Icm. Maclennan

The Evolution of B-Cell Clones

This chapter identifies three forms of B-cell memory: (a) B blasts which characterize the established stage of the follicular response to TD antigens, (b) recirculating memory B cells, and (c) non-recirculating memory B cells of the marginal zones of the spleen and equivalent areas of other secondary lymphoid organs. The follicular B blasts show sustained proliferation driven by small amounts of antigen bound to FDCs. The probable relationships between these cells is summarized diagrammatically in Fig. 4. It is probable that follicular B blasts generate both the recirculating and marginal zone memory cells. The chapter by Gray and Leanderson in this volume cites data which indicate that the recirculating memory pool is not sustained for more than a few weeks in the absence of antigen. Data leading to the same conclusion for marginal zone memory B cells is set out in Sect. 5.1 of this chapter. Marginal zone memory B cells do not appear to move spontaneously to follicles for periodic renewal. They will only leave the marginal zone if a fresh supply of antigen reaches them in that site. Recirculating B cells are able to respond to antigen already held on FDCs. It is not known if they are able to displace B blasts of equivalent affinity for antigen which already occupy antigen-holding sites on FDCs. This could be a mechanism by which B blasts with high antigen affinity produced in one follicle could displace blasts of lower affinity in other follicles. Little is known of the factors which regulate the numbers of marginal zone and recirculating follicular memory B cells. In responses to hapten-protein conjugates, hapten-binding cells may approach 10% of marginal zone B cells but comprise well under 1% of recirculating follicular cells. The numbers of these memory cells do not increase if the recirculating pool of lymphocytes is depleted, indicating that the factors which regulate the number of memory B cells are independent of those which regulate the total size of the recirculating B-cell pool. A depleted peripheral B-cell pool can only be fully reconstituted by recruitment of newly produced virgin B cells. Data cited in Sect. 5.2 support the concept that this recruitment is at least partially independent of antigen-driven B-cell proliferation. Consequently, substantial proportions of the peripheral B-cell pools are likely to be either virgin cells or cells which have been recruited by antigen or anti-idiotype without entering cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparative Analysis of the Development of B Cells in Marginal Zones and Follicles

Two distinct B cell compartments can be identified in rat spleen. The follicles which contain small recirculating B cells which express surface IgM and IgD and the marginal zones, which are populated with static IgM+ve IgD-ve B cells (1,2). Increasing evidence implicates marginal zone B cells in responses to T cell-independent type 2 (TI-2) antigens and recirculating cells in responses to T cell-dependent antigens (3). This paper describes observations which give insight into the development and maturation of the recirculating follicular (RF) B cells and marginal zone (MZ) B cells.

The Origin of Marginal-Zone Cells

The marginal zone is the outer compartment of the white pulp of mammalian spleens. Most cells found in this area are intermediate-sized lymphocytes (1,2). Surface marker analyses (3–6) have provided strong evidence that these cells are B lymphocytes. Also marginal zone lymphocytes are absent from the spleens of rats treated with heterologous anti-μ antibody from birth (6). In rat spleens marginal-zone B cells outnumber the B lymphocytes found in the follicles by a factor of two to three (6,7).

CBA/N mice have marginal zone B cells with normal surface immunoglobulin phenotype.

Antigens can be broadly classified into those which require T cell help to evoke an immune response and those which do not (1). However, the T cell-independent (TI) antigens can be further subdivided on the basis of time during ontogeny when they can evoke antibody respones (2). In man the TI-1 group of antigens can activate B cells in vivo before birth while TI-2 antigens fail to evoke normal responses until some months after birth. Another criterion which allows the TI-1 and TI-2 antigens to be distinguished is the relative capacity of these two groups of antigens to generate antibody responses in CBA/N mice (3). TI-1 antigens evoke responses in these mice while TI-2 antigens do not. TI-2 antigens are mostly based upon a core structure of polysaccharide. Haptens linked to polysaccharides such as Ficoll or hydroxyethyl starch, behave as TI-2 antigens.

Evidence that static but not recirculating B cells are responsible for antibody production against dinitrophenol on neutral polysaccharide, a TI-2 antigen.

The peripheral B cell pool of the rat can be divided into a subpopulation that recirculates between follicles in secondary lymphoid organs and one that is static (1). A large component of the non-recirculating population is found in the marginal zones (MZ) of the spleen. MZ B cells differ from recirculating follicular (RF) B cells in lacking surface membrane (Sm) IgD. Both express Sm IgM (2). Recently, several lines of indirect evdence have emerged that infer the involvement of MZ B cells in responses to thymus-independent type-2 (TI-2) antigens: i) Rats suppressed from birth with anti-IgD antibodies develop intact MZ, but not RF B cell populations (3). ii) This treatment results in an elevation of of serum IgG2c with loss of IgG2a (3). IgG2c is the predominant isotype elicited in response to TI-2 antigens, while IgG2a is dominant in T cell-dependent responses (4). iii) Approximately 20% of MZ B cells express Sm IgG2c (3). iv) Dendritic cells within the marginal zone selectively localise neutral polysacccharides which are TI-2 antigens (5,6). v) Adult splenectomy causes a profound depression of the response to the the TI-2 antigen dinitrophenylated hydroxyethyl starch (DNP-HES), but not to DNP-haemocyanin, a thymus-dependent antigen (7).

