Diversity of immunoglobulin lambda light chain gene usage over developmental stages in the horse

Abstract

To further studies of neonatal immune responses to pathogens and vaccination, we investigated the dynamics of B lymphocyte development and immunoglobulin (Ig) gene diversity. Previously we demonstrated that equine fetal Ig VDJ sequences exhibit combinatorial and junctional diversity levels comparable to those of adult Ig VDJ sequences. Herein, RACE clones from fetal, neonatal, foal, and adult lymphoid tissue were assessed for Ig lambda light chain combinatorial, junctional, and sequence diversity. Remarkably, more lambda variable genes (IGLV) were used during fetal life than later stages and IGLV gene usage differed significantly with time, in contrast to the Ig heavy chain. Junctional diversity measured by CDR3L length was constant over time. Comparison of Ig lambda transcripts to germline revealed significant increases in nucleotide diversity over time, even during fetal life. These results suggest that the Ig lambda light chain provides an additional dimension of diversity to the equine Ig repertoire.

Hematopoiesis in the equine fetal liver suggests immune preparedness

“…5′-rapid amplification of cDNA ends (RACE) library was constructed with the SMARTer™ RACE cDNA Amplification Kit (Clontech, Mountain View, CA) following manufacturer’s instructions. The 5′-RACE PCR was performed with 5′ RACE primers for the heavy chain VDJ sequence that spans the last 24 nucleotides conserved among IGHJ segments, and 5′ RACE primers lambda light chain sequence that spans a conserved sequence in the lambda constant segments, as previously described (Tallmadge et al 2013; Tallmadge et al 2014). The lambda chain was chosen as the horse uses primarily this light chain in circulating antibodies (Ford et al 1994).…”

“…B lymphopoiesis can be readily detected at the molecular level in the equine fetus around 90 to 120 days of gestation but perhaps even earlier in the yolk sac of the embryo (Tallmadge et al 2009; Tallmadge et al 2013; Tallmadge et al 2014). Endogenous antibodies are first detected midway (around 180 days) through gestation and, when challenged in utero, the equine fetus generates an antigen-specific IgM and IgG antibody response by at least 200 days of gestation (DG) (Martin and Larson 1973; Morgan et al 1975).…”

“…Unlike the heavy chain nomenclature, the subgroup number is not followed by an “S” but a “-”, and the gene number based on chromosomal location from 3′ to 5′ in the locus (as annotated by Sun et al 2010). Accordingly, “Vλ5” was renamed IGLV4-66, indicating subgroup 4 and the 66 th IGLV gene from the 3′ end of the locus (Tallmadge et al 2014). IGLJ genes names do not include a subgroup designation, and are named based on chromosomal location from 5′ to 3′ in the locus (Lefranc 2001).…”

“…Our previous studies have described the generation of B cells and production of the Ig repertoire in the lymphoid tissues of the horse during phases of development (Tallmadge et al 2009; Tallmadge et al 2013; Tallmadge et al 2014). We found that some aspects of Ig diversity were already established early in development while others appeared to be developmentally programed.…”

“…We found that some aspects of Ig diversity were already established early in development while others appeared to be developmentally programed. For example, bias in IGHV segments present in the equine adult repertoire were also present in the fetal spleen (Tallmadge et al 2013), while IGLV segment utilization was less restricted in the fetal compared to adult Ig repertoire (Tallmadge et al 2014). However, it was unknown the degree of Ig diversity originated in the primary (liver and bone marrow) lymphoid tissues of the fetus, and further diversification in secondary lymphoid tissues (e.g.…”