Norman R. Klinman

Decreased Frequency of Somatic Hypermutation and Impaired Affinity Maturation but Intact Germinal Center Formation in Mice Expressing Antisense RNA to DNA Polymerase ζ

To examine a role of DNA polymerase ζ in somatic hypermutation, we generated transgenic mice that express antisense RNA to a portion of mouse REV3, the gene encoding this polymerase. These mice express high levels of antisense RNA, significantly reducing the levels of endogenous mouse REV3 transcript. Following immunization to a hapten-protein complex, transgenic mice mounted vigorous Ab responses, accomplished the switch to IgG, and formed numerous germinal centers. However, in most transgenic animals, the generation of high affinity Abs was delayed. In addition, accumulation of somatic mutations in the VH genes of memory B cells from transgenic mice was decreased, particularly among those that generate amino acid replacements that enhance affinity of the B cell receptor to the hapten. These data implicate DNA polymerase ζ, a nonreplicative polymerase, in the process of affinity maturation, possibly through a role in somatic hypermutation, clonal selection, or both.

The B‐cell biology of aging

Summary: Although both the number and responsiveness of peripheral B cells in aged mice remain relatively intact, there are dramatic changes in B‐cell generation. Alterations in B‐cell development include both a skewing of V‐gene utilization, especially in cells responsive to phosphorylcholine, and a decrease in the generation of various developmental B‐cell sub‐sets. The altered representation of these subsets appears to be the consequence of two developmental blocks. The first developmental block occurs during the maturation of pro‐B cells and is evidenced by a decrease in the number of pre‐B cells. The second developmental block occurs at the earliest stage of slg+‐cell maturation (slgMvery lo). Because of this block in B‐cell maturation, it spite of a decrease in incoming pre‐B cells, the number of sIgMvery lo cells appears to increase in aged mice. Additionally, the time of residence of cells within this maturational stage increases dramatically, While the proportion of cells in more mature (sIgMhi) stages of bone marrow development are decreased. In addition to the decreased number of maturing bone marrow B cells, the population of splenic B cells that represent recent bone marrow émigrés (HSAvery hi) is markedly decreased. In the face of this decrease in newly emerging cells from the bone marrow, the population of mature splenic B cells is maintained by their increased longevity.

The “Clonal Selection Hypothesis” and Current Concepts of B Cell Tolerance

The fundamental precepts of the clonal selection hypothesis have stood well the test of four decades of subsequent research. Thus, self–nonself-discrimination does, as predicted, rely on the unipotentiality of B cells and the elimination of self-reactive B cells as they mature via signals induced through the sIg receptors of the cells. Furthermore, many of the logical extensions of this theory have been confirmed. For example, if tolerance were to apply to the spectrum of self-antigens, tolerance induction of immature B cells should ideally be applicable to an array of self-antigenic structures over a broad concentration range. Indeed, tolerance induction of immature B cells can be induced even by extremely low concentrations of antigen and appears to be independent of the form of the antigen (58xMetcalf, E.S and Klinman, N.R. J. Exp. Med. 1976; 143: 1327–1340Crossref | PubMedSee all References, 61xMetcalf, E.S, Schrater, A.F, and Klinman, N.R. Immunol. Rev. 1979; 43: 143–183CrossrefSee all References).However, because much of the details of immune responsiveness had not yet been defined, the reality of self–nonself-discrimination by B cells is far more complex than anticipated by the clonal selection hypothesis. Interestingly, most of the exceptions to the clonal selection hypothesis result in a higher frequency of self-reactive B cells. Thus, for example, neither B cells whose sIg receptors bind soluble self-antigens with low affinity nor those that recognize antigens that present determinants monovalently are eliminated. Additionally, T cell help can circumvent B cell tolerance. Even though T cell help would rarely be available in the adult bone marrow or neonatal spleen, in some instances B cells might escape tolerance by receiving T cell or mitogenic signals. This would not be trivial during the generation of memory B cells wherein T cell help would likely be available. Thus, for newly generating memory B cells whose V region mutations create anti-self reactivity, the fate of the cells could depend on a competition for receptors by self-antigen, for which Th would not be available, and the immunizing antigen, wherein Th and positive selection would be available (Linton et al. 1991xLinton, P.J, Rudie, A, and Klinman, N. J. Immunol. 1991; 146: 4099–4104PubMedSee all ReferencesLinton et al. 1991).As a result of these exceptions to clonal selection expectations, and the absence of some self-antigens from the bone marrow, many self-reactive B cells are present in the peripheral B cell pool (62xMoller, G, Gronowicz, U, Persson, A, Coutinho, A, Moller, Hammerstron, L, and Smith, E. J. Exp. Med. 1976; 143: 1429–1438Crossref | PubMed | Scopus (24)See all References, 33xHarris, D.E, Cairns, L, Rosen, F.S, and Borel, Y. J. Exp. Med. 1982; 156: 567–584Crossref | PubMedSee all References, 18xCooper, H.M, Klinman, N.R, and Paterson, Y. Eur. J. Immunol. 1988; 19: 315–322CrossrefSee all References, 85xStockinger, B and Hausmann, B. Eur. J. Immunol. 1988; 18: 249–253Crossref | PubMedSee all References). Although, in some instances, the presence of such B cells may enable auto-antibody production, the systemic immune system has several safeguards. Most important, stimulation by most antigens requires Th, and Th specific for self-antigens are rare. Additionally, several safeguards appear to be built into the B cell tolerance mechanism per se. First, as described above, many anti-self antibodies would exhibit low affinity for self-antigens whether the B cells were stimulated by a cross-reactive foreign antigen or the self-antigen. Second, there is a second window of tolerance susceptibility that prohibits the generation of anti-self memory B cells. This eliminates the possibility of either expanded clones of anti-self B cells or the generation of high affinity anti-self B cells. Indeed, it appears that a breakdown in this mechanism may be responsible for much of autoimmune disease (Radic et al. 1989xCold Spring Harbor Symp. Radic, M.Z, Mascelli, M.A, Erikson, J, Shan, H, Shlomchik, M, and Weigert, M. Quant. Biol. 1989; 54: 933–946Crossref | PubMedSee all ReferencesRadic et al. 1989). Third, there are modes of B cell innactivation, such as anergy, that may be applicable to more mature B cells that may disfavor B cell longevity or stimulation and thus may supplement the tolerance mechanisms that eliminate newly generating anti-self cells. Finally, certain antigenic determinants, such as those present on cell surfaces, may lead to the elimination of even mature B cells, thus ensuring a lack of reactivity to certain crucial self-components. Therefore, although the mechanisms that are available to ensure the absence of auto-antibody production are far more complex than anticipated by the clonal selection hypothesis, the absence from the repertoire of mature B cells with the capacity to respond to self-constituents and generate high affinity auto-antibodies is precisely the predicted outcome of the theory.

