Dissecting affinity maturation: a model explaining selection of antibody-forming cells and memory B cells in the germinal centre

Until recently, the relationship between apoptosis, selection in the germinal centre (GC) and production of high-affinity antibody-forming cells (AFCs) and memory B cells has been unclear. Here, Tarlinton and Smith present a model that accounts for the switch in GC production from high-affinity AFCs to memory B cells, and explain how Bcl-2, an inhibitor of apoptosis, can influence memory cells but not bone marrow AFCs.     

B cell receptor signaling in germinal centers prolongs survival and primes B cells for selection

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High frequency of germinal centre derivation in diffuse large B cell lymphoma from Asian patients

BACKGROUND: Recent reports have divided diffuse large B cell lymphoma (DLBCL) into germinal centre B cell-like and activated B cell-like subgroups with implicated differences in prognosis.AIMS: To delineate the germinal centre B cell derivation group from an Asian series of DLBCLs. METHODS: Fifty four cases were analysed by polymerase chain reaction to detect the t(14;18) translocation and immunohistochemistry for BCL2, CD10, BCL6, and E2F1 expression. RESULTS: Eighteen of 54 cases had bcl2 gene rearrangement, 36 of 52 expressed BCL2, 29 of 52 expressed BCL6, 20 of 53 expressed CD10, and 18 of 53 expressed E2F1. There was a significant association between bcl2 gene rearrangement and the expression of both BCL2 and CD10. Using the minimally acceptable criteria of t(14;18) rearrangement and/or CD10 expression, 26 of 54 cases were probably germinal centre derived, in agreement with other reports. A higher proportion of cases had t(14;18) translocation, suggesting that they may be derived from transformed follicular lymphomas. E2F1 positivity did not correlate with the known germinal centre markers, even though it has recently been suggested that it may be a new GC marker. CONCLUSIONS: It may be possible to stratify patients for treatment using markers for specific lineages of B cell differentiation.     

Lymphotoxin acts as an autocrine growth factor for Epstein-Barr virus-transformed B cells and differentiated Burkitt lymphoma cell lines

A critical event in B cell immortilization by Epstein-Barr virus (EBV) is the establishment of an autocrine loop where cells produce a growth factor which supports their own proliferation. We investigated the potential of lymphoblastoid cell lines (LCL) and Burkitt lymphoma (BL) cell lines to produce and respond to the cytotoxins, tumor necrosis factor-α (TNF-α) and lymphotoxin (LT). Transformation in vitro of peripheral blood B cells by EBV from seven different donors resulted in spontaneous production of both LT (11542 pg/ml ± 7546, mean ± SD) and, to a lesser extent, TNF-α (197 pg/ml ± 174). Similarly BL cell lines derived from in vivo transformation which developed a ‘LCL-like’ phenotype in vitro (group III) produced more LT (1990 pg/ml ± 1740) than the ‘group I’ BL cell lines (< 40 pg/ml LT) which had maintained the original BL biopsy cell phenotype in vitro. Transformation of periphreal blood B cells to generate LCL also resulted in an increase in surface p75 (p < 0.02) and to a lesser extent p55 (not significant, ns) TNF receptor (TNF-R) expression. Similar increases in surface TNF-R (p75 p < 0.02, p55 ns) were observed on the ‘group III’ BL cell lines compared with the ‘group I’ BL cell lines. Proliferation of an LCL and a ‘group III’ BL cell line in vitro was via an autocrine loop since inhibition of LT reduced proliferation. This proliferation could also be blocked in the presence of the antagonistic anti-p55 TNF-R antibody, H398, but not the antagonistic antibody anti-p75 TNF-R antibody UTR-1. Furthermore, proliferation could be induced with the p55 agonistic antibody, HTR-9. In contrast to these observations with p55 TNF-R antibodies, two out of six of the ‘group III’ BL lines (Jijoye and Oba) only expressed the p75 TNF-R and proliferation of these cells could only be blocked by the antagonistic anti-p75 TNF-R antibody UTR-1. These data suggest that LT is an autocrine growth factor for lymphoblastoid cells, and BL cell lines which display an LCL phenotype. Furthermore, although both TNF-R are increased on the surface of these cells, this autocrine growth signal is mediated principally through binding to the p55 TNF-R.     

Follicular dendritic cells and T cells: nurses and executioners in the germinal centre reaction.

