Ellen Hsu

What limits affinity maturation of antibodies in Xenopus--the rate of somatic mutation or the ability to select mutants?

Although the Xenopus immunoglobulin heavy chain locus is structurally and functionally similar to mammalian IgH loci, Xenopus antibodies are limited in heterogeneity, and they mature only slightly in affinity during immune responses. During the antibody response of isogenic frogs to DNP‐KLH, mu and upsilon cDNA sequences using elements of the VH1 family were cloned, sequenced and compared with germline counterparts. There were zero to four mutations per sequence, mostly single base substitutions, in the framework and CDRs 1 and 2 of VH. No mutations were found in JH. Since the point mutation rate was only 4‐ to 7‐fold lower than that calculated for mice, affinity maturation does not seem to be limited by mutant availability. Because of a relatively low ratio of replacement to silent mutations in the CDRs and a very high ratio of GC to AT base pairs altered by mutation, it is suggested that the problem results from the absence of an effective mechanism for selecting mutants, which in turn might be related to the absence of germinal centers in Xenopus.

Studies on Xenopus immunoglobulins using monoclonal antibodies

Monoclonal antibodies raised against Xenopus Ig recognize antigenic determinants on IgM and low mol. wt Ig (LMW Ig or IgY ). On SDS-PAGE two forms of mu were distinguished in the supernatant and cell lysate of tunicamycin-treated spleen cell culture. The two bands of v observed in serum appear to be the result of carbohydrate heterogeneity. The light chains resolved into two distinct bands which differed in peptide maps; the more slowly migrating band of light chain was preferentially associated with v. Patterns of cross-reactivity between the immunoglobulins of 11 species of Xenopus and three subspecies of Xenopus laevis were obtained; the polymorphism of mu and v antigenic determinants and its implications for species divergence are discussed.

Light chain heterogeneity in the amphibian Xenopus

Three subpopulations of light chains in Xenopus can be distinguished by monoclonal antibodies as well as by electrophoretic mobility on SDS-PAGE, peptide map and cell surface distribution. Analysis of these proteins from LPS-stimulated lymphocytes culture supernatants by two-dimensional gel electrophoresis showed a heterogeneity comparable to that observed for mouse kappa light chains. However, evidence from the selective expression of light chain subpopulations, as well as highly restricted light chain representation in anti-DNP antibodies, supports earlier findings that an antibody response in Xenopus is greatly limited in heterogeneity.

25 – Methods Used to Study the Immune System of Xenopus (Amphibia, Anura)

Publisher Summary This chapter describes the methods used to study the immune system of Xenopus. The classical distinction between T and B cells can be made in Xenopus where in vitro collaboration experiments have unequivocally proved the participation of carrier-specific helper T cells in the immune response of hapten-specific B cells. This collaboration, as in mammals, is controlled by major histocompatibility complex (MHC)-encoded molecules present on the lymphocyte surface. For in vivo responses, animals, larvae, or adults can be immunized with dinitrophenylated keyhole limpet hemocyanin or cellular antigens. For in vitro secondary responses, lymphocytes are prepared from primed animals and cultured with antigen at a final concentration of 1–10 μg/mL. Conditions for a primary response in vitro to this antigen have not yet been well defined. In many instances and especially when using chimeras, it is necessary to know which cells are contributed by which part of the chimera. In Xenopus, a good nuclear marker has been described between Xenopus borealis and Xenopus laevis. In some instances, when MHC compatibility is necessary, such markers cannot be used. In those cases, the best marker remains the ploidy marker.

Effector and regulator functions of splenic and thymic lymphocytes in the clawed toad Xenopus

B cells in Xenopus, as characterized by surface immunoglobulin, nylon-wool adherence and ability to produce antibody in vitro, were found in both major lymphoid organs: 10-25% Ig-positive cells for spleen and 0.5-4% for thymus. The splenic and thymic B cells were able to produce specific antibody of both isotypes, IgM and low molecular weight (LMW) Ig (IgG equivalent). While for a T-cell-dependent antigen, DNP-KLH, both required T-cell help, thymic B cells for a specific antigen produced far more LMW Ig than splenic B cells. Thymic T cells had the same ability as splenic T cells to elicit an allogenic GvH, but with much less efficiency, probably as a result of fewer mature cells. In contrast to splenic T cells, thymic T cells provided little or no help for B cells and, in certain instances, demonstrated suppressor activity.

Ontogeny of the Immune System in Anuran Amphibians

The immune system of anuran amphibians is established during the first 2–3 weeks postfertilization. The lymphoid organs are populated by cells of lateral plate mesoderm. Around day 12 the larval immune system starts to function and exhibits the following characteristics: low titers of antibody, poor IgG analog production, an antibody heterogeneity lower and different from the adult, presence of class II MHC molecules, lack of class I MHC molecule expression, and a not fully differentiated capacity of allograft rejection. The transition to the adult system occurs at metamorphosis when a second histogenesis of the lymphoid system occurs.

Pathogen-host interactions: antigenic variation v. somatic adaptations

Host and pathogen engage in a constant evolutionary struggle known as a “Red Queen Paradigm”. In this struggle, natural selection favours the pathogen which evolves effective virulence mechanisms and the host which is able to field adequate resistance strategies. A number of factors limit what each side can do. These include the fact that the elaboration of virulence or resistance mechanisms results in costs in genetic fitness and requires the use of ever more of the limited number of genes available in the genome. In addition, since the pathogen usually has a very much shorter generation time than the host, it can fix new virulence mutations much more quickly than the host can evolve matching resistance mechanisms. Finally, the host must ensure that its defence system does not result in unacceptable levels of collateral damage to its own tissues. This chapter briefly outlines how these considerations shape host–pathogen interactions.