Germinal center development

Summary: Using a set of surface markers Including igD and CD38, human tonsillar B cells were classified into discrete subpopulations. Molecular and functional analysis allowed us to identify: i) two sets of naive B cells (Bml and Bm2); ii) germinal center founder cells (Bm2'); iii) an obscure population of germinal center B cells, displaying a high load of somatic mutations in IgV genes, Cμ to Cδ switch and preferential Igλ light chain usage: these cells may represent the precursors of normal and malignant IgD-secreting plasma cells; iv) the centroblasis (Bm3) in which somatic mutation machinery is activated; v) the centrocytes (Bm4) in which isotype switch occurs; vi) the memory B cells. The characterization of these sub-populations showed that: i) programmed ceil death is set before somatic mutations, possibly providing an efficient way for affinity maturation; ii) only high affinity centrocytes are allowed to switch isotype; iii) CD40-hgation inhibits plasmacytic differentation of mature B lymphocytes; Iv) memory B cells preferentially differentiate into plasma cells; v) IgD isotype switch occurs in normal B cells; vi) receptor editing may be induced by somatic mutations in germinal centers. We also characterized two types of antigen -presenting cells in germinal centers: follicular dendritic cells that select high affinity B cells, and a new subset of germinal center dendritic cells that activate germinal center T cells.     

Germinal center responses to complex antigens

Germinal centers (GCs) are the primary sites of antibody affinity maturation, sites where B-cell antigen-receptor (BCR) genes rapidly acquire mutations and are selected for increasing affinity for antigen. This process of hypermutation and affinity-driven selection results in the clonal expansion of B cells expressing mutated BCRs and acts to hone the antibody repertoire for greater avidity and specificity. Remarkably, whereas the process of affinity maturation has been confirmed in a number of laboratories, models for how affinity maturation in GCs operates are largely from studies of genetically restricted B-cell populations competing for a single hapten epitope. Much less is known about GC responses to complex antigens, which involve both inter- and intraclonal competition for many epitopes. In this review, we (i) compare current methods for analysis of the GC B-cell repertoire, (ii) describe recent studies of GC population dynamics in response to complex antigens, discussing how the observed repertoire changes support or depart from the standard model of clonal selection, and (iii) speculate on the nature and potential importance of the large fraction of GC B cells that do not appear to interact with native antigen.     

Germinal center kinases in immune regulation

Germinal center kinases (GCKs) participate in a variety of signaling pathways needed to regulate cellular functions including apoptosis, cell proliferation, polarity and migration. Recent studies have shown that GCKs are participants in both adaptive and innate immune regulation. However, the differential activation and regulatory mechanisms of GCKs, as well as upstream and downstream signaling molecules, remain to be fully defined. It remains unresolved whether and how GCKs may cross-talk with existing signaling pathways. This review stresses the progresses in research of GCKs relevant to the immune system.     

Germinal center derived malignant lymphoma in cystadenolymphoma

Summary Two cases of malignant non-Hodgkin's lymphoma arising in an yAlbrecht-Arzt-tumour are reported. In the first case a centroblastic-centrocytic lymphoma in a palatinal cystadenolymphoma of a 64-year-old female is described. In the other case a centroblastic lymphoma developed in an Albrecht-Arzt-tumor of the submandibular region in an 82-year-old man. The occurence of a high-grade malignant lymphoma in cystadenolymphoma has not been reported in the literature so far.     

Germinal center B cells and mixed leukocyte reactions

The present study was undertaken to determine if germinal center (GC) B cells are sufficiently activated to stimulate mixed leukocyte reactions (MLR). Percoll density fractionation and a panning technique with peanut agglutinin (PNA) were used to isolate GC B cells from the lymph nodes of immune mice. The GC B cells were treated with mitomycin C or irradiation and used to stimulate allogeneic or syngeneic splenic T cells in the MLR. Controls included high-density (HD) B cells prepared from spleens of the same mice and HD B cells activated with lipopolysaccharide (LPS) and dextran sulfate. GC B cells bound high amount sof PNA (i.e., PNAhi). Similarly, the LPS-dextran sulfate-activated B cells were PNAhi. Treatment with neuraminidase rendered the PNAlo HD B cells PNAhi. GC B cells and the LPS-dextran sulfate-activated HD B cells stimulated a potent MLR, while the untreated HD B cells did not. However, following neuraminidase treatment, the resulting PNAhi HD B cell population was able to induce an MLR. The PNA marker appeared to be an indicator of stimulatory activity, but incubating the cells with PNA to bind the cell surface ligand did not interfere with the MLR. GC B cells were also capable of stimulating a syngeneic MLR in most experiments although this was not consistently obtained. It appears that germinal centers represent a unique in vivo microenvironment that provides the necessary signals for B cells to become highly effective antigen-presenting cells.     