The humoral immune response constitutes an efficient system to protect the organism against diseases caused by invading pathogens. To guarantee a highly efficient defence, the humoral immune system has to be tightly regulated. Two cell subsets in particular, T cells and follicular dendritic cells (FDCs), contribute to the success of these regulation processes. Whereas the particular role of T cells is the elimination of autoreactive clones, the main role of FDCs is to present unprocessed antigen and check B-cell clones for higher affinity. B-cell clones unsuited for improved humoral immune response will be specifically killed. Involvement of Fas-mediated apoptosis might be an additional tool not only in T-cell-mediated regulation, but also in FDC-B cell interaction in the germinal centre.     

Follicular dendritic cells restrict interleukin-4 availability in germinal centers and foster memory B cell generation

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Triple check for antigen specificity of B cells during germinal centre reactions

Somatic hypermutation of the immunoglobulin variable genes during germinal reaction might permit the expansion of B-cell clones with unwanted (e.g. autoreactive) specificity. Here, Ernst Lindhout and colleagues propose three antigen-specific checkpoints that ensure the appropriate antigen specificity of activated B cells is maintained by regulating the activation, selection and further differentiation of B cells.     

Alpha6-integrin is expressed on germinal centre B cells and modifies growth of a B-cell line

The production of high‐affinity antibodies requires diversification of the antibody repertoire by somatic hypermutation followed by selection of those B cells bearing the highest affinity antibodies. Whilst many surface molecules that mediate the cell–cell interactions required for germinal centre formation have been identified, little is known of the importance of interactions with components of the extracellular matrix, i.e. fibronectin, collagen and laminin. We demonstrate that the laminin‐binding α6‐integrin is expressed on germinal centre B cells and is induced during the in vitro activation of naïve splenic B cells. A laminin network is demonstrated within the germinal centre. Analysis of an α6‐integrin‐expressing mouse B‐cell line, A20, demonstrates that this molecule is essential for binding to laminin, and that blocking by anti‐α6‐integrin immunoglobulin causes loss of adhesion associated with an increase in proliferation. There is no correlation with changes in BCL‐6 or Blimp‐1 expression, suggesting that α6‐integrin does not play a role in differentiation.     

An analysis of B cell selection mechanisms in germinal centers.

Affinity maturation of antibodies during immune responses is achieved by multiple rounds of somatic hypermutation and subsequent preferential selection of those B cells that express B cell receptors with improved binding characteristics for the antigen. The mechanism underlying B cell selection has not yet been defined. By employing an agent-based model, we show that for physiologically reasonable parameter values affinity maturation can be driven by competition for neither binding sites nor antigen--even in the presence of competing secreted antibodies. Within the tested mechanisms, only clonal competition for T cell help or a refractory time for the interaction of centrocytes with follicular dendritic cells is found to enable affinity maturation while generating the experimentally observed germinal centre characteristics and tolerating large variations in the initial antigen density.     

Effects of growth regulation on conditionally-transformed alginate-entrapped insulin secreting cell lines in vitro

The ability to control cell growth is an issue of critical importance for the use of transformed beta-cell lines within a bioartificial pancreas. Such control can be achieved either by entrapping the cells in a biomaterial that can inhibit cell proliferation or by genetically modifying the cells to regulate growth. Integrating tetracycline-off or -on operon systems into murine insulinoma cell lines (betaTC-tet and R7T1, respectively) allows cell growth regulation upon exposure to tetracycline (TC) or its derivative doxycycline (Dox), respectively. However, the effects of this regulatory approach on the long-term phenotypic metabolic and secretory stability of alginate-entrapped cells have yet to be thoroughly investigated. In this study, cultures of betaTC-tet and R7T1 cells entrapped in alginate beads were allowed to grow freely, or were growth-regulated, either at the onset, or after 20 days of growth. The data show that growth regulation of alginate-entrapped cells is achievable with chronic administration of the regulatory compound in a concentration-dependent manner. However, as these cultures age, the amount of insulin released does not always reflect the metabolic and histological characteristics of the cultures. This change, coupled with a loss of glucose stimulated insulin secretion in the Dox treated R7T1 cell line, indicate a phenotypic shift of cells with an activated tet-operon. These observations have implications on the selection and long-term function of three-dimensional bioartificial pancreatic constructs that include conditionally transformed beta-cell lines.     

