Cholinergic regulation of thymocyte negative selection

Acetylcholine (ACh) and its receptors (AChRs) are widely produced by various kinds of immune cells and fine-tune both innate and adaptive immune responses. However, whether cholinergic signaling regulates immune cell development is unclear. Here, we show that mouse CD4+CD8+ double-positive (DP) thymocytes express high levels of a9 nicotinic (n) AChR, and that this receptor controls the negative selection of thymocytes. Knock-out mice lacking a9 nAChR show an altered TCR repertoire and reduced CD4+ and CD8+ T cells in a mixed bone marrow chimera setting. Although thymic tuft cells, B cells, and a portion of T cells were all found to express choline acetyltransferase (ChAT) and so are sources of ACh in mouse thymus, T cell-derived ACh plays a more important regulatory role. Our results thus reveal a new mechanism of immune cell development control that involves lymphocyte-mediated cholinergic signaling. Overall design: CD4 SP thymocytes (1x106 cells/sample) and CD8 SP thymocytes (3.3x105 cells/sample) from WT or Chrna9-/- mice (two pairs of males and one pair of females) were FACS-sorted. RNA was extracted using an RNeasy microkit from Qiagen. TCR libraries were prepared using a SMARTer Mouse TCR a/b Profiling Kit and sequenced using the MiSeq platform (Illumina). Data were analyzed using MiXCR and VDJtools. Statistical analyses were performed using Prism 5.0 (GraphPad), with the two-tailed paired t-test employed for comparison of repertoire properties.