A novel monoclonal antibody specific for canine CD25 (P4A10): selection and evaluation of canine Tregs.
A monoclonal antibody (mAb), P4A10, was made to the canine interleukin-2 receptor alpha chain (IL-2Ralpha; p55; Tac antigen; CD25) to facilitate studies of canine regulatory T-cells (Treg). By non-reduced Western blot, P4A10 bound to a 55kDa protein, the size of human IL-2Ralpha. In flow cytometry assays, it reacted with a minor population of circulating dog CD3(+)CD4(+) T-cells and the majority (>60%) of in vitro PMA-Ionomycin (PMA-IO)-activated canine CD3(+) T-cells. P4A10 recognized a hematopoietic cell population enriched for FoxP3+ cells as measured by flow cytometry. The P4A10-selected fractions of T-cells had significantly increased copy numbers of CD25, FoxP3, IL-10, and TGFbeta as detected by RT-PCR (reverse transcriptase-PCR) compared to the negative fractions. The P4A10-selected cells inhibited (3)H (tritiated) thymidine incorporation in a mixed leukocyte reaction (MLR) containing responders of the same origin. P4A10-selected T-cells from fresh peripheral blood mononuclear cells had less FoxP3 (p=0.07) by qRT-PCR (quantitative RT-PCR) and were less suppressive (p=0.01) than in vitro alloantigen-activated Treg. The mAb P4A10 is specific for canine CD25 and can be used to facilitate studies of CD25+FoxP3+ Treg in this clinically relevant large animal model.
Abrams
V Kraig
VK
Fred Hutchinson Cancer Research Center, Seattle, WA 98109-102, United States. kabrams@fhcrc.org
Hwang
Billanna
B
Lesnikova
Marina
M
Gass
M John
MJ
Wayner
Elizabeth
E
Castilla-Llorente
Cristina
C
Georges
George E
GE
Nash
Richard A
RA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D004285:Dogs; D005260:Female; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D066298:In Vitro Techniques; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000276:immunology*; D008213:Lymphocyte Activation; D008297:Male; D050378:T-Lymphocytes, Regulatory / Q000145:classification / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D051858:Forkhead Transcription Factors; D007074:Immunoglobulin G; D053645:Interleukin-2 Receptor alpha Subunit