Somatic hypermutation of VHS107 genes is not associated with gene conversion among family members

Murine B cells proliferating in the germinal centers of peripherallymphoid tissue accumulate mutations in their rearranged variableregions, a diversification process which contributes to affinitymaturation of the antibody response. The highly targeted natureof the hypermutation process could be explained by a somaticgene conversion mechanism. Well characterized examples of suchan activity in B cells are seen during diversification of thechicken and rabbit Ig repertoires. The genomic organization,low complexity and high degree of homology exhibited by thefour members of the murine V_HS107 gene family suggested thatthese gene segments may be suitable candidates for the searchof gene conversion events derived from upstream V_HS107 counterparts.After an immune response to a complex T cell-dependent antigen(sheep red blood cells), rearranged V13, V11 and V1 genes wereisolated from splenic extrafollicular and germinal center Bcells. Extensive somatic mutation was evident in V11 and V1sequences. When these sequences were examined, as well as V1sequences isolated from phosphorylcholine-specific hybridomas,the observed nucieotide changes were not associated with anygene conversion between family members, suggesting instead thatthey arose by a mechanism which introduces point mutations.     

Germinal center checkpoints in B cell tolerance in 3H9 transgenic mice

Regulation throughout B cell maturation and activation prevents autoreactive B cells from entering germinal center (GC) reactions. This study shows that a subset of autoreactive B cells in V(H)3H9 micro IgH transgenic mice escapes these serial checkpoints and proceeds into splenic GC. GC B cells isolated from these mice all express the transgenic V(H)3H9 micro heavy chain, some co-express light chains that yield an anti-dsDNA specificity and some have somatic mutations, consistent with their GC origin. Nonetheless, B cell tolerance is ultimately preserved as serum titers of anti-dsDNA antibodies are not elevated. These observations suggest that those autoreactive GC B cells that escaped earlier checkpoints and possibly also those cells that acquire autoreactivity de novo by mutating their antigen receptor are arrested within the splenic GC before differentiating further into antibody-secreting plasma cells.     

Analysis of individual immunoglobulin λ light chain genes amplified from single cells is inconsistent with variable region gene conversion in germinal-center B cell somatic mutation

Responding B cells in specific immune responses diversify their immunoglobulin genes and are selected on their variant antigen receptors in the microenviroment of the germinal center. The patterns of mutations previously reported for immunglobulin (Ig) genes have supported mechanistic hypotheses of either error-prone DNA synthesis or templated variable region gene conversion as the underlying mechanism in the generation of these mutations. To assess the role of gene conversion in germinal-center somatic mutation, we chose to examine nucleotide changes in mouse λ light chain genes which arose in response to a specific antigen. Laboratory mice possess three Vλ subexons, two of which differ from one another by only seven nucleotides, making these two subexons ideal for gene conversion. In the current study, we used six-parameter flow cytometry to isolate single λ light chain-expressing germinal-center B cells from two different time points in a primary immune response. We then individually amplified and sequenced individual V_λ1 genes from these single cells for mutational analysis. None of the 32 V_λ1 genes, containing a total of 40 mutations, showed evidence of gene conversion from either of the other Vλ subexons. Features such as the replacement to silent ratio of the mutations documented at the earlier time point indicate an absence of antigen-driven selection. These data indicate that V region gene conversion does not contribute to germinal-center somatic mutation and that gene conversion is not responsible for targeting mutation specifically to rearranged Ig genes. The biological implications are discussed.     

Molecular single-cell analysis of Hodgkin- and Reed-Sternberg cells harboring unmutated immunoglobulin variable region genes

Hodgkin- and Reed-Sternberg (H/RS) cells in classical Hodgkin's disease of the B lineage are the clonal progeny of antigen-experienced B cells harboring highly mutated immunoglobulin variable (V) region genes. Based on the detection of obviously destructive somatic mutations in a fraction of cases, we speculated that H/RS cells may be derived from a pre-apoptotic germinal center B cell. Seemingly contradicting this speculation, we present here the first case of classical Hodgkin's disease with H/RS cells harboring unmutated, potentially functional V region genes, which may indicate the derivation of the H/RS clone from a naive B cell. However, germinal center founder cells, which have not yet acquired somatic mutations, already have the intrinsic propensity to die by apoptosis. Thus, the rare occurrence of H/RS cells with unmutated V genes is expected if the H/RS cells are derived from the pool of pre-apoptotic germinal center B cells.     

