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Microbes & Immunity Anti-mouse CXCR5 monoclonal antibody

pathways, which modulates immune cells to promote limpet hemocyanin (KLH) was conjugated at the
lymphocyte infiltration, activation, and differentiation, C-terminus of the peptide.
thereby enhancing the antitumor immune response. Cell lines, including P3X63Ag8U.1 (P3U1), Chinese
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Furthermore, CXCR5 is also expressed in cancer cells, hamster ovary (CHO)-K1, and LN229 were purchased
which makes pivotal contributions to the development from the American Type Culture Collection (Manassas,
and progression. 8,43-47 Therefore, monoclonal antibodies VA, USA). CHO-K1, P3U1, and each chemokine receptor-
(mAbs), which specifically target CXCR5 would be expressed CHO-K1 were cultured in a Roswell Park
useful for cancer therapy and elucidation of the disease Memorial Institute (RPMI)-1640 medium (Nacalai Tesque,
progression.
Inc., Kyoto, Japan), supplemented with 10% heat-inactivated
The Cell-Based Immunization and Screening fetal bovine serum (FBS, Thermo Fisher Scientific Inc.,
(CBIS) method includes the immunization of antigen- Waltham, MA, USA), 100 units/mL penicillin, 100 μg/mL
overexpressed cells and high-throughput hybridoma streptomycin, and 0.25 μg/mL amphotericin B (Nacalai
screening using flow cytometry. We have developed specific Tesque, Inc., Kyoto, Japan). LN229 and LN229 cells
mAbs against mouse CCR1 (mCCR1; clone C Mab-6), expressed mCXCR5 (LN229/mCXCR5) were cultured in a
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mouse CCR3 (mCCR3; clone C Mab-3), mouse CCR5 Dulbecco’s Modified Eagle Medium (Nacalai Tesque, Inc.,
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(mCCR5; clone C Mab-2), mouse CCR8 (mCCR8; clone Kyoto, Japan), supplemented with 10% heat-inactivated
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C Mab-2), mouse CXCR1 (mCXCR1; clone Cx Mab-1), FBS (Thermo Fisher Scientific Inc., Waltham, MA, USA),
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mouse CXCR3 (mCXCR3; clone Cx Mab-4), and 100 units/mL penicillin, 100 μg/mL streptomycin, and 0.25
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mouse CXCR4 (mCXCR4; clone Cx Mab-1) using μg/mL amphotericin B (Nacalai Tesque, Inc., Kyoto, Japan).
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the CBIS method. Furthermore, we established specific
mAbs against mouse CCR2 (mCCR2; clone C Mab-6), 2.2. Animals
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mCCR3 (clones C Mab-6 and C Mab-7), mouse CCR4 A 5-week-old female Sprague-Dawley rat was purchased
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(mCCR4; clone C Mab-1), mouse CCR6 (mCCR6; clone from CLEA Japan (Tokyo, Japan). There is no influence of
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C Mab-13), mouse CCR9 (mCCR9; clone C Mab-24), sex on the results of the study. The animal was housed under
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and mouse CXCR6 (mCXCR6; clone Cx Mab-1) using specific pathogen-free conditions. All animal experiments
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the N-terminal peptide immunization. In this paper, we were performed according to the National Institutes of
report the successful development of a novel anti-mouse Health guide for the care and use of Laboratory animals
CXCR5 (mCXCR5) mAb using the N-terminal peptide (NIH Publications No. 8023, revised 1978) and approved
immunization method. by the Animal Care and Use Committee of Tohoku
2. Materials and methods University (Permit number: 2022MdA-001).
2.1. Plasmids, peptides, and cell lines 2.3. Development of transfectants
The synthesized DNA encoding mCXCR5 (Accession No.: The plasmids were transfected into LN229 and CHO-
NM_007551.3), mouse XCR1 (mXCR1; Accession No.: K1 cells using the Neon transfection system (Thermo Fisher
NM_011798), and mouse CX3CR1 (mCX3CR1, Accession Scientific Inc., Waltham, MA, USA). Stable transfectants
No.: BC012653.1) were purchased from Eurofins Genomics were established by staining with the following mAbs: anti-
KK (Tokyo, Japan). The complementary DNAs (cDNAs) of mCXCR5 mAb (clone L138D7; BioLegend, San Diego, CA,
mouse CCR10 (mCCR10; Accession No.: NM_007721.4; USA), anti-PA tag mAb (clone NZ-1 for mCCR10), anti-
Catalog No.: MR224922), and mouse CXCR2 (mCXCR2; mCXCR2 mAb (clone SA045E1; BioLegend, San Diego,
Accession No.: NM_009909.3; Catalog No.: MR227587) CA, USA), anti-mXCR1 mAb (clone ZET; BioLegend,
were purchased from OriGene Technologies, Inc. San Diego, CA, USA), and anti-mCX3CR1 mAb (clone
(Rockville, MD, USA). The mCXCR5, mXCR1, and SA011F11; BioLegend, San Diego, CA, USA). The cells
mCX3CR1 cDNAs were cloned into the pCAGzeo vector were sorted using a cell sorter (SH800; Sony Corp., Tokyo,
(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). After sorting, the cells were cultured in medium
Japan). The mCCR10 cDNA was cloned into a pCAGzeo_ supplemented with 0.5 mg/mL of Zeocin (InvivoGen,
ssnPA16 vector. The mCXCR2 cDNA was cloned into a San Diego, CA, USA) or 0.5 mg/ml of G418 (Nacalai
pCMV6neo vector. Tesque, Inc., Kyoto, Japan). These chemokine receptors-
overexpressed CHO-K1 or LN229 (e.g., CHO/mCXCR5)
A partial sequence of the N-terminal extracellular 58
region of mCXCR5 ( -MNYPLTLDMGSITYNMDDL- ), clones were successfully established.
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with a C-terminal cysteine was obtained from Eurofins Stable transfectants of the following chemokine
Genomics KK (Tokyo, Japan). Furthermore, the keyhole receptors were previously established: CHO/mCCR1,
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Volume 2 Issue 1 (2025) 102 doi: 10.36922/mi.5664