Origin and properties of soluble CD21 (CR2) in human blood

By analysis with a panel of CD21 MoAbs it is shown that a large part of the soluble CD21 in human blood plasma is of the long isoform (CD21L), as judged by comparison with antigen produced by mouse L cells transfected with CD21L‐cDNA and reactivity with the restricted CD21 MoAb R4/23. This is compatible with the hypothesis that soluble CD21 in the blood is mainly derived from follicular dendritic cells (FDC). Cells from a human keratinocyte cell line transfected with cDNA from the Burkitt lymphoma cell line Raji also produced soluble CD21L (sCD21L), whereas the short form of sCD21 (sCD21S) was the major component of sCD21 produced by the B lymphoblastoid cell line LICR‐LON‐HMy and the T cell line Jurkat. Confocal studies of FDC isolated from human tonsil revealed that CD21 was present in the cytoplasm. On gel filtration sCD21 from untreated serum has an apparent size considerably greater than the 130 kD found by SDS–PAGE analysis. This may be partly accounted for by the non‐globular shape of the molecule, but may also indicate, as reported by others, that in its native state sCD21 is complexed with other proteins. However, no evidence of complexing with sCD23 or C3d could be found.

Marginal Zones - the Largest B-cell Compartment of the Rat Spleen

The marginal zone of rat spleens consist of a massive sinusoidal network fed by the terminal branches of the splenic artery (1,2). Within the zone are large numbers of intermediate sized lymphocytes. In this report we summarize the characteristics of these cells and compare them with the lymphocytes found in the inner small lymphocytic zones of the splenic white pulp.

The Paradox of High Rates of B Cell Production in Bone Marrow and the Longevity of Most Mature B Cells

In mammals, the bone marrow is the principal site of primary B cell generation in post-natal life (1). Studies in mice indicate the the physiological rate of B cell production in bone marrow is sufficient to replenish the peripheral B lymphocyte pool within 4 days (2). The generative potential of B cell precursors is further emphasised by the ability of rats treated from birth with anti-IgM and anti-IgD antibodies, to fully repopulate secondary lymphoid organs with B cells, within a span of eight days following the cessation of antibody-induced suppression (3). These observations alone are consistent with the view that mature B cells have a relatively short half-life of a few days (4,5). However, only 25% of recirculating and static B cells of the splenic white pulp of rats are labelled by five days of 3H-thymidine infusion, indicating that most B cells within secondary lymphoid organs are not newly formed cells (6). The studies of Sprent and Basten (7) also support such a viewpoint. These data (6, 7), taken together with the observations on the rate of B cell production in bone marrow (2), imply that only a small proportion of B cells produced in adult bone marrow become part of the mature lymphocyte pool.

In Which Cells Does Neoplastic Transformation Occur in Myelomatosis

Myelomatosis is strictly a neoplasm of plasma cells in bone marrow. It does not involve other sites of antibody production; thus, even when the monoclonal protein produced by the neoplastic clone is IgA1 or IgA2 the neoplastic cells are not found in the lamina propria of the gut (Leonard et al 1979). Importantly the production of an IgM paraprotein by the neoplastic cells is vanishingly rare, amounting to only 0.2 % of 2011 successive patients admitted to MRC myelomatosis trials. IgG paraproteins were found in 57.2 % (0.15% with IgG with a minor IgM paraprotien of the same light chain isotype), IgA in 27% (0.10% with IgA with a minor IgM paraprotein of the same light chain isotype), IgD in 1.5%, IgE 0.1%. Free light chains alone were produced by the neoplastic cells of 14% of patients and 1.8% had no paraprotein. Physiologically in the established phase of those T cell-dependent antibody responses where B cells are being activated in the spleen antibody production takes place in the bone marrow (Benner et al 1981). This also applies to responses in those lymph nodes which do not receive lymph from mucosal surfaces. It has been shown in rodents that the B cells are activated in the follicles of these secondary lymphoid tissues (Tew et al. 1992) and migrate via the lymph and/or blood to the marrow. Migrant plasmablasts can be found in the blood of healthy humans. Analysis of the life-span of bone marrow plasma cells in rats indicates that IgG-or IgA-secreting plasma cells survive for about a month (Ho et al 1986). Conversely the IgM-producing plasma cells in the marrow like most plasma cells in the spleen and lymph nodes, irrespective of the class of Immunoglobulin produced, live for only 3 days (Ho et al 1986). It may be that these IgM-producing cells have been generated by local activation of B cells at the surface of macrophages by T cell-independent antigens (Corbel and Melchers 1983). Normal human bone marrow does not contain obvious secondary lymphoid tissue so it seems likely that the IgG and IgA plasma cells in this tissue are also derived from distant secondary lymphoid tissues. (Studies of plasma cells from the bone marrow from patients with rheumatoid arthritis as a means for investigating the physiological equivalent of neoplastic cells in myelomatosis could be misleading; for the marrow in this disease contains well-developed secondary lymphoid tissue).

Marginal Zone B Cells and the Localisation of Antigen on Follicular Dendritic Cells

The mechanism of uptake of antigen by follicular dendritic cells (FDC) is poorly understood, but complement and preformed antibody have been shown to be necessary for this to process (1). Brown et al (2) suggested that lymphocytes may be involved in the active transport of antigen into follicles. More recent studies (3,4) have led to the suggestion that it may be marginal zone B cells which transport antigen into splenic follicles.