Long-term follow-up of