Human splenic marginal zone B cells lack expression of activation-induced cytidine deaminase

It has been speculated that somatic hypermutation of rearranged immunoglobulin variable (V) region genes does not only take place in the germinal center (GC) microenvironment, but also in the marginal zone (MZ) of the spleen, and that human peripheral blood IgM-positive B cells with somatically mutated V region genes may derive from mutating MZ B cells. As somatic hypermutation is strictly dependent on the enzyme activation-induced cytidine deaminase (AID), we used an AID-specific monoclonal antibody that is suitable for immunohistochemical staining to analyze human splenic MZ cells for AID expression. Analysis of tissue sections from 29 spleens revealed only very rare MZ cells (approx. 0.05%) showing AID staining, whereas in 25 of the spleen samples strong AID staining of GC B cells was observed. Thus, there are virtually no AID-expressing MZ B cells, indicating that somatic hypermutation does not take place at a significant level in the MZ. Consequently, it appears unlikely that the somatically mutated IgM B cells are generated in the splenic MZ. Moreover, the lack of AID-positive MZ B cells questions the recent speculation that B cell chronic lymphocytic leukemias with mutated V genes are derived from mutating MZ B cells.     

Marginal zone and germinal center development in the spleens of neonatally thymectomized and nonthymectomized young rats

Spleens from neonatally thymectomized and nonthymectomized young rats were studied histologically and histochemically to elucidate the development of the splenic immune system with and without thymus. In intact animals primitive germinal center activity could be elicited with antigen as early as 13 days of age. More definitive germinal centers lacking tingible body macrophages were observed at 18 days of age. Germinal centers containing tingible body macrophages did not develop until 35 days of age in response to antigenic stimulation. This coincided with maximal development of the marginal zone of medium-sized lymphocytes and the mature development of nodular macrophages possessing strong acid phosphatase activity. Neonatally thymectomized rats developed marginal zones and germinal centers similar to control littermates when the young animals were maintained on tetracycline. Thymectomized animals not given tetracycline showed disturbances in splenic development. These are discussed. The results suggest that the thymus may be critical to the immune system in rats from birth to about 30 days of age but is not essential to its function beyond this period. Marginal zone lymphocytes and germinal center cells proliferate normally and mature to the plasma cell stage in the absence of a thymus if the animals are maintained on tetracycline beyond this critical age.     

Clonal proliferation of B lymphocytes in the germinal centers of human reactive lymph nodes: possibility of overdiagnosis of B cell clonal proliferation.

Clonal expansion of the germinal center B cells of human reactive lymph nodes was analyzed. By micromanipulation, 28 germinal centers were microdissected from three nonneoplastic lymph nodes that had been fixed with formalin. Immunoglobulin heavy chain variable (V) region gene rearrangement was examined by seminested polymerase chain reaction (PCR) using two sets of primers (FR2-J and FR3A-J). An oligoclonal development (one to five clones) was found in each germinal center. Depending on the primer used, four or five (16%) of the germinal centers showed a single rearrangement band. The average number of B-cell clones in each germinal center was approximately 2.5. Next, the authors analyzed 50 endoscopic biopsy specimens from 6 patients with non-mucosa-associated lymphoid tissue (MALT) type gastric lymphoma, 25 patients with chronic gastritis, and 19 patients with nonspecific colitis. In addition to the samples from the 6 patients with malignant lymphoma, 8 of 44 biopsy samples (18.2%) from patients diagnosed as having chronic gastritis or nonspecific colitis showed one or two amplified bands. These results indicate that PCR analysis of immunoglobulin heavy chain V region gene rearrangement in small biopsy specimens could be misleading, causing overdiagnosis of reactive lymphoid tissue as B-cell clonal proliferation.     

Follicular exclusion of autoreactive B cells requires FcgammaRIIb

In non-autoimmune mice, the 3H9 transgenic Ig heavy chain can pair with endogenous Iglambda1 light chains to generate B cells with specificity for DNA. These autoreactive cells are actively regulated in vivo, as indicated by the exclusion of lambda1 cells from the splenic B cell follicle and the absence of auto-antibody production. To study the role of Fcgamma receptor IIb (FcgammaRIIb) in peripheral B cell tolerance, FcgammaRIIb(-/-) mice were crossed with C57BL/6 mice bearing a site-directed knock-in of the 3H9 transgene. 3H9FcgammaRIIb(-/-) mice become autoreactive, lose the follicular exclusion of anti-DNA B cells and instead have lambda1 B cells located within splenic germinal centers. They have increased frequencies of splenic auto-antibody-producing cells and elevated titers of IgG anti-DNA auto-antibody. The data implicate an FcgammaRIIb-dependent checkpoint that can exclude autoreactive B cells from splenic follicles. By restricting their participation in germinal center reactions, this putative checkpoint helps attenuate the production of potentially pathogenic auto-antibodies. The data further suggest that this FcgammaRIIb-dependent regulation is B cell autonomous.     

