However, two other rearranged transcripts for productive IgLkG1 and IgLkF1 located in chromosome 15 were recognized in this cell. V(D)J gene recombination, which generates diversity by assembling these gene segments into VHDJH and VLJL genes. To ensure that one B cell only expresses one antibody, VHDJH rearrangement occurs only in one IgH locus (allelic exclusion), whereas VLJL rearrangement only in either the or locus (isotype exclusion). However, teleosts express multiple IgLs encoded by unique CL genes. Using single-cell transcriptomics, we have exhibited the transcription of unique rearranged VLJLCL genes in single rainbow trout B cells. Our results spotlight the laxity of isotype exclusion in teleosts and strongly suggest that fish B cells can produce antibodies of different specificities. Subject areas: Immunology, Cell biology, MYLK Transcriptomics Graphical abstract Open in a separate window Highlights ? Single teleost B cells transcribe different IgL chain sub-isotypes ? In many cells, more than one transcript is usually productively rearranged as VLJLCL ? These differently rearranged VLJLCL genes render IgL chains with variable CDR3 ? This suggests that single fish B cells can produce Igs of different specificities Immunology; Cell biology; Transcriptomics Introduction Antibodies, also termed immunoglobulins (Igs), are tetrameric molecules that include two identical heavy (H) chains and two identical light (L) chains. While IgH chains comprise one variable (V) domain name and two to four constant (C) domains, IgL chains comprise one V domain name and one C domain name. V domains from paired IgH and IgL chains mediate antigen binding and account for the specificity of a given antibody. A recombined VHDJH gene encodes each VH domain name and results from the rearrangements of V, diversity (D) and joining (J) gene segments located in the IgH locus. In contrast, a recombined VLJL gene encodes each VL domain name and emerges Caspase-3/7 Inhibitor I from your rearrangement of V and J gene segments placed in the IgL locus. These V(D)J recombination events sequentially involve random selection of Caspase-3/7 Inhibitor I individual gene segments, generation of double-strand breaks in each gene segment by rearrangement activation gene 1 (RAG1) and RAG2 endonucleases, deletion of the intervening DNA, and ligation of the remaining gene segments (Rodgers, 2017). This complex process occurs in the bone marrow and generates antibody recognition diversity in an antigen-independent manner (Schroeder and Cavacini, 2010). According to the nature of their C domain name, encoded by CH genes from your IgH locus, teleost express three unique isotypes named IgM, IgD, and IgT, this last being specific to teleosts. When expressed as surface signal-transducing B cell receptors (BCR), specific IgM, IgD, or IgT combinations define discrete B cell subsets. Like in mammals, IgM+IgD+ cells constitute the main B cell subset in systemic immune tissues (Fillatreau et?al., 2013; Simon et?al., 2019). In these cells, IgM and IgD receptors are produced by option splicing of a long mRNA that includes the VHDJH segment in addition to C and C and therefore are thought to express the same variable region (Geisberger et?al., 2006). Upon activation by antigen, IgM+IgD+ B cells transcriptionally down-regulate surface IgD expression to become IgM+IgD? B cells, also defined as IgM+ B cells, which include systemic IgM-secreting plasmablasts/plasma cells (Granja and Tafalla, 2019). Additionally, some IgM+IgD+ B cells drop surface IgM through a yet not well-defined class switch recombination event, generating IgM?IgD+ B cells, including IgD-secreting plasmablasts/plasma cells (Edholm et?al., 2010; Perdiguero et?al., 2019). Finally, IgT+ B cells, including IgT-secreting plasmablasts/plasma cells, preferentially inhabit mucosal surfaces (Zhang et?al., 2010). Consequently, these cells represent the main responders to mucosal antigens such as those from commensal bacteria, although systemic IgT responses have also been reported (Abos et?al., 2018b; Castro et?al., 2013). At a genomic level, three IgT genes (IgT1, IgT2 and IgT3) have been recognized in teleost species such as rainbow trout (Zhang et?al., 2017), but whether each IgT+ B cell express only one or more of these IgTs is currently unknown. As for IgL chains, most mammals analyzed to date express two isotypes located in two individual gene loci, which are known as Ig and Ig (Das et?al., 2008). In mammals, these IgL gene loci have a translocon arrangement which Caspase-3/7 Inhibitor I comprised multiple VL and JL segments situated upstream of a single CL gene segment. In teleosts, IgL gene loci have a multi-clustered orientation, with small clusters made up of VL, JL, and CL scattered over multiple chromosomes (Daggfeldt et?al., 1993; Ghaffari and Lobb, 1993; Zimmerman et?al., 2008). In teleost species such as rainbow trout (genome (v1.0). As predicted, the results obtained revealed that in a high proportion of cells (approximately 46%), no rearrangement of any IgL cluster was observed. However, the comparison of the CB:UMI combinations in the gene expression libraries.