Germinal center structure and function: lessons from CD19

The germinal center is a critical locus in the production of protective immunity, but its function is poorly understood. Studies of mutant forms of CD19 revealed differences in signaling in different compartments inside the germinal center, and structural findings indicate a selective role in the interaction with follicular dendritic cells in the GC. Loss of these signals leads to surprising changes in germinal center B cells that challenge previous models of GC function.     

Germinal center T cells are distinct helper-inducer T cells.

Germinal centers (GCs) contain a significant number of CD4+ T cells, but what role these T cells may play in the development of GC B cells has not been determined. To gain insight into their role, we studied the phenotype of GC T cells and the lymphokines secreted by GC T cells isolated from human tonsils obtained after tonsillectomies. In addition to confirming that a large fraction of GC T cells are Leu-7(CD57)+ and Leu-8-, we found that they have no binding sites for peanut agglutinin. Furthermore, we found that they are CD45RA- and CD45R0+, the phenotype of helper-inducer T cells. We also found that Leu-7(CD57)+ cells display CD69, a phenotypic marker of very early cell activation, but do not display three other markers of cell activation: CD25 [interleukin-2 (IL-2) receptor], CD71 (transferrin receptor), and DR. When isolated, Leu-7(CD57)+ cells were stimulated in vitro with a mitogen that can induce peripheral blood T cells with the helper-inducer phenotype to produce various cytokines, Leu-7(CD57)+ cells did not produce IL-2, interleukin-4 (IL-4), interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha) in significant amounts. Taken together, GC T cells from a distinct subpopulation of T cells with helper-inducer phenotype by their histologic location, by their surface phenotype, and by their ability to produce lymphokines. This finding is consistent with the possibility that GC T cells have been selectively recruited to actively help B cells develop in GCs.     

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.     

Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5

Germinal center (GC) dark and light zones segregate cells undergoing somatic hypermutation and antigen-driven selection, respectively, yet the factors guiding this organization are unknown. We report here that GC organization was absent from mice deficient in the chemokine receptor CXCR4. Centroblasts had high expression of CXCR4 and GC B cells migrated toward the CXCR4 ligand SDF-1 (CXCL12), which was more abundant in the dark zone than in the light zone. CXCR4-deficient cells were excluded from the dark zone in the context of a wild-type GC. These findings establish that GC organization depends on sorting of centroblasts by CXCR4 into the dark zone. In contrast, CXCR5 helped direct cells to the light zone and deficiency in CXCL13 was associated with aberrant light zone localization.     

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.     

Germinal center kinetics in lymph nodes of primed mice stimulated with complexed as opposed to free antigen

Primed mice with low titers of circulating tetanus antitoxin (AB) were stimulated via the hind footpads with either fluid tetanus toxoid alone (AG) to create in vivo complexes in AG excess, or the same dose of toxoid complexed at equivalence with isologous antibody (AB-AG CPX), to have in vivo complexes in AB excess. All experimental animals reacted with three topically distinct consecutive waves of enhanced proliferative activity in popliteal lymph nodes, i.e., in the T-zone (peak on day 2), in the medullary area, the main site of plasmocytopoiesis (day 3), and in lymphoid follicles (day 5-6). Maximum serum AB titers following injection of AG-AB CPX were only about 25% of those found in animals boosted with AG alone. This suppressive effect was best reflected in a comparable reduction in plasmocytopoiesis, and to an lesser extent in the proliferative activity within the T-zone, and not at all in the overall magnitude of germinal center formation and/or expansion. However, the patterns of germinal center kinetics differed markedly between the two groups: a high sharp peak of development on day 5, followed by a marked drop on day 6 characterized the response in mice given AG alone, and a broad peak around day 6 that of those receiving AG-AB CPX. These differences could not adequately be accounted for by variations in centroblast/centrocyte proliferation rate vs. pycnotic indices, so that different patterns of lymphoid cell emigration from the centers may be considered. The results suggest that immune complexes, fixed on follicular dendritic cells, with different antigen-to-antibody ratios have divergent effects on the development and kinetics of germinal centers, the principal sites of memory B cell generation.