_weblog/
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We also have a paper that looked at the in vivo activation of CD8+ T
cells in an allograft model: Clin Immunol. 1999 90(3):323-33.
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References suggested:
1. Bercovici N, Givan AL, Waugh MG, Fisher JL, Vernel-Pauillac F, Ernstoff MS, Abastado
JP, Wallace PK. Multiparameter precursor analysis of T-cell responses to antigen. 2003.
276:5-17
2. Bercovici N, Duffour MT, Agrawal S, Salcedo M, Abastado JP. New methods for assessing
T-cell responses. 2000. 7:859-864
3. Suchin EJ, Langmuir PB, Palmer E, Sayegh MH, Wells AD, Turka LA. Quantifying the
frequency of alloreactive T cells in vivo: new answers to an old question. 2001.
166:973-981
4. Gudmundsdottir H, Wells AD, Turka LA. Dynamics and requirements of T cell clonal
expansion in vivo at the single-cell level: effector function is linked to proliferative
capacity. 1999. 162:5212-5223
5. Hanekom WA, Hughes J, Mavinkurve M, Mendillo M, Watkins M, Gamieldien H, Gelderbloem
SJ, Sidibana M, Mansoor N, Davids V, Murray RA, Hawkridge A, Haslett PA, Ress S, Hussey
GD, Kaplan G. Novel application of a whole blood intracellular cytokine detection assay
to quantitate specific T-cell frequency in field studies. 2004. 291:185-195
6. Oughton JA, Kerkvliet NI. Novel phenotype associated with in vivo activated CTL
precursors. 1999. 90:323-333
