Germinal centres and the origin of the B-cell system. I. Germinal centres in the rabbit appendix

Regeneration of follicular structures in the rabbit spleen after a single dose of 450 rads whole body X-irradiation was shown to be dependent upon the presence of the appendix.Results obtained were in favour of a stream of cells—probably derived from appendix germinal centres—to follicular structures in the spleen, in particular to the lymphocyte corona and marginal zone but excluding germinal centres.Recovery of the antibody forming potential following X-irradiation was equally shown to be dependent upon the presence of the appendix.It was concluded that following 450 rads whole body X-irradiation the appendix contributes to the recovery of the antibody forming capacity of the spleen by the production of antibody-forming cell precursors; these latter cells are presumably identical with the follicle replenishing class of lymphocytes derived from appendix germinal centres.     

Localization of 125I-labelled antigen in germinal centres of mouse spleen: effects of competitive injection of specific or non-cross-reacting antigen

Studies were performed on localization of ¹²⁵I-human γ-globulin in spleen lymphatic tissue germinal centres during the primary and secondary immune response as influenced by competitive injections of specific or non-cross-reacting antigens. Isologous mouse 7S serum protein labelled with ¹²⁵I was used as the control. The results of these studies support the following conclusions:

(1) Antigen retention in germinal centres during the primary immune reaction is a dynamic process. For some antigens there may be opsonins available at the the time of injection which promote initial localization in germinal centres. However, the continued localization of antigen over weeks and months is a function of specific antibody production.

(2) For some period of time, germinal centres are specific to the antigen that stimulated their development, and eventually these centres will respond to a different antigen.

(3) Antigen persisting in germinal centres is functional in the development of the secondary immune potential.

     

A defect of B-lymphocyte transport of aggregated HGG into germinal centres in NZB and NZB×NZW F1 hybrid mice

Previous experiments have shown (i) that the localization of intravenously injected heat aggregated human γ-globulin (HGG) in the germinal centres of normal mice provides a model for studying the natural uptake of circulating immune complexes in these areas, and (ii) that the aggregated HGG is carried into germinal centres by lymphocytes which have receptors for altered γ-globlin.

Evidence from thymus-cell depletion experiments is now presented which suggests that the lymphocytes concerned are bone-marrow-derived B cells. Defective localization was found in NZB and NZB × NZW F₁ hybrids at different ages and the onset of the autoimmunity and the appearance of histological abnormalities in the spleen. As disease develops it progresses to a complete inability to localize complexes in germinal centres. It is concluded that a functional defect of the bone marrow-derived lymphocyte population exists in these mice.

     

Life and death within germinal centres: a double-edged sword

Within germinal centres, B lymphocytes are destined to die by apoptosis via Fas signalling, unless they are positively rescued by antigen and by signals initiated by CD40-CD154 interactions. Thus, while the germinal centre microenvironment can become a virtual graveyard for most B lymphocytes that fail to bind antigen with high affinity, it concomitantly provides the necessary stimuli for the survival of cells that successfully accomplish affinity maturation. Such dichotomy in the physiology of germinal centre reaction that results in survival of the functional B-cell repertoire and the elimination of abnormal cells, dictates the fate towards B-cell homeostasis or disease. Consequently, the death and survival-signalling arms within germinal centres predominantly reside on the timely and controlled expression of Fas and its ligand (FasL), and CD40 and CD154, respectively. In keeping with this notion, lymphoproliferation or deficient immunity are documented landmarks of inactivation of either the Fas/FasL or CD40/CD154 signalling pathways. The present review considers two different scenarios in the control of B-cell survival and death within germinal centres. The first is an idealistic scenario, in which a discriminatory and co-ordinate signalling initiated by the CD40/CD154 and Fas/FasL pairs, respectively, leads the rescue of the functional B-cell repertoire and the elimination of the abnormal phenotype. The second is a gloomy scenario in which both the lack and the hyperexpression of either receptor/ligand pairs, are seen as equally deleterious.     

Thymic germinal centres in myasthenia gravis: a correlative study

The density of germinal centres in the thymic medulla in twenty-three cases of myasthenia gravis correlated inversely with age but did not correlate with sex, the duration of symptoms of myasthenia gravis, the presence of thymoma, or the result of tests for antibody to striated muscle and thymic epithelial cells.

The number of germinal centres formed in lymphoid tissues during an immune response in man is known to decrease with increasing age and this probably applies to autoimmune reactions. The inverse correlation of thymic germinal centres and age in myasthenia gravis is thus consistent with an autoimmune reaction occurring in the thymus in all cases of myasthenia gravis.

