Single cell immunophenotyping identifies CD8⁺GZMK⁺IFNG⁺T cells as a key immune population in cutaneous Lyme disease

The skin lesion erythema migrans (EM) is the first clinical sign of Lyme disease, an infection due to the tick-transmitted bacteriumBorrelia burgdorferi(Bb). Previously, we used scRNA-Seq to characterize the cutaneous immune response in the EM lesion, focusing on B cells. Here, with an expanded sample size, we profiled T cell responses in EM lesions compared to autologous uninvolved skin. In addition to CD4⁺T cell subsets known to be abundant in the EM, we identified clonal expansion of CD8⁺GZMK⁺IFNG⁺T cells that exhibited significant differential expression of interferon-regulated genes. This subset included IFNG⁺cells with low cytotoxic gene expression, which may promote inflammation. While FOXP3⁺regulatory T cells were also increased in EM, they exhibited little IL10 expression. In contrast, a CD4⁺FOXP3^-tissue-resident T cell subset contained the largest population of cells with IL10 expression. Fibroblasts, endothelial cells, and pericytes were the principal cells that significantly differentially expressed key T cell-recruiting chemokines. These studies represent the first comprehensive interrogation of the cutaneous T cell response toBbinfection using single cell transcriptomics with adaptive immune receptor sequencing, providing insight into the skin barrier defense and orchestration of the immune response to this vector-borne pathogen.