Characterization of Plasma Cell Lineages in the Nurse Shark

Abstract

IgM was the first immunoglobulin (Ig) isotype to arise in evolution. Cartilaginous fish, including sharks, skates, and rays, are the oldest animals with an Ig-based adaptive immune system. Sharks express a germline-joined isotype, IgM<sub>1gj<sub>, as well as two forms of IgM, the typical pentameric (19S) form, which is attached to J chain, and a monomeric (7S) form devoid of J chain. Preliminary evidence suggests that 19S IgM consists of low affinity antibodies, whereas the 7S monomers can represent high affinity antibodies that function similar to mammalian IgG. Germline-joined IgM<sub>1gj<sub>, and some 19S IgM are secreted early in ontogeny, while adult sharks produce 19S and 7S IgM. The developmental relationship among the B-cells that produce these different isotypes is unknown. In one model, 19S producers "switch" to secrete 7S IgM by silencing J chain expression after activation by antigen and T cells. In another model, there may be two or more completely separate lineages that give rise to early 19S, late 19S and 7S IgM-secreting B-cells. In order to study the developmental relationship between 19S and 7S IgM-producing B cells in the nurse shark, we examined differences in transcription factor expression, Ig gene usage, heavy/light chain pairings, and sequence signatures from neonatal and adult IgM. In these studies we found a population of Blimp1-negative 19S-secreting cells in neonatal and adult nurse sharks, and unique pairing of a particular IgM H with a σ' L chain in neonatal sharks. In addition to 19S IgM, neonatal nurse sharks express the germline-joined isotype, IgM<sub>1gj<sub>. Therefore, we also examined L chain pairings in IgM<sub>1gj<sub>-secreting cells. Our findings suggest that a κ IgL pairs with IgM<sub>1gj<sub>, and that IgM<sub>1gj<sub> binds to self-molecules in neonatal gill and other tissue. Lastly, we attempted to devise a protocol for the separation of 19S- from 7S-secreting cells in order to examine the IgM repertoire in these populations. These studies provide greater insight into the function of early/innate antibodies in sharks, and we predict that they will also be relevant to mammalian models of early antibody secretion.