Fig. 3.

In nurse sharks, AID is proposed to catalyze somatic hypermutation (SHM) of TCR alpha chain genes (α chain) during the late double-positive stage of thymocyte development, likely producing new TCR paratopes capable of passing thymic selection. a Single-molecule RNA fluorescence in situ hybridization (FISH) probing fixed shark thymus sections simultaneously for AID (probes labeled with Quasar 670; pseudo colored red) and TCR α chain (probes labeled with CalFluor Red 610; pseudo colored green) and counterstained with DAPI (blue). TCRα is highly expressed within thymic cortical regions near the cortico-medullary junction (CMJ), suggesting that late TCR α chain gene rearrangement is occurring here. Coincidently, cells expressing high levels of AID encircle groups of cells expressing high levels of the TCR α chain, suggesting that AID is involved in SHM of the α chain V region during late stages of positive selection or early stages of negative selection (Ott et al. 2018) (scale bars: 150 μm, 75 μm, and 30 μm at 10×, 20×, and 63× magnification, respectively. White box indicates the magnified regions of the 20× image shown in the 63× image). b Theoretical model illustrating putative rearrangement of TCR beta chain (β chain) and α chain in nurse shark thymus, based on what is known in mammals. CD4/CD8 double negative (DN) thymocytes utilize RAG to rearrange β chain in the sub-capsular region (SCR) and outer cortex. Cells with productive β chain arrangements then proliferate, expressing both CD4 and CD8 as double positive (DP) thymocytes. The strong distinction between small thymocytes in the cortex and larger cells in the medulla has been shown in sharks, as well as RAG and TCR β expression in the sub-capsular regions (Criscitiello et al. 2010). As DP thymocytes move toward the inner cortex and CMJ where RAG-mediated α chain re-rearrangement (editing) has been shown to occur in mice, shark (but not mouse) thymocytes begin to express AID, rescuing nonproductive receptor rearrangements from apoptosis through receptor editing and/or receptor salvaging via SHM. In the latter case, AID-catalyzed SHM can produce TCR with improved affinity to MHC: Ag complexes (to pass positive selection) or reduce recognition of selfpeptide, rescuing self-reactive thymocytes from apoptosis (to pass negative selection). Salvaged thymocytes then express either CD4 or CD8 on their surface as single-positive (SP) cells [AID: activation-induced cytidine deaminase; RAG: recombination activating genes]. Figure 3 b created with BioRender.com