Abstract Increasing evidence of brain-immune crosstalk raises expectations for the efficacy of novel immunotherapies in Alzheimer’s disease (AD), but the lack of methods to understand brain tissues make it difficult to examine therapeutics. Here, we investigated the changes of spatial transcriptomic signatures and brain cell type using the 10x Genomics Visium platform in immune modulated AD models by various treatments. To proceed with an analysis suitable for a single spot-based transcriptomics, we first organized a workflow for segmentation of neuroanatomical regions, establishment of appropriate gene combinations, and comprehensive review of altered brain cell signatures. Ultimately, we investigated spatial transcriptomic changes following administration of immunomodulators, NK cell supplements and anti-CD4 antibody, that ameliorate behavior impairment, and designated brain cells and regions showing probable associations with behavior changes. We provided the customized analytic pipeline into an application named STquantool. Thus, we anticipate that our approach can help researchers to interpret real action of drug candidate by simultaneously investigating the dynamics of all transcripts for development of novel AD therapeutics.