Toll-like receptor 7/8 agonist stimulation rapidly skews the antibody repertoire of B cells in non-human primates

Shiyu Wang¹Yifei Wu²Yuting Xu¹Rui Deng²Fangzheng Liu²Michael Citron³Lihua Luo⁴I-Ming Wang³Xiao Liu⁵Wei Zhang^2*

• ¹Department of cell biology and neurology, School of Life Sciences, Xuzhou Medical University, Xuzhou, China
• ²Department of Biomedical Informatics, School of Life Sciences, Central South University, Changsha, China
• ³Merck & Co. Inc., Kenilworth, United States
• ⁴BGI Group, Shenzhen, China
• ⁵Tsinghua University, Beijing, China

Toll-like receptors 7 and 8(TLR7/8) recognize purine-rich single-stranded RNA from pathogens, triggering immune responses. Although TLR7/8 agonists are emerging as potential novel adjuvants, their impact on the adaptive B cell response, particularly antibody repertoire, remains unclear. Here, six Indian rhesus macaques(IRMs) and six African green monkeys(AGMs) were stimulated with a TLR7/8 agonist. Peripheral blood samples were collected pre-stimulation and multiple timepoints within one week post-stimulation for flow cytometry and antibody repertoire sequencing. B cell activation was observed at 24 hours. Significant increases in antibody repertoire diversity were detected at 48 and 72 hours in both species; however, diversity remained elevated at one week in IRMs but returned to baseline in AGMs. Analysis of antibody lineage distributions revealed a marked increase in increased lineages at 48 and 72 hours, characterized by higher frequency, a greater number of antibody clonotypes, and increased mutation rates. The identified expanded lineages, induced by the TLR7/8 agonist, exhibited λ chain bias and divergent antibodies across individuals, and primarily originated from highly mutated antibodies, likely corresponding to memory/effector-like B cells. Our findings demonstrate that the TLR7/8 agonist rapidly induces a divergent expansion of B cells, establishing an immune foundation supporting vaccine responses and offering new insights into the dynamic B cell modulation.