Targets of somatic hypermutation within immunoglobulin light chain genes in zebrafish

Abstract

Summary In mammals, somatic hypermutation (SHM) of immunoglobulin (Ig) genes is critical for the generation of high‐affinity antibodies and effective immune responses. Knowledge of sequence‐specific biases in the targeting of somatic mutations can be useful for studies aimed at understanding antibody repertoires produced in response to infections, B‐cell neoplasms, or autoimmune disease. To evaluate potential nucleotide targets of somatic mutation in zebrafish (Danio rerio), an enriched IgL cDNA library was constructed and > 250 randomly selected clones were sequenced and analysed. In total, 55 unique VJ‐C sequences were identified encoding a total of 125 mutations. Mutations were most prevalent in VL with a bias towards single base transitions and increased mutation in the complementarity‐determining regions (CDRs). Overall, mutations were overrepresented at WRCH/DGYW motifs suggestive of activation‐induced cytidine deaminase (AID) targeting which is common in mice and humans. In contrast to mammalian models, N and P addition was not observed and mutations at AID hotspots were largely restricted to palindromic WRCH/DGYW motifs. Mutability indexes for di‐ and trinucleotide combinations confirmed C/G targets within WRCH/DGYW motifs to be statistically significant mutational hotspots and showed trinucleotides ATC and ATG to be mutation coldspots. Additive mutations in VJ‐C sequences revealed patterns of clonal expansion consistent with affinity maturation responses seen in higher vertebrates. Taken together, the data reveal specific nucleotide targets of SHM in zebrafish and suggest that AID and affinity maturation contribute to antibody diversification in this emerging immunological model.

Unique Features of Fish Immune Repertoires: Particularities of Adaptive Immunity Within the Largest Group of Vertebrates

“…Although fishes lack conventional class switch (Stavnezer and Amemiya 2004), fish AID has similar biochemical functions and deaminates cytosine, thus inducing point mutations; it even mediates class switch in mouse B cells (Barreto et al 2005). The mutational pattern observed in Ig sequences from catfish (Yang et al 2006), zebrafish (Marianes et al 2011), and also in nurse shark (Diaz et al 1999) reminds actually the one described in mammals, with a preference for transitions but no major bias for mutation of G:C or A:T pairs (Diaz et al 2001). …”

“…In contrast, the distribution of mutations focused on CDRs in immunoglobulin kappa-like light chain in medaka suggested selection and affinity maturation; additionally, the dS/dN ratio was consistent with an antigen-driven selection in 5 out of 13 mutated sequences (Magadán-Mompó et al 2013). In zebrafish, the expansion of VJC clonal lineages with an increased frequency of mutation might also suggest selection of B cells expressing hypermutated Ig (Marianes et al 2011). …”

Perspectives on antigen presenting cells in zebrafish

“…Rainbow trout IgM+ and IgT+ B cells undergo splenic clonal expansion in response to systemic viral infection (Castro et al, 2013). Although teleost B cells do not class switch, SHM of immunoglobulin genes is evident in zebrafish (Marianes and Zimmerman, 2011; Jiang et al, 2011) and channel catfish (Yang et al, 2006). Additionally, affinity maturation has been measured in rainbow trout, although antibody affinities increase marginally compared to the logarithmic increases that are measured in mammals (Cain et al, 2002; Kaattari et al, 2002; Ye et al, 2011).…”

Differential compartmentalization of memory B cells versus plasma cells in salmonid fish

“…Primary immunization can, thus, lead to increased immunogen sensitivity [1], enhanced in vivo [2][3][4][5][6] and in vitro [6][7][8] antibody responsiveness, expansion of antigen-sensitive precursor pools [9] and, most importantly, anamestic responses to pathogens [4,5,10,11] and immunoprophylaxis [4,[12][13][14]. Anamestic features absent from teleost responses include isotype switching [15] (although they possess multiple isotypes [16]), relatively modest somatic mutaCorrespondence: Dr. Stephen L. Kaattari e-mail: kaattari@vims.edu tion [17][18][19] and affinity maturation [20][21][22] as compared with the IgG responses of mammals. Intriguingly, characteristics of teleost memory B cells (MBCs) [9] parallel those associated with a class of mammalian MBCs.…”