Infection with Toxoplasma gondii Alters Lymphotoxin Expression Associated with Changes in Splenic Architecture

B cell responses are required for resistance to Toxoplasma gondii; however, the events that lead to production of class-switched antibodies during T. gondii infection have not been defined. Indeed, mice challenged with the parasite exhibited an expansion of T follicular helper cells and germinal center B cells in the spleen. Unexpectedly, this was not associated with germinal center formation and was instead accompanied by profound changes in splenic organization. This phenomenon was transient and was correlated with a decrease in expression of effector proteins that contribute to splenic organization, including lymphotoxins α and β. The importance of lymphotoxin was confirmed, as the use of a lymphotoxin β receptor agonist results in partial restoration of splenic structure. Splenectomized mice were used to test the splenic contribution to the antibody response during T. gondii infection. Analysis of splenectomized mice revealed delayed kinetics in the production of parasite-specific antibody, but the mice did eventually develop normal levels of parasite-specific antibody. Together, these studies provide a better understanding of how infection with T. gondii impacts the customized structures required for the optimal humoral responses to the parasite and the role of lymphotoxin in these events.     

Splenomegaly and immune complex splenitis in rabbits with experimentally induced chronic serum sickness: immunopathological findings

This study describes the morphological and immunocytochemical aspects of the spleen in rabbits with experimentally induced chronic serum sickness. Thirty-seven rabbits were immunized with daily injections of bovine serum albumin (BSA) and six served as non-immunized controls. The most significant lesions were found in rabbits with chronic serum sickness induced by high doses of BSA. The spleens were increased in size and in weight. Granular deposits of BSA, rabbit IgG and C3, presumably immune complexes (IC), were found in the basement membranes of the venous sinuses and of the capillaries in the marginal zone, in the walls of splenic arterioles and, occasionally, between the macrophages in the splenic cords and lymphoid cells in lymphatic follicles. An increased number of degranulated polymorphonuclear leukocytes, macrophages and giant cells, degenerative changes of dendritic cells and, in some instances, splenic fibrosis were also seen. These splenic lesions developed when the concentration of BSA-antibodies in the sera decreased. The spleens of rabbits receiving high doses of BSA in a stage between acute and chronic serum sickness were also increased in size and in weight. The red pulp was enlarged, and immune deposits were observed within macrophages but not in splenic structures. The spleens of non-responder rabbits had a slight decrease in number of lymphatic follicles and germinal centers only. The spleens of non-immunized rabbits were consistently normal. The results indicate that in rabbits receiving multiple injections of high doses of BSA, chronic serum sickness is associated with splenomegaly and IC-splenitis and that these lesions occur when the level of circulating BSA antibody declines. IC-splenitis could impair the clearance of IC and influence the immune function of the spleen. These findings could have implications in the pathogenesis of splenomegaly and of defective splenic function in human IC-mediated diseases.     

Predominance of a novel splenic B cell population in mice expressing a transgene that encodes multireactive antibodies: support for additional heterogeneity of the B cell compartment

We generated IgHmudelta transgenic mice using a V(H) gene that in A/J mice encodes multireactive BCR in the preimmune B cell compartment and is predominantly expressed by a memory B cell subpopulation. Most primary splenic B cells in these mice have a size, cell-surface phenotype and in vitro response profile distinct from mature follicular (B2), marginal zone (MZ) or B1 B cells, but are long-lived and appear to be slowly cycling. They reside in conventional B cell areas of the spleen and mount robust foreign antigen-driven germinal center responses, but do not efficiently differentiate to secretory phenotype. We propose that these qualities result from ongoing, low-avidity BCR-self-ligand interactions and promote entry into the memory pathway. Given these data, and the enormous diversity and characteristic multireactivity of the preimmune antibody repertoire, we also suggest that it may be more appropriate to view the primary B cell compartment as a continuum of functional and phenotypic 'layers', rather than as a group of discrete B1, B2 and MZ subsets.     

