Ilsa M. Kaattari

Plasmablast and Plasma Cell Production and Distribution in Trout Immune Tissues

These studies describe the in vitro and ex vivo generation of plasmablasts and plasma cells in trout (Oncorhynchus mykiss) peripheral blood and splenic and anterior kidney tissues. Cells were derived either from naive trout and cultured with the polyclonal activator, Escherichia coli LPS, or from trout that had been immunized with trinitrophenyl-keyhole limpet hemocyanin. Hydroxyurea was used to resolve populations of replicating (plasmablast) and nonreplicating (plasma cell) Ab-secreting cells (ASC). Complete inhibition of Ig secretion was only observed within the PBL. Both anterior kidney and splenic lymphocytes possessed a subset of ASCs that were hydroxyurea resistant. Thus, in vitro production of plasma cells appears to be restricted to the latter two tissues, whereas peripheral blood is exclusively restricted to the production of plasmablasts. After immunization with trinitrophenyl-keyhole limpet hemocyanin, specific ASC could be isolated from all immune organs; however, the anterior kidney contained 98% of all ASC. Late in the response (>10 wk), anterior kidney ASC secreted specific Ab for at least 15 days in culture, indicating that they were long-lived plasma cells. Cells from spleen and peripheral blood lost all capacity to secrete specific Ab in the absence of Ag. Late in the Ab response, high serum titer levels are solely the result of Ig secretion from anterior kidney plasma cells.

Affinity maturation in trout: clonal dominance of high affinity antibodies late in the immune response.

The ability of an animal to develop a highly specific antibody response through affinity maturation has been considered an integral part of the adaptive immune response. However, much of the literature dealing with teleostean antibody responses suggests that little or no affinity maturation may occur within these taxa. As trout antibodies are similar to multimeric mammalian IgM, it has been reasoned that affinity maturational shifts in intrinsic affinity might be similarly small. Such small increases in affinity can, however, lead to potentially great avidity changes for multimeric antibodies. We therefore employed a partition-based immunoassay that permits the dissection of a single antiserum into discrete, affinity-based antibody subpopulations. Such partitioning assays provide for enhanced sensitivity and resolution of these affinity subpopulations over that which can be obtained by fluorescence quenching or equilibrium dialysis. Through the use of the partition-based immunoassay, we were able to detect a consistent increase in affinity within trout anti-TNP antisera. Furthermore, it was determined that trout are capable of generating new, higher affinity antibodies relatively late in the antibody response, which then come into dominance. Such evidence suggests that either somatic mutation does occur or that a unique form of affinity-based regulation of antibody expression is employed.

Plasmablasts and plasma cells: Reconsidering teleost immune system organization

Comparative immunologists have expended extensive efforts in the characterization of early fish B cell development; however, analysis of the post-antigen induction stages of antibody secreting cell (ASC) differentiation has been limited. In contrast, work with murine ASCs has resolved the physically and functionally distinct cells known as plasmablasts, the short-lived plasma cells and long-lived plasma cells. Teleost ASCs are now known to also possess comparable subpopulations, which can greatly differ in such basic functions as lifespan, antigen sensitivity, antibody secretion rate, differentiative potential, and distribution within the body. Understanding the mechanisms by which these subpopulations are produced and distributed is essential for both basic understanding in comparative immunology and practical vaccine engineering.

The differential dynamics of antibody subpopulation expression during affinity maturation in a teleost

A compositional analysis of the antibody response in rainbow trout was conducted using an affinity-based immunopartitioning assay. Trout were immunized with TNP-keyhole limpet hemocyanin (TNP-KLH) and individual serum titers and their affinity distributions analyzed over a period of 27 weeks. The kinetics of antibody affinity subpopulation development revealed certain key features: 1) the lowest affinity subpopulation (log aK, 3.5-3.99) appears early, does not achieve high titer, and was more transient than the higher affinity subpopulations; 2) intermediate affinity subpopulations (log aK, 5.0-5.99) appear later (week 5), achieve relatively high titers and persist longer; and 3) the highest affinity subpopulations (log aK, 6.0-7.49) emerge much later (post week 15), and have comparable titers to the intermediate affinity group. We find that the affinity maturation of the serum antibody response can be resolved into each affinity subpopulations contribution both in quantity and timing.

Transduction of binding affinity by B lymphocytes: A new dimension in immunological regulation

To date, immunologists have operated with two primary paradigms governing the antibody response: (1) that affinity maturation is primarily dependent upon antigen-driven selection of both the germline and somatically amended repertoires, and (2) that antibody effector function is isotypically determined. The teleost model now suggests that these classical paradigms should be broadened to incorporate the ability of the B cell to transduce the strength of antigen recognition (affinity) into structural modifications of its antibody product, which, in turn, modulates the antibodys effector function. Although this relationship, thus far, has only been examined and demonstrated in the teleost, we find a number of the individual elements of this structural/functional relationship have been reported for mammalian IgM, which prompts future investigations into its universality. In sum, these findings suggest a heretofore unrecognized feature of B lymphocyte affinity discrimination, which transduces the affinity of antigen recognition into functionally modified antibodies.

Two new Ig VH gene families in Oncorhynchus mykiss

Genes encoding the immunoglobulin heavy-chain variable region (Ig VH) in rainbow trout (Oncorhynchus mykiss) have been grouped into 11 families. While obtaining a baseline assessment of the various gene families utilized by trout in the production of secreted antibody, we discovered two new families. These proposed Ig VH families, Families XII and XIII, were rarely observed; only two VH sequence types were detected for each new family, suggesting that they may not be commonly used in response to antigens, or that the captive environment may not lead to typical exposures seen in the wild. Additionally, unlike preceding studies, we found at least one representative gene sequence for each of the 11 reported Ig VH gene families, possibly indicating that the repertoire of trout Ig VH gene families may be more universal among different stocks than previously realized.