     

Light-chain-restricted germinal centres in reactive lymphadenitis: report of eight cases

Aims:  Light-chain-restricted germinal centres are generally associated with the existence of a neoplastic lymphoproliferative disorder. The aim was to present a series of cases with persistent lymph node enlargement that featured some germinal centres showing light chain immunoglobulin restriction.
Methods and results:  A series of six reactive lymphadenitis and two Castleman's disease cases was analysed by immunohistochemistry, IgH-polymerase chain reaction (PCR) and microdissected PCR. In all cases some germinal centres contained a population of plasma cells and plasmacytoid germinal centre cells showing light chain immunoglobulin restriction. In three cases the monotypic cells also showed distinct Bcl-2 expression. Two of the cases showed a predominant IgH rearrangement on a florid polyclonal background and one had an IgH monoclonal rearrangement, as revealed by PCR. Microdissected germinal centre PCR revealed a dominant repeated band in one of three cases and in another case a non-repeated clonal peak was observed. One of the patients developed a follicular lymphoma, which became evident from a subsequent biopsy.
Conclusions:  These findings may be a manifestation of an underlying disorder in the regulation of the immune response, or an exaggeration of the germinal centre oligoclonal nature. This should be taken into account in the differential diagnosis of follicular hyperplasia.     

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.     

Germinal centre proliferation in response to mitogenic lymphokines

Lymphokines which were mitogenic for cultured lymphocytes caused germinal centre enlargement within the regional lymph node following their intralymphatic injection. It was found that 17 μg of a lymphokine preparation, produced by 2·5 × 10⁴ peritoneal exudate lymphocytes, resulted in a 14-fold increase in germinal centre area and a 7-fold increase in the labelled cell content of these centres on the 3rd day after injection.

Since the daily rate of lymphocyte recirculation through the regional node would supply sufficient numbers of antigen-sensitive lymphocytes to generate this amount of mitogenic lymphokine following antigenic stimulation, it is argued that lymphokine-induced germinal centre enlargement plays a physiological role in immunoregulation.

     

Lack of Shiga-like toxin binding sites in germinal centres of mouse lymphoid tissues

B cells in germinal centres are known to express carbohydrate antigen CD77 in human lymphoid tissues. The CD77 antigen is specifically recognized by Shiga-like toxins (SLTs) that are produced by enterohaemorrhagic Escherichia coli O157:H7. To determine whether the binding subunits of Shiga-like toxin-1 (SLT-1B) could have adverse effects on the murine immune system when used as an immunogen, we investigated whether SLT-1B could bind to germinal centres of mouse lymphoid tissues. Frozen sections of peripheral lymph nodes and Peyer's patches from immunized mice were tested for the presence of SLT-1B-binding sites by immunohistological methods. Germinal centres were not stained with SLT-1B, while they were intensely stained with peanut agglutinin (PNA), another marker of germinal centres. On the other hand, SLT-1B specifically bound to renal tubules and collecting ducts in frozen sections of mouse kidney. This is consistent with results from human tissues. We also demonstrated that B220/PNA double-positive populations in lymph nodes from immunized mice exhibited only marginal staining with SLT-1B. The present results suggest that SLTs would not impede germinal centre functions of the murine immune system.     

A novel monoclonal antibody (FUN-1) identifies an activation antigen in cells of the B-cell lineage and Reed—Sternberg cells

The characterization of a new monoclonal antibody (MoAb) recognizing a human B-cell activation antigen, designated FUN-1, is described in this paper. Immunoprecipitation revealed that FUN-1 recognizes an antigen with a molecular weight (MW) of 75 kD. FUN-1 reacts with pokeweed mitogen-activated B lymphocytes and monocytes of peripheral blood, but not with unstimulated lymphocytes or granulocytes. It also reacts with large lymphoid cells in germinal centres, Epstein–Barr virus-transformed B cell lines, large B-cell lymphomas, Ki-1-positive anaplastic largecell lymphomas, and Reed–Sternberg cells of Hodgkin's disease, but not with Concanavalin A-activated T cells, acute lymphoblastic leukaemias, T-cell lymphomas, or low-grade B-cell leukaemias. These findings indicate that FUN-1 recognizes a previously unreported B-cell activation antigen. This MoAb appears to be useful for the study of maturation and differentiation in the B-cell lineage as well as for the immunohistochemical diagnosis of B-cell lymphomas and Hodgkin's disease.     

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.     

The localization of aggregated human γ-globulin in the spleens of normal mice

The localization of aggregated human γ-globulin in the germinal centres of the white pulp of the spleens of normal mice was found to be dependent on the ability of the spleen to concentrate this material from the blood and of lymphoid cells in the mantle layer of the Malpighian bodies to take it up on their surface membranes. The fact that lymphoid cells resident in the spleen perform this function was shown by the inability of lymphoid cells transferred to recipient animals to transport the aggregated material to the spleens of the latter.

Ionizing radiation in relatively high doses prevented the localization of aggregated human γ-globulin and was effective only when given before or less than 6 hours after administration of the aggregated material. Spleens shielded during whole body irradiation were capable of localizing the material in germinal centres, but local irradiation of the spleens with protection of the rest of the body prevented splenic localization.

These results support the conclusion that localization of altered heterologous antibody is a function of lymphoid cells already present in the spleens at the time of injection of this material and is not dependent on its transport into the white pulp by migrating cells which pick it up in the blood or elsewhere.

Antilymphocytic serum also prevented localization of aggregated human γ-globulin, suggesting a direct effect of this antiserum on non-circulating lymphoid cells. However, the manner of its action requires further study.

     

De novo expression of MECA-79 glycoprotein-determinant on developing B lymphocytes in gut-associated lymphoid tissues

Rabbit is one of several species that depend on development of B lymphocytes in gut-associated lymphoid tissues for primary immunoglobulin-repertoire diversification. The rabbit appendix is an important site of early B-lymphocyte development. We previously reported that peripheral lymph node addressin detected by monoclonal antibody (mAb) MECA-79 played a role in recruitment of immature blood-borne B cells into neonatal rabbit appendix. Here, we report expression of an approximately 127 000 MW O-linked sulphated proteoglycan on developing B cells in appendix and Peyer's patches recognized by the mAb MECA-79. Binding of the mAb to B lymphocytes was sensitive to enzyme treatment with O-sialoglycoprotease and expression was partially inhibited by sodium chlorate, a metabolic inhibitor of sulphation. The proportions of MECA-79(+) B lymphocytes gradually increased from < 0.5% at 3 days to > 70% at 6 weeks in appendix and Peyer's patches. The proportions of MECA-79(+) B lymphocytes in spleen and peripheral blood were very low (0.5-2%). However, the MECA-79 determinant was detected on B cells in splenic germinal centres after immunization. In situ labelling of appendix cells showed that the MECA-79 determinant was expressed on fluorescein-labelled B lymphocytes that migrated from appendix into mesenteric lymph nodes. B-cell MECA-79 may be involved in interactions with T cells and/or dendritic cells. Alternatively, because we found that lymphatic endothelium in the thymus-dependent area of appendix, a site for lymphocyte exit, expressed P-selectin (CD62P), interaction of the MECA-79 determinant on B cells with CD62P may have a role in the exit of B lymphocytes from rabbit appendix.     

Oligoclonal Development of Germinal Centre Cells as Detected by Anti-Idiotype Antibodies

Experimental models using immunization with known haptens have shown that germinal centres develop from only a limited number of precursor cells which expand in an oligoclonal fashion. Using a panel of antibodies raised against the idiotype of follicle centre cell lymphomas and known to be reactive only with a few normal B cells, we could demonstrate that individual clones can be identified within germinal centres of individual follicles of human lymph nodes and tonsils. Therefore also in humans in a non-experimental, natural situation, germinal centres expand in an oligoclonal fashion.     

Phylogenetic studies on the immune response: I. Localization of antigens and immune response in the toad, Bufo marinus

Toads of the species Bufo marinus were injected subcutaneously with ¹²⁵I-labelled flagella from Salmonella adelaide. Observations were made on the ensuing serum antibody response, the antigen localization pattern and sequential cellular changes in lymphatic and other tissues.

The serum antibody findings confirmed the work of previous investigators in showing a good primary response, prolonged synthesis of mercaptoethanol sensitive antibody and little or no evidence of secondary responsiveness.

Antigen became localized in the jugular bodies and spleen where proliferation of pyroninophilic cells could be observed after 5 days. Both the antigen-trapping cells and the first pyroninophilic blasts were scattered randomly throughout the jugular bodies. There was no clear-cut separation into cortex and medulla. Nothing resembling the antigen-trapping web of rat lymph node follicles was observed, nor were there any germinal centres. In the spleen, antigen was trapped in the red pulp and some degree of concentration around the islands of white pulp could be noted 1 day later. However, unlike in the rat, entry of antigen into the white pulp did not occur.

Both focal and diffuse collections of lymphoid and pyroninophilic cells were found in the kidney after antigenic stimulation. It seems likely that the kidney is a major antibody-forming organ in the toad.

The hypothesis is advanced that the absence of immunological memory may be due to the absence of the follicular antigen-trapping web and of resultant germinal centres.

     

Complement activation in human lymphoid germinal centres

The presence of complement activation products has been studied in morphologically normal human lymphatic tissue from tonsil, spleen and lymph node. Newly established monoclonal antibodies (mAbs) with reactivity against the C4 cleavage fragments C4a, C4b, C4c and C4d were applied on cyrostat sections in the indirect immunoperoxidase staining technique. Irrespective of organ type, C4d activation product could be detected in germinal centres of all secondary lymphoid follicles. To substantiate this finding, the complete sequence of complement activation products was investigated by a series of mono- and polyclonal antibodies to the complement proteins C1, C2, C3, factor B, C5, C9 to C5b-9 neoantigens and to the regulatory complement proteins C4 binding protein (C4bp), factor I, factor H and properdin. Similar to C4d, all secondary follicles exhibited a strong staining reaction for C3d antigens restricted to germinal centres. At the same site, albeit with distinctly weaker intensity, components of the membrane attack complex (MAC) C5b-9 were found. The simultaneous deposition of C1, C4b and C4bp in certain germinal centres indicates that complement activation is induced via the classical pathway. Concomitant deposition of IgM suggests IgM-antigen complexes that have been trapped on follicular dendritic cells (FDC) during normal immune response as the most likely candidates for activators of the classical pathway. Our data demonstrate that human lymphoid germinal centres as important sites of immune regulation closely interrelate with the complete cascade of complement-activation products, including the membrane attack complex (MAC).