PMID- 33237936 OWN - NLM STAT- MEDLINE DCOM- 20210104 LR - 20210104 IS - 1932-6203 (Electronic) IS - 1932-6203 (Linking) VI - 15 IP - 11 DP - 2020 TI - Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs. PG - e0242572 LID - 10.1371/journal.pone.0242572 [doi] LID - e0242572 AB - 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. FAU - Ohshima, Shino AU - Ohshima S AD - Division of Basic Medical Science, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan. FAU - Matsubara, Tatsuya AU - Matsubara T AD - Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, Gifu, Gifu, Japan. FAU - Miyamoto, Asuka AU - Miyamoto A AD - Division of Basic Medical Science, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan. FAU - Shigenari, Atsuko AU - Shigenari A AD - Division of Basic Medical Science, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan. FAU - Imaeda, Noriaki AU - Imaeda N AD - Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, Gifu, Gifu, Japan. FAU - Takasu, Masaki AU - Takasu M AD - Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, Gifu, Gifu, Japan. FAU - Tanaka, Masafumi AU - Tanaka M AD - Division of Basic Medical Science, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan. FAU - Shiina, Takashi AU - Shiina T AD - Division of Basic Medical Science, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan. FAU - Suzuki, Shingo AU - Suzuki S AD - Division of Basic Medical Science, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan. FAU - Hirayama, Noriaki AU - Hirayama N AD - Institute of Advanced Biosciences, Tokai University, Hiratsuka, Kanagawa, Japan. FAU - Kitagawa, Hitoshi AU - Kitagawa H AD - Department of Veterinary Medicine, Faculty of Veterinary Medicine Okayama University of Science, Imabari, Ehime, Japan. FAU - Kulski, Jerzy K AU - Kulski JK AD - Division of Basic Medical Science, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan. AD - Faculty of Health and Medical Sciences, UWA Medical School, The University of Western Australia, Crawley, WA, Australia. FAU - Ando, Asako AU - Ando A AD - Division of Basic Medical Science, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan. FAU - Kametani, Yoshie AU - Kametani Y AUID- ORCID: 0000-0002-7417-1836 AD - Division of Basic Medical Science, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan. AD - Institute of Advanced Biosciences, Tokai University, Hiratsuka, Kanagawa, Japan. LA - eng PT - Journal Article DEP - 20201125 PL - United States TA - PLoS One JT - PloS one JID - 101285081 RN - 0 (Antibodies, Monoclonal) RN - 0 (CD4 Antigens) RN - 0 (CD8 Antigens) RN - 0 (Histocompatibility Antigens Class I) RN - 0 (swine leukocyte antigen) SB - IM MH - Amino Acid Sequence MH - Animals MH - Antibodies, Monoclonal/*immunology MH - Antibody Specificity MH - CD4 Antigens/analysis/chemistry/*immunology MH - CD8 Antigens/analysis MH - Cell Line, Tumor MH - Female MH - Genotype MH - HEK293 Cells MH - Histocompatibility Antigens Class I/genetics/immunology MH - Humans MH - Leukocytes, Mononuclear/immunology MH - Lymphocyte Activation MH - Male MH - Mice MH - Mice, Inbred BALB C MH - Models, Molecular MH - Protein Conformation MH - Sequence Alignment MH - Sequence Homology, Amino Acid MH - Specific Pathogen-Free Organisms MH - Swine MH - Swine, Miniature/genetics/*immunology MH - Transfection PMC - PMC7688132 COIS- Thee authors have declared that no competing interests exist. EDAT- 2020/11/26 06:00 MHDA- 2021/01/05 06:00 PMCR- 2020/11/25 CRDT- 2020/11/25 17:25 PHST- 2020/05/19 00:00 [received] PHST- 2020/11/04 00:00 [accepted] PHST- 2020/11/25 17:25 [entrez] PHST- 2020/11/26 06:00 [pubmed] PHST- 2021/01/05 06:00 [medline] PHST- 2020/11/25 00:00 [pmc-release] AID - PONE-D-20-14895 [pii] AID - 10.1371/journal.pone.0242572 [doi] PST - epublish SO - PLoS One. 2020 Nov 25;15(11):e0242572. doi: 10.1371/journal.pone.0242572. eCollection 2020.