The effects of cryopreservation on the expression of canine regulatory T-cell markers.
BACKGROUND
Regulatory T (Treg) cells have been described as key regulators in various immunological processes and are of growing interest in veterinary allergy. Cryopreservation of immune cells is performed routinely in human basic science research and in clinical studies. As such, it allows batch testing of collected samples at a single time point, resulting in a significant reduction in sample variability. Data which describe the effects of cryopreservation on Treg cell frequency and functionality in the canine species are important to inform future research.


OBJECTIVE
The purpose of this study was to establish a robust freeze/thaw procedure and flow cytometric staining protocol for canine Treg cells, and to compare the frequencies of different canine Treg cell phenotypes before and after cryopreservation.


METHODS
Nine privately owned dogs.


METHODS
Peripheral blood mononuclear cells were isolated and Treg cells stained and analysed by flow cytometry, before and after three months of cryopreservation. The recovery percentages and the corresponding correlations (fresh versus cryopreserved) for CD4+CD25+, CD4+FOXP3+and CD4+CD25+FOXP3+cell populations were calculated.


RESULTS
A high recovery rate of 97.2 (r = 0.94, P < 0.0001), 93.9 (r = 0.77, P < 0.01) and 101.7% (r = 0.99, P < 0.0001) for CD4+CD25+, CD4+FOXP3+and CD4+CD25+FOXP3+cell populations, respectively, was observed.


CONCLUSIONS
This study demonstrates an optimized protocol for freezing, thawing and quantifying canine Treg cells. These results indicate that cryopreservation does not substantially affect the expression of surface and intracellular markers of canine Treg cells; however, additional studies will be necessary to assess whether functionality of the cells is also maintained.
Clinic for Small Animal Internal Medicine, Vetsuisse Faculty University of Zurich, Winterthurerstrasse 260, 8057, Zurich, Switzerland.
Noemi
N
Tarpataki
Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, 7270, Davos Platz, Switzerland.
Marcin
M
Wawrzyniak
Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, 7270, Davos Platz, Switzerland.
Cezmi A
CA
Akdis
Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, 7270, Davos Platz, Switzerland.
Beate
B
Rückert
Clinical Laboratory and Center for Clinical Studies, Vetsuisse Faculty, University of Zurich, Winterthurerstrasse 260, 8057, Zurich, Switzerland.
Marina L
ML
Meli
Clinic for Small Animal Internal Medicine, Vetsuisse Faculty University of Zurich, Winterthurerstrasse 260, 8057, Zurich, Switzerland.
Nina M
NM
Fischer
Clinic for Small Animal Internal Medicine, Vetsuisse Faculty University of Zurich, Winterthurerstrasse 260, 8057, Zurich, Switzerland.
Claude
C
Favrot
Clinic for Small Animal Internal Medicine, Vetsuisse Faculty University of Zurich, Winterthurerstrasse 260, 8057, Zurich, Switzerland.
Ana
A
Rostaher
D000818:Animals; D015925:Cryopreservation / Q000662:veterinary*; D004285:Dogs / Q000097:blood*; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D016131:Lymphocyte Subsets; D008297:Male; D050378:T-Lymphocytes, Regulatory / Q000378:metabolism*