Immunoglobulin gene hypermutation in germinal centers is independent of the RAG-1 V(D)J recombinase

Summary: Antigen-driven somatic hypermutation in immunoglobulin genes coupled with stringent selection leads to affinity maturation in the B-lymphocyte populations present in germinal centers. To date, no gene(s) has been identified that drives the hypermutation process. The site-specific recombination of antigen-receptor gene segments in T and B lymphocytes is dependent on the expression of two recombination activating genes, RAG-1 and RAG-2, The RAG-1 and RAG-2 proteins are essential for the cleavage of DNA at highly conserved recombination signals to make double-strand breaks and their expression is sufficient to confer V(D)J recombination activity to non-lymphoid cells. Until very recently, expression of the V(D)J recombinase in adults was believed to be restricted to sites of primary lymphogenesis. However, several laboratories have now demonstrated expression of RAG-1 and RAG-2 and active V-to-(D)J recombination in germinal center B cells. This observation of active recombinase in germinal centers raises the issue of RAG-mediated nuclease activity as a component of V(D)J hypermutation. Here, we show that a tratisgenic K-light chain gene in a RAG-1^-/- genetic background can acquire high frequencies of mutations. Thus, the RAG-1 protein is not essential for the machinery of immunoglobulin hypermutation. The genetic approaches to identifying the genes necessary for sotnatic hypermutation will require further studies on DNA-repair and immunodeficient models.     

Continued expression of recombination-activating genes and TCR gene recombination in human peripheral T cells

It has been reported that a small population of peripheral T lymphocytes are capable of expressing V(D)J recombinase and initiating secondary V(D)J rearrangements. To determine whether RAG-expression and secondary TCR gene recombination in peripheral T cells are an antigen-driven process in secondary lymphoid tissues, we examined naive CD4(+) T cells, activated/memory CD4(+) T cells, and germinal center T cells from human tonsils. Our results showed that low levels of RAG-1 and RAG-2 mRNA were present in all T cell subpopulations except CD3(+)/CD4(-) T cells. LM-PCR analyses for double-strand DNA breaks showed that all the T cell subsets expressing RAG genes contain double-strand signal break ends, indicating ongoing TCR gene recombination. Continued RAG gene expression, introducing and repairing of double-strand DNA breaks at the TCR loci in the periphery may have significant implications in the development of some T cell neoplasms.     

Somatic hypermutation of the T cell receptor Vβ gene in microdissected splenic white pulps from HIV-1-positive patients

Somatic mutation of rearranged immunoglobulin V genes occurs in germinal centers (GC), resulting in affinity maturation of the immune response. Rearranged T cell receptor (TCR) genes were thought to be excluded from this process despite similarities in their gene structure. Somatic mutations were found among TCR Vα (TCRAV) chains of antigen-specific T cells localized in GC of mice. Here, somatically mutated TCR Vβ (TCRBV) chains are identified among microdissected splenic white pulps from HIV-positive individuals. Both the frequency and the nature of the base substitutions were found to be similar to those of mutated immunoglobulin VH genes. This was true for intrinsic mutations in the TCR framework regions as well as for mutations underlying selective pressures in the TCRBV5 gene segment. The concentration of mutations and a preference for replacement mutations in complementarity determining regions of expanded clones were indicative of a positive selection process.     

Developmentally programmed expression of AID in chicken B cells

In mice, activation induced deaminase, AID, is expressed only in germinal center B cells. It is required for the initiation of somatic hypermutation and class switch recombination. In chickens and most mammals immunoglobulin gene rearrangement generates limited diversity and the primary immunoglobulin repertoire depends on subsequent somatic hypermutation or gene conversion. Immunoglobulin gene conversion in chickens starts in the embryonic bursa, before antigen exposure. The demonstrated requirement for AID for gene conversion in the bursal lymphoma cell line, DT40, implies developmental regulation of AID expression. To test this prediction, we examined the timing and location of AID mRNA expression. An abrupt increase in AID mRNA coincided with the onset of extensive Ig gene conversion in the bursa. Expression was also detected at earlier stages, implying either that expression of AID is not the only controlling factor for gene conversion, or that gene conversion can precede the formation of bursal follicles.     

The germinal center response

The germinal center reaction is the foundation of T-cell-dependent humoral responses. Antigen-specific B cells recruited into germinal centers undergo a complex cellular program that allows for extensive expansion, isotype switching, somatic hypermutation, and differentiation into antibody-forming cells and memory cells. Importantly, the germinal center environment filters the repertoire of differentiating B cells such that high affinity variants are preferentially selected while low affinity or self-reactive clones are eliminated by apoptosis. The present article reviews the many processes that govern germinal center B-cell differentiation, as well as the cellular and molecular elements necessary to initiate and sustain a germinal center response. The major histologic features of the germinal center are also discussed, as well as the current dominant models of the germinal center reaction in humans and mice. Finally, a new model of murine B-cell differentiation is described on the basis of a multiparameter flow cytometric kinetic analysis of germinal center B cells.     

Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells

After immunization with T cell-dependent antigens, the high-affinity B cells selected in germinal centers differentiate into memory B cells or long-lived antibody-forming cells. However, a role for germinal centers in development of these B lineage cells is still controversial. We show here that Bcl6-deficient B cells, which cannot develop germinal centers, differentiated into IgM and IgG1 memory B cells in the spleen but barely differentiated into long-lived IgG1 antibody-forming cells in the bone marrow. Mutation in the V-heavy gene was null in these memory B cells. Therefore, Bcl6 and germinal center formation are essential for somatic hypermutation, and generation of memory B cells can occur independently of germinal center formation, somatic hypermutation, and Ig class switching.     

Germinal centers and the B cell system. VIII. Functional characteristics and cell surface markers of germinal center cell subsets differing in density and in sedimentation velocity

Germinal center cells from the rabbit appendix were fractionated by velocity sedimentation and isopycnic gradient centrifugation. Subsets were analysed with respect to cell size and surface markers, and were functionally characterized by testing the capacities for primary antibody synthesis, memory cell production, and formation of new germinal centers in an autologous transfer system. The migratory behaviour of the germinal center cell subsets within the spleen of homologous recipients was also studied using autoradiography. Both cell fractionation methods yielded a separation of large and small cells. Surface immunoglobulin and C3 receptors were equally expressed on germinal center cells differing in size and density. The different subsets were also equally capable in giving rise to IgM-antibody-forming cells and memory cells upon antigenic stimulation. Furthermore, large germinal centers were newly formed in the spleen of the recipients, irrespective of the cell subset injected. It was concluded that the results do not support the hypothesis that, inside germinal centers, the differentiation of large lymphoid cells (centro-blasts) into small centrocytes also implies a maturation process. Subsets of germinal center cells, however, showed a different and characteristic migratory behaviour; while small cells migrated preferentially to the corona of lymphocytes in spleen follicles, large, light cells showed an affinity for the germinal center area. We postulate that, upon stimulation, immature B cells develop an affinity for the germinal center microenvironment, to participate in a germinal center reaction.     

Human immunodeficiency resulting from a maturational arrest of germinal center B cells

Common variable immunodeficiency (CVI) is an acquired human disorder involving a striking and heterogeneous maturational defect of B lymphocytes. In this study, we used a recently developed VH gene utilization assay to analyze the abundance of developmentally restricted and unrestricted V genes in blood B cells from nine CVI patients. Unrestricted clones (bearing rearranged VH5, VH4, or VH6 genes) were present in normal abundance in this group of CVI patients. However, clones bearing VH3L, a subgroup of the VH3 family normally abundant in blood B cells but absent in B cells at the germinal center stage, were deficient in seven of nine CVI patients. Based on these findings and a reconsideration of previously reported B cell features in CVI, we propose that the disorder represents in most cases a maturational arrest of B cells at the germinal center stage.     

Germinal center cells express bcl-2 protein after activation by signals which prevent their entry into apoptosis

B cells undergo selection within germinal centers on the basis of their capacity to be activated by antigen held on follicular dendritic cells. Isolated germinal center B cells in culture kill themselves by apoptosis but this is prevented if their receptors for antigen are cross-linked. In this study it is confirmed that almost all germinal center B cells, unlike other B cells, do not express the 25-kDa protein encoded by the bcl-2 oncogene. Cross-linking the surface Ig of isolated germinal center cells causes them to express bcl-2 protein. Two other stimuli which inhibit the entry of germinal center cells to apoptosis result in the expression of bcl-2 protein. These stimuli are: (a) CD40 antibody and (b) recombinant 25-kDa fragment of the CD23 protein plus recombinant interleukin 1 alpha. Respectively, these induce germinal center cells to differentiate to resting B cells or plasmablasts. Dual-fluorescence studies on small lymphocytes confirm the presence of bcl-2 protein in mitochondria but show that this is also present in other extra-nuclear areas. Burkitt lymphoma cells have a phenotype which indicates that they are neoplastic cells of germinal center origin. The expression of bcl-2 protein by Burkitt lymphoma lines was also studied. Burkitt lines which retain the phenotype of fresh Burkitt lymphoma cells can be induced to enter apoptosis on culture with the Ca2+ ionophore ionomycin. These cells were found not to express bcl-2 protein. By contrast, Burkitt lines which have drifted towards a lymphoblastoid cell line phenotype and are resistant to the induction of apoptosis express high levels of the bcl-2 protein. The findings support the concept that the susceptibility of germinal center cells to entering apoptosis is associated with their lack of expression of bcl-2 protein. Aberrant expression of bcl-2 protein by some neoplastic germinal center cells may allow survival in situations where their normal counterparts die.