Th2 single-cell heterogeneity and clonal interorgan distribution in helminth-infected mice

Th2 cells provide effector functions in type 2 immune responses to helminths and allergens. Despite substantial knowledge about molecular mechanisms of Th2 cell differentiation, there is little information on Th2 cell heterogeneity and clonal distribution between organs mainly due to technical limitations. To address this issue, we performed combined single-cell transcriptome and TCR clonotype analysis on murine Th2 cells in mesenteric lymph nodes (MLN) and lung after infection with Nippostrongylus brasiliensis (Nb) as a model of human hookworm infection. We identified strong organ-specific expression profiles, but also found populations with conserved effector or migration signatures. A substantial MLN subpopulation with an interferon response signature suggests a role for interferon-signaling in Th2 cell differentiation or diversification. RNA-inferred developmental directions further implied proliferation as a hub for differentiation decisions. Although the TCR repertoire appeared to be highly heterogeneous, we identified expanded Th2 clones and CDR3 motifs. Clonal relatedness between distant organs confirmed the effective exchange of Th2 effector cells. However, locally expanded clones dominated the response, as the most expanded clones in MLN and lung did not overlap. This new insight in Th2 cell subsets and clonal relatedness in distant organs demonstrates their heterogeneity and suggests that they serve distinct effector functions. We performed combined transcriptome and TCR clonotype analysis using the chromium 10xGenomics and Illumina single cell sequencing platform with Th2 cells isolated from lung and MLN of two IL-4eGFP reporter (4get) mice (Mohrs et al. Immunity 2001) that had been infected 10 days before with Nb. Th2 cells (CD4+IL-4eGFP+) were sorted from single cell suspensions of both organs and were directly subjected to scRNA library preparation. Additionally, we compared the transcriptional profile of the lung draining mediastinal LN and MLN in a second single cell sequencing run.