Taiki Sato Ippei Ikegami Masahiro Yanagi Takeshi Ohyu Taiki Sugaya Shotaro Shirato Masanobu Tanemoto Shiori Kamiya Kohei Kikuchi Yuka Kamada Takehito Nakata Ryuta Kamekura Akinori Sato Kenichi Takano Masahiro Miyajima Atsushi Watanabe Shingo Ichimiya ^*

• Sapporo Medical University, Sapporo, Japan

Antigen-specific humoral responses are orchestrated through complex interactions among immune cells in lymphoid tissues, including the collaboration between B cells and T follicular helper (Tfh) cells. Accumulating evidence indicates a crucial role for interleukin-9 (IL-9) in the formation of germinal centers (GCs), enhancing the generation of class-switched high-affinity antibodies. However, the exact function of IL-9 in Tfh cell regulation remains unclear. In this study, we examined the humoral immune responses of CD4 Cre/+ Il9ra fl/fl mice, which lack an IL-9-specific receptor in Tfh cells. Upon intraperitoneal immunization with sheep red blood cells (SRBCs), CD4 Cre/+ Il9ra fl/fl mice displayed diminished levels of SRBC-specific IgG antibodies in their sera, along with reduced levels of GC B cells and plasma cells. Notably, Il9ra-deficient Tfh cells in the spleen exhibited decreased expression of their signature molecules such as B-cell lymphoma 6, C-X-C chemokine receptor 5, IL-4, and IL-21 compared to control mice. In models of allergic asthma induced by house dust mite (HDM) inhalation, CD4 Cre/+ Il9ra fl/fl mice failed to elevate serum levels of HDM-specific IgE and IgG. This was accompanied by reductions in Tfh cells, GC B cells, and plasma cells in mediastinal lymph nodes. Furthermore, group 2 innate lymphoid cells (ILC2s) were identified as producers of IL-9 under immunizing conditions, possibly induced by leukotrienes released by activated IgD + B cells around the T-B border. These observations may indicate the critical role of IL-9 receptor signaling in the activation of Tfh cells, with ILC2s potentially capable of supplying IL-9 in organized lymphoid tissues.