Page 109 - MI-2-1

Microbes & Immunity

COMMUNICATION
Establishment of a high-affinity anti-mouse

CXCR5 monoclonal antibody for flow cytometry

Kenichiro Ishikawa, Hiroyuki Suzuki , Tomohiro Tanaka, Mika K. Kaneko ,
and Yukinari Kato*
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1,
Seiryo-machi, Aoba-ku, Sendai, Miyagi, Japan

Abstract

The CXC chemokine receptor 5 (CXCR5) is a member of the G protein-coupled receptor
family that is highly expressed in B cells and a subset of T cells, such as T follicular helper
cells. Various types of cancers, including non-small cell lung cancer, breast cancer,
and prostate cancer, also express CXCR5. Therefore, antibodies that specifically bind
to CXCR5 could be useful for clarification of the mechanisms of cancer progression.
In this study, we aimed to develop high-affinity monoclonal antibodies targeting
mouse CXCR5 (mCXCR5) for flow cytometry. The established anti-mCXCR5 mAb
(Cx Mab-3; rat IgG , kappa), demonstrated reactivity with mCXCR5-overexpressed
5
2b
Chinese hamster ovary (CHO)-K1 (CHO/mCXCR5) in flow cytometry. Kinetic analyses
using flow cytometry indicated that the dissociation constants (K ) of Cx Mab-3 for
5
D
−10
*Corresponding author: CHO/mCXCR5 cell is 7.2 × 10 M. Furthermore, Cx Mab-3 did not cross-react with
5
Yukinari Kato other mouse CC, CXC, CX3C, and XC chemokine receptors. These results indicate that
(yukinari.kato.e6@tohoku.ac.jp) Cx Mab-3 is useful for detecting mCXCR5 in flow cytometry with high affinity and
5
Citation: Ishikawa K, Suzuki H, specificity.
Tanaka T, Kaneko MK, Kato Y.
Establishment of a high-
affinity anti-mouse CXCR5 Keywords: Mouse CXC chemokine receptor 5; Monoclonal antibody; Peptide
monoclonal antibody for flow immunization; Flow cytometry
cytometry. Microbes & Immunity.
2025;2(1):101-113.
doi: 10.36922/mi.5664
Received: October 29, 2024 1. Introduction
Revised: December 1, 2024
Chemokines are classified into four different subfamilies: CC, CXC, CX3C, and XC,
Accepted: December 10, 2024 which depend on the position and number of cysteine residues in their N-terminus.
1-9
Published Online: December 26, The chemokine receptors belong to seven-transmembrane G-protein-coupled
2024 receptor families, and trigger intracellular signal transductions through binding to
Copyright: © 2024 Author(s). the ligands. 1,8,10-19 They play fundamental roles in development, homeostasis, immune
This is an Open-Access article system, cell proliferation, angiogenesis, and lymphocyte differentiation. 20-39
distributed under the terms of the
Creative Commons Attribution The CXC chemokine receptor Type 5 (CXCR5) is predominantly expressed on the
License, permitting distribution, 40
and reproduction in any medium, surface of B cells and a subset of T cells, such as T follicular helper (Tfh) cells. The
provided the original work is CXCR5+ Tfh cells interact with germinal center B cells to promote their differentiation
properly cited. into plasma cells or memory B cells formation through somatic hypermutation and
41
Publisher’s Note: AccScience class-switch. Therefore, CXCR5+ Tfh cells play an important role in secondary
Publishing remains neutral with lymphoid tissue orchestration and lymphoid neogenesis in the spleen, lymph nodes,
regard to jurisdictional claims in 8,40 42
published maps and institutional and Peyer’s patches. CXCL13 is one of the ligands of CXCR5. The CXCL13/
affiliations. CXCR5 axis activates downstream signaling, including PI3K/Akt, MEK/ERK, and Rac

Volume 2 Issue 1 (2025) 101 doi: 10.36922/mi.5664