Assembly of a spatial circuit of T-bet-expressing lymphocytes is required for antiviral humoral immunity

Effective antiviral immunity requires generation of T and B lymphocytes expressing transcription factor T-bet, a regulator of type 1 inflammatory responses. Using T-bet expression as an endogenous marker for cells participating in a type 1 response, we report coordinated interactions of T-bet-expressing T and B lymphocytes based on their dynamic co-localization at T cell zone and B follicle boundary (T-B boundary) and germinal centers (GC) within the draining lymph node during lung influenza infection. We demonstrate that the assembly of this circuit takes place in distinct anatomical niches, guided by CXCR3, which enables positioning of TH1 cells at the T-B boundary. The encounter of B and TH1 cells at T-B boundary enables IFN produced by the latter to induce IgG2c class switching. Within GCs, T-bet+ TFH cells represent a specialized stable sub-lineage required for GC growth, but dispensable for IgG2c class switching. Our studies show that during respiratory viral infection, T-bet-expressing T and B lymphocytes form a circuit assembled in a spatio-temporally controlled manner that acts as a functional unit enabling a robust and coherent humoral response tailored for optimal antiviral immunity. Cells from mediastinal lymph nodes were isolated from Tbx21tdTomato-T2Acre, Bcl6-YFP, Foxp3Thy1.1 reporter mice 14 days after influenza infection. Populations were FACS-sorted on an Aria II cell sorter (BD Bioscience). TH1 cells were defined as Thy1.1-RFP+YFP-; T-bet+TFH cells defined as Thy1.1-RFP+YFP+; and T-bet-TFH cells defined as Thy1.1-RFP-YFP+. Three replicates of each cell subset were generated. Cells were resuspended in Trizol™. RNA was extracted from Trizol™ and prepared for sequencing by the Integrated Genomics Core at Memorial Sloan Kettering Cancer Center. 10-30 million 50bp paired-end reads were acquired on an Illumina HiSeq 4000.