Increased expression of TACI on NOD B cells results in germinal centre reaction anomalies,enhanced plasma cell differentiation and immunoglobulin production

B cells have an important pathogenic role in the development of type 1 diabetes in the non‐obese diabetic (NOD) mouse. We have previously reported that NOD mice display an increased percentage of TACI^high‐expressing B cells compared with C57BL/6 mice and this trait is linked to chromosomes 1 and 8. In this paper the genetic association of the transmembrane activator, calcium modulator and cyclophilin ligand interactor (TACI) trait was confirmed using double congenic NOD.B6C1/Idd22 mice. TACI ligation by a proliferation‐inducing ligand (APRIL) has been shown to influence plasma cell differentiation, immunoglobulin production and isotype switch. Hence, the functional consequence of the up‐regulation of TACI on NOD B cells was analysed both in vitro and in vivo. NOD B cells stimulated with APRIL showed an enhanced plasma cell differentiation and class switch to IgG and IgA compared with B cells from C57BL/6 mice. Moreover, flow cytometry analyses revealed that germinal centre B cells in NOD failed to down‐regulate TACI. Availability of the TACI ligand B‐cell activating factor (BAFF) has been shown to be a limiting factor in the germinal centre reaction. In line with this, upon immunization with 4‐hydroxy‐3‐nitrophenylacetyl hapten‐conjugated hen egg lysozyme, NOD mice produced higher titres of low‐affinity antibodies compared with C57BL/6 mice. This observation was supported by the detection of increased levels of BAFF in NOD germinal centres after immunization compared with C57BL/6 by immunofluorescence. Our results support the hypothesis that increased TACI expression on NOD B cells contributes to the pathogenesis of type 1 diabetes in the NOD mouse.     

Follicular fluid as a favourable environment for endometrial and endometriotic cell growth in vitro

Follicular fluid from women with endometriosis has been shown to induce a higher endometrial cell proliferation than that derived from women without the disease. To elucidate this issue further, the aims of the present study were to compare the ability of follicular fluid and peritoneal fluid to stimulate both endometrial and endometriotic cell proliferation and to verify whether the mitogenic effect was merely sex steroid-dependent. Endometrial and endometriotic cells were cultured in follicular fluid or peritoneal fluid diluted in serum-free media; the growth induced in these conditions was compared with that obtained by culturing these cells in medium supplemented with charcoal stripped calf serum and a correspondent content of 17-beta-oestradiol and progesterone. Follicular fluid was able to induce significantly higher cell proliferation than peritoneal fluid from controls, patients with endometriosis stage I-II and women with endometriosis stage III-IV (P < 0.05). Moreover, the growth in control media containing a corresponding amount of steroid hormones was significantly lower than that obtained with follicular or peritoneal fluids. This finding indicates that the stimulating effect is not simply related to the concentrations of 17-beta-oestradiol and progesterone present in these fluids. Finally, based on these results and on other previous observations, the hypothesis that follicular fluid may be involved in the development of endometriotic ovarian cysts is discussed.     

Recirculation of germinal center B cells: a multilevel selection strategy for antibody maturation

Summary:  Optimization of antibody affinity is a hallmark of the humoral immune response. It takes place in hundreds of transient microstructures called germinal centers (GCs). Their function and time-dependent behavior are subjects of active investigation. According to a generally accepted notion, their individual kinetics follows the average kinetics of all GCs present in the observed lymphatic tissue. In this review, we challenge this view and point out, with the help of mathematical simulations, that inferring the kinetics of individual GCs from cross-sectional evaluation of GC kinetics is virtually impossible. Thus, the time course of individual GCs is open to conjecture. For instance, one possible interpretation is that GCs exist for a time span considerably shorter than that of the observed average kinetics. We explore the implications of different temporal organizations of GCs in the light of the hypothesis that GC B-cell emigrants recolonize GC niches. This assumption leads to a view where GCs work in parallel but are linked by recirculation of B-cell emigrants. In this view, interleaved global and local competition provide for an implementation of multiple levels of B-cell selection in affinity maturation. The concepts of iteration, all-or-none behavior, and phasic mutation schedule are discussed in the light of this hypothesis.     

T cell interactions with B cells during germinal center formation,a three‐step model

Establishment of effective immunity against invading microbes depends on continuous generation of antibodies that facilitate pathogen clearance. Long‐lived plasma cells with the capacity to produce high affinity antibodies evolve in germinal centers (GCs), where B cells undergo somatic hypermutation and are subjected to affinity‐based selection. Here, we focus on the cellular interactions that take place early in the antibody immune response during GC colonization. Clones bearing B‐cell receptors with different affinities and specificities compete for entry to the GC, at the boundary between the B‐cell and T‐cell zones in lymphoid organs. During this process, B cells compete for interactions with T follicular helper cells, which provide selection signals required for differentiation into GC cells and antibody secreting cells. These cellular engagements are long‐lasting and depend on activation of adhesion molecules that support persistent interactions and promote transmission of signals between the cells. Here, we discuss how interactions between cognate T and B cells are primarily maintained by three types of molecular interactions: homophilic signaling lymphocytic activation molecule (SLAM) interactions, T‐cell receptor: peptide‐loaded major histocompatibility class II (pMHCII), and LFA‐1:ICAMs. These essential components support a three‐step process that controls clonal selection for entry into the antibody affinity maturation response in the GC, and establishment of long‐lasting antibody‐mediated immunity.     

Prognostic significance of CD44 expression in diffuse large B cell lymphoma of activated and germinal centre B cell-like types: a tissue microarray analysis of 90 cases

BACKGROUND: Gene expression profiling of diffuse large B cell lymphoma (DLBCL) revealed three disease types: germinal centre B cell-like (GC), activated B cell-like (ABC), and a "third" type. Expression of CD44 variant isoforms (CD44v) is associated with an unfavourable clinical outcome in DLBCL, but previous studies did not consider the clinicopathological heterogeneity of this disease. AIMS: To analyse the expression and prognostic significance of CD44 in DLBCL types. METHODS: A tissue microarray (TMA) comprising 90 DLBCLs was constructed. CD10, CD20, bcl-2, bcl-6, CD44 standard isoform (CD44s), and CD44v4, CD44v6, and CD44v9 were analysed immunohistochemically and correlated with clinical follow up. RESULTS: TMA expression of CD10, CD20, bcl-2, and bcl-6 showed 100% concordance with results from conventional sections in 60 cases. Samples were segregated into 22 GC (bcl-6+/CD10+/bcl-2-), 25 ABC (bcl-6-/CD10-/bcl-2+), and 35 unclassifiable DLBCLs. Overall survival (OS) at 30 months was 89%, 44%, and 58% in GC, ABC, and unclassified types, respectively. CD44v6 was coexpressed with bcl-2, appeared predominantly on bcl-6 negative cases, and correlated with disease stage. Cases negative for CD44s could be separated into CD44v6 negative (OS, 82% at 70 months) and CD44v6 positive (OS, 58%). CONCLUSIONS: TMA technology is useful for immunophenotyping and clinicopathological analysis of large lymphoma populations. The GC phenotype of DLBCL is of independent prognostic significance for OS. Expression of CD44v6 correlates with disease stage, and might contribute to lymphoma dissemination. CD44v6 is expressed predominantly in ABC DLBCL, and in CD44 negative cases is associated with worse OS.     

Disodium cromoglycate inhibits immunoglobulin production in vitro without affecting cell growth in human B cell lines

The effect of disodium cromoglycate (DSCG) on human B cell lines (IM-9, GM-1056, and AF-10) was studied. DSCG inhibited immunoglobulin production by these B cell lines without affecting thymidine uptake or cell number. Thus, in addition to its antiallergic function, DSCG may also act as a B cell-modulating reagent in vitro.     

Regulation of VH gene repertoire and somatic mutation in germinal centre B cells by passively administered antibody

Immunization with T-dependent antigens induces a rapid differentiation of B cells to plasmacytes that produce the primary immunoglobulin M (IgM) and IgG antibodies with low affinities for the immunogen. It is proposed that the IgG antibody forms immune complexes with the residual antigen which provide an important stimulus for the formation of germinal centres (GC) and the activation of somatic mutation. This hypothesis was tested by passive administration of hapten-specific antibody into mice shortly after the immunization with nitrophenyl (NP) coupled to chicken gamma globulin (NP-CGG) in an environment of limited T-cell help. Athymic mice that received normal T helper cells at 72 hr after the administration of antigen produced low levels of anti-NP antibody and the splenic GC formation was delayed until day 12 after the antigen administration. The analysis of VDJ segments from NP-reactive GC B cells showed very few mutations in the VH genes. Passive injection of anti-NP IgG1 monoclonal antibody - but, not IgM - stimulated the GC formation up to normal levels and the somatic mutation activity in the GC B cells was fully restored. In addition, GC B cells in the recipients of IgG1 antibody demonstrated a change in the usage of germline-encoded VH genes which was not apparent among the primary antibody-forming cells. These results suggest the existence of a specific feedback mechanism whereby the IgG antibody regulates the GC formation, clonotypic repertoire and somatic mutation in GC B cells.     

Human B cell colony growth from pre-B cells in vitro.

We describe a simple one-step technique for the growth of human B cell colonies in semi-solid agar in vitro. This method used conditioned medium from the human plasmacytoma cell line LICR-LON-H My 2 as a source of stimulating activity. A linear relationship exists between the number of B cells seeded and the number of colonies formed (r = 0.95). Most colony forming cells, approximately 1 in 500 of B cells seeded, lack surface immunoglobulin, possess Fc receptors and mark with the Leu 12 monoclonal antibody. Cells within developing colonies are found to have cytoplasmic IgM, IgA and IgG depending on the length of time in culture.