Effector Memory CD4+ T cells from the spleens of Interleukin1-receptor antagonist knockout mice

Interleukin-1, a pro-inflammatory cytokine, plays a crucial role in inflammatory disease pathogenesis. Interleukin-1 receptor antagonist knockout (IL-1Ra KO) mice spontaneously develop aortitis, arthritis, and dermatitis, and are employed as a model for human inflammatory diseases. Previous studies have shown that transferring total T cells from IL-1Ra KO mice into nude mice induces aortitis and arthritis; however, the roles of specific T cell subsets in these inflammatory responses remain unclear. In this study, we aimed to investigate the T cell subsets in IL-1Ra KO mice. We found that the proportion of PD-1+CD44+CD62L˗CD4+ T cells in the spleen and lymph nodes of IL-1Ra KO mice was significantly higher than that of wild type mice. RNA sequencing revealed elevated expression of basic helix-loop-helix family member e40 and granulocyte macrophage colony stimulating factor (GM-CSF) in splenic CD44+CD62L˗CD4+ T cells from IL-1Ra KO mice. In addition, GM-CSF production from splenic CD4+ T cells of IL-1Ra KO mice was significantly higher than that of wild type mice when stimulated with PMA and ionomycin in vitro. Notably, immunohistochemical staining showed infiltration of GM-CSF+CD4+ T cells at inflammatory sites in IL-1Ra KO mice. Our results suggest that a subset of GM-CSF+CD4+T cells emerges under IL-1 signal-enhanced inflammatory conditions. Total RNA was isolated from sorted live effector memory CD4+ T cells with the FACSAria III (BD Biosciences) cell sorter, using a miRNeasy micro kit (QIAGEN, Venio, The Netherlands). The integrity and quantity of the total RNA was measured with an Agilent 2100 Bioanalyzer RNA 6000 Pico kit (Agilent Technologies, Santa Clara, CA, USA). A 10-ng aliquot of the total RNA obtained from each sample was used to construct a sequencing library with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, MA, USA) with the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer’s protocols. The quality of the libraries was assessed using the Agilent 2200 TapeStation High Sensitivity D1000 (Agilent Technologies). The pooled libraries of the samples were sequenced using the NovaSeq 6000 (Illumina, Inc., San Diego, CA, USA) in 150-base pair (bp) paired-end reads.