Defining subsets of naive and memory B cells based on the ability of their progeny to somatically mutate in vitro.

The increased affinity of memory antibody responses is due largely to the generation and selection of memory B cells that accumulate somatic mutations after initial antigenic stimulation. Further affinity maturation and mutation also accompany subsequent immunizations. Previous studies have suggested that, like primary antibody-forming cell (AFC) clones, secondary AFC do not accumulate further mutations and, therefore, the origins of progressive affinity maturation remain controversial. Here, we report the generation of somatically mutated memory B cell clones in vitro. Our findings confirm the existence of a naive B cell subset whose progeny, rather than generating AFC, somatically mutate and respond to subsequent antigenic stimulation. Interestingly, upon stimulation, a subset of memory B cells also generates antigen-responsive cells that accumulate further somatic mutations.

Relative roles of somatic and darwinian evolution in shaping the antibody response

The need for a highly specific system of recognition in immunity has resulted in the evolution of several somatic mechanisms such as V(D)J recombination, to diversify the repertoire of B cells. Therefore, repertoire diversification is the driving force for the cells that constitute the bulk of the response to unpredictable pathogens, the B2 naïve B cells. Predictability of antigen, on the other hand, has played a major role in shaping the neonatal repertoire, in which evolution to recognize commonly encountered pathogens has driven the germline sequence of several VH segments that are used frequently in the neonatal repertoire. A third population, the memory B cell population, is generated to respond to a known pathogen, but predictability of the pathogen is not acquired until after a first exposure. Therefore, it is somatic evolution in germinal centers that drives the generation of high-affinity memory B cells.

Selection in the expression of functionally distinct B-cell subsets

Although the isolation and characterization of functionally distinct B-cell subsets has been a major preoccupation of immunologists for the past two decades, only recently has it been recognized that the various B-cell subsets may be disparately selected during their development and maturation on the basis of their expressed Ig V regions. Thus, pre-B cells are selected for clonal expansion and maturation by virtue of the amino-acid sequence of their nascent H chains and newly emerging B cells may be selected for longevity and subset distribution on the basis of both clonotype-specific and relatively non-specific interactions of their surface Ig receptors.

Interrelating B Cell Subpopulations and Environmental Regulation with the Expression of Three Tiers of Repertoire Diversity

The B cell repertoire consists of three tiers of clonotype diversity. One tier, which is the product of H chain V region rearrangements in the absence of N additions, is of limited diversity (less than 10(8) clonotypes) so that clonotypes of this tier would be expected to recur within and among B cells of individuals of an inbred strain. These clonotypes, therefore, could be subjected to, and conserved by, evolutionary selective pressures such as those imposed by ubiquitous bacterial pathogens. The second tier of clonotypes is created by H chain V region rearrangements that include N additions, and is, therefore, exceedingly diverse. Clonotypes of this tier would be unlikely to recur; however, by providing maximal diversity they would ensure protection against a wide spectrum of pathogens. The third tier of diversity is that which is generated by the superimposition of somatic mutations on clonotypes of the other two tiers. This tier of clonotypes is reflective of the refinement of specificities that are destined for expression in memory B cells. B cells exists as three distinct subpopulations, Ly-1 B cells, conventional primary B cells and memory B cells. These subpopulations differ functionally, developmentally, and by the extent to which they are impacted by immunoregulatory processes. Furthermore, B cells of these subpopulations differentially express the three tiers of clonotype diversity.

Production of antibodies of monoclonal specificity without the use of hybridoma cell lines

Reproducible immunodiagnostic tests require antibodies of standardized specificity and affinity. A significant step toward this goal has been achieved with the development of the methodology of growing clonal populations of cells secreting antibodies with a defined specificity (Kohler and Milstein, 1975). In this technique an antibody-secreting plasma cell, isolated from an immunized animal, is fused with an immortal myeloma cell. The products of this fusion are called hybridoma cells and are the source of monoclonal antibodies which currently provide the most reproducible and well characterized antibodies for immunodiagnostics.