Reproducibility of Pentamer and Streptamer staining of CMV-specific CD8+ T-cells: defining the optimal multimer concentration and incubation conditions
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1
Navarrabiomed - Fundacion Miguel Servet, Spain
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2
Complejo Hospitalario de Navarra, Spain
Pentamers (PM) and Streptamers (ST) are widely used to detect CMV-specific CD8+ T-cells. However, the optimal staining conditions remain to be established. We analysed different concentrations of PM/ST and temperatures for PM staining, and assessed the reproducibility of both techniques.
PBMCs were isolated from six HLA-A*02:01 CMV-seropositive healthy donors. After multimeric staining one million of PBMCs, anti-human CD3, CD8 and CD45 were added. Cells were acquired in a FACSCalibur and CMV-specific T-cell percentage was examined.
PM/ST titration: For PM, 2.5, 5 or 10 µl of HLA-A*02:01/PMpp65495-503 were incubated with PBMCs for 10 minutes at room temperature (RT). For ST, 0.75 µg PE-labelled Strep-Tactin were mixed with 0.25, 0.5, 0.75 or 1 µg of HLA-A*02:01/STpp65495-503 to form the ST complex. From those, 0.1, 0.2, 0.3 or 0.4 µg were incubated with PBMCs for 45 minutes at 4ºC.
PM staining temperature: PBMCs were stained with 5 µl of HLA-A*02:01/PMpp65495-503 for 30 minutes at 37 ºC, 10 minutes at 4ºC or 10 minutes at RT.
PM/ST staining reproducibility: Per donor, six tubes per multimer (PM or ST) were incubated with PBMCs. The percentage of variance associated with the technique was estimated using a random effects model, separately for PM or ST.
The best results for PM were obtained using 5 µl (median = 0.56%; IQR = 0.41 - 0.99) during 10 minutes at RT (median = 0.48%; IQR = 0.13 - 0.93), while 0.2 µg of ST complex gave the best results for ST (median = 0.51%; IQR = 0.44 - 0.88), although statistical significance was not reached (Mann-Whitney test, p>0.05). Furthermore, the percentage of variance showed low values of variability (PM: 0.3%; ST: 0.7%), proving the reproducibility of both techniques.
In summary, staining conditions do not seem to alter the reproducibility of PM/ST quantification of CMV-specific CD8+T-cells.
Keywords:
CMV infection,
CMV specific CD8 T cells,
MHC-multimers,
Pentamers,
Streptamers
Conference:
15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.
Presentation Type:
Abstract
Topic:
Adaptive Immunity
Citation:
Beloki
L,
Ciaurriz
M,
Ramirez
N,
Mansilla
C,
Zabalza
A,
Bandres
E,
Rodriguez Calvillo
M and
Olavarria
E
(2013). Reproducibility of Pentamer and Streptamer staining of CMV-specific CD8+ T-cells: defining the optimal multimer concentration and incubation conditions.
Front. Immunol.
Conference Abstract:
15th International Congress of Immunology (ICI).
doi: 10.3389/conf.fimmu.2013.02.00913
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Received:
28 Jun 2013;
Published Online:
22 Aug 2013.
*
Correspondence:
Ms. Lorea Beloki, Navarrabiomed - Fundacion Miguel Servet, Pamplona, Spain, lorea.beloki.bes@navarra.es