A Novel Monoclonal Antibody Specific for Canine CD25 (P4A10): Selection and Evaluation of Canine Tregs

Vet Immunol Immunopathol. 2010 Jun 15;135(3-4):257-65. doi: 10.1016/j.vetimm.2009.12.006. Epub 2009 Dec 29.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibody Specificity
  • Base Sequence
  • Binding, Competitive
  • Blotting, Western
  • DNA Primers / genetics
  • Dogs
  • Female
  • Flow Cytometry
  • Forkhead Transcription Factors / metabolism
  • Humans
  • Immunoglobulin G / immunology
  • In Vitro Techniques
  • Interleukin-2 Receptor alpha Subunit / genetics
  • Interleukin-2 Receptor alpha Subunit / immunology*
  • Lymphocyte Activation
  • Male
  • T-Lymphocytes, Regulatory / classification
  • T-Lymphocytes, Regulatory / immunology*
  • T-Lymphocytes, Regulatory / metabolism

Substances

  • Antibodies, Monoclonal
  • DNA Primers
  • Forkhead Transcription Factors
  • Immunoglobulin G
  • Interleukin-2 Receptor alpha Subunit