摘要Gut microbes exhibit complex interactions with their hosts and shape an organism's immune system throughout its lifespan.As the largest secondary lymphoid organ,the spleen has a wide range of immu-nological functions.To explore the role of microbiota in regulating and shaping the spleen,we employ scRNA-seq and Stereo-seq technologies based on germ-free(GF)mice to detect differences in tissue size,anatomical structure,cell types,functions,and spatial molecular characteristics.We identify 18 cell types,9 subtypes of T cells,and 7 subtypes of B cells.Gene differential expression analysis reveals that the absence of microorganisms results in alterations in erythropoiesis within the red pulp region and congenital immune deficiency in the white pulp region.Stereo-seq results demonstrate a clear hierarchy of immune cells in the spleen,including marginal zone(MZ)macrophages,MZ B cells,follicular B cells and T cells,distributed in a well-defined pattern from outside to inside.However,this hierarchical structure is disturbed in GF mice.Ccr7 and Cxcl13 chemokines are specifically expressed in the spatial locations of T cells and B cells,respectively.We speculate that the microbiota may mediate the structural composition or partitioning of spleen immune cells by modulating the expression levels of chemokines.