Human Interleukin-4-Dependent Facilitation of Human IgG Production in PBL-NOG-hIL-4-Tg mice

Yoshie Kametani^1*Shino Oshima¹Ryoji Ito²Yusuke Ohno²Soga Yamada¹Yuki Hoshino¹Asuka Miyamoto¹Mao Suzuki¹Nagi Katano¹Banri Tsuda³Mariko Miyazawa⁴Hirofumi Kashiwagi⁴Daiki Kirigaya¹Tomoka Shimizu¹Mika Kojima¹Yusuke Kikuchi¹Shunsuke Nakada¹Rentaro Ohki¹Atsushi Yasuda⁵Ayako Hirota⁶Yukio Nakamura⁷Jerzy K Kulski^8,9Tomotaka Mabuchi¹⁰Hitoshi Ishimoto⁴Takashi Shiina¹

• ¹Department of Molecular Life Science, Tokai University Shool of Medicine, Isehara, Japan
• ²Central Institute for Experimental Medicine and Life Science, Kawasaki, Japan
• ³Department of Palliative Medicine, Tokai University School of Medicine Tokai University School of Medicine, Isehara, Japan
• ⁴Department of Obstetrics and Gynecology, Tokai University School of Medicine, Isehara, Japan
• ⁵Department of Internal Medicine, Division of Nephrology, Endocrinology and Metabolism, Tokai University School of Medicine,, Isehara, Japan
• ⁶Department of Dermatology, Tokai University School of Medicine, Isehara, Japan
• ⁷Repertoire Genesis Inc, Osaka, Japan
• ⁸The University of Western Australia Faculty of Health and Medical Sciences, Perth, Australia
• ⁹Faculty of Health and Medical Sciences, School of Biomedical Science, The University of Western Australia, Crawley, Australia
• ¹⁰Department of Dermatology, Tokai University School of Medicine,, Isehara, Japan

Immune-humanized mice provide valuable experimental models for evaluating immune-based therapies, yet the induction of human antigen-specific IgG production remains limited due to species-specific incompatibilities. Our previous work demonstrated that NOG-hIL-4-Tg mice, which express human interleukin-4 (IL-4), support human T and B cell maintenance and enable antigen-specific IgG production following transplantation of human peripheral blood mononuclear cells (PBMCs). In this study, we investigated how IL-4 enhances antibody responses in this model. Flow cytometry and histological analysis revealed that human B cell maintenance was associated with specific plasma hIL-4 concentration ranges at supraphysiological levels. T and B cell receptor repertoire analysis using next-generation sequencing showed that clonal diversity remained largely conserved for one This is a provisional file, not the final typeset article month and decreased to three months post-engraftment. Immunoglobulin repertoire profiling confirmed IgG class switching in an IL-4-concentration-dependent manner. Among the IgG subclasses, IgG3 increased during the first and second months and then decreased thereafter. In contrast, IgG1 tended to increase over time; however, the proportion of IgG subclass varied among individual donors. Following immunization with two distinct peptide antigens, the mice produced enhanced levels of antigen-reactive IgG. However, many B cell clones also exhibited weak responses to unrelated third-party antigens, likely reflecting insufficient affinity maturation. Histological evaluation showed tertiary lymphoid structure (TLS)-like accumulations of B and T cells in the spleen, although fully developed germinal centers were absent. Taken together, these findings demonstrate that NOG-hIL-4-Tg mice maintain human B cells within a regulated IL-4 environment, promote early formation of splenic tertiary lymphoid structures, and support IgG production characterized by clonal expansion, class switching, and somatic hypermutation. These results confirm that human IL-4 expression supports human antibody responses in the PBL-NOG-hIL-4-Tg mouse system. (278)