Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs.

Shino Ohshima,Masafumi Tanaka,Shingo Suzuki,Hitoshi Kitagawa,Yoshie Kametani,Noriaki Hirayama,Noriaki Imaeda,Atsuko Shigenari,Asuka Miyamoto,Jerzy K Kulski,Masaki Takasu,Takashi Shiina,Asako Ando,Tatsuya Matsubara

doi:10.1371/journal.pone.0242572

Shino Ohshima, Masafumi Tanaka + Show 12 more

Open Access

PDF Available

https://doi.org/10.1371/journal.pone.0242572

Copy DOI

Export

Save

Cite

Abstract
Highlights/Summary
Full-Text PDF
Similar Papers

Abstract

Listen

Cluster of differentiation 4 (CD4) molecule expressed on the leukocytes is known to function as a co-receptor for class II major histocompatibility complex (MHC) binding to T cell receptor (TCR) on helper T cells. We previously identified two CD4 alleles (CD4.A and CD4.B) in a Microminipig population based on nucleotide sequencing and PCR detection of their gene sequences. However, CD4.B protein expression was not examined because of the unavailability of a reactive antibody to a CD4.B epitope. In this study, we have produced two swine-specific monoclonal antibodies (mAbs) against CD4.B molecules, one that recognizes only CD4.B (b1D7) and the other that recognizes both the CD4.A and CD4.B alleles (x1E10) and that can be used to distinguish CD4 T cell subsets by flow cytometry and immunohistochemistry. Using these two mAbs, we identified CD4.A and CD4.B allele-specific proteins on the surface of CD4.A (+/+) and CD4.B (+/+) T cells at a similar level of expression. Moreover, stimulation of peripheral blood mononuclear cells (PBMCs) derived from CD4.A (+/+) and CD4.B (+/+) swine with toxic shock syndrome toxin-1 (TSST-1) in vitro similarly activated both groups of cells that exhibited a slight increase in the CD4/CD8 double positive (DP) cell ratio. A large portion of the DP cells from the allelic CD4.A (+/+) and CD4.B (+/+) groups enhanced the total CD4 and class I swine leukocyte antigen (SLA) expression. The x1E10 mAb delayed and reduced the TSST-1-induced activation of CD4 T cells. Thus, CD4.B appears to be a functional protein whose expression on activated T cells is analogous to CD4.A.

Highlights

Cluster of differentiation 4 (CD4), a glycoprotein expressed on immune competent cells such as lymphocytes, monocytes, macrophages and dendritic cells, is widely used as a marker of T cell subsets for the functional analysis of the immune response
In a previous study we found that the CD4.A protein was recognized by available monoclonal antibodies (mAbs), whereas the CD4.B protein was not recognized by these mAbs [17]
The diverged amino acid sequences in CD4.A and CD4.B of Microminpigs were located on N;54–80, which is a little upstream of the predicted sites for human CD4 and class II major histocompatibility complex (MHC) binding

Summary

Introduction

Cluster of differentiation 4 (CD4), a glycoprotein expressed on immune competent cells such as lymphocytes, monocytes, macrophages and dendritic cells, is widely used as a marker of T cell subsets for the functional analysis of the immune response. The 10 amino-acid substitutions in CD4.2 NIH minipigs were found in the same positions as those in CD4.B [18] These regions of amino-acid substitution overlapped with the site of class II MHC recognition by CD4 indicating that the allelic polymorphism may affect the acquired immune response. We developed two mAbs which recognize Microminipig CD4.B, one specific to CD4.B and the other to CD4.A and CD4.B in order to analyze CD4.A and CD4.B protein expression and function We used these two antibodies to evaluate (1) the level of CD4 protein expression and T cell activation, and (2) the association of T cell activation with class I SLA protein expression levels in response to in vitro T cell stimulation with theTSST-1

Materials and methods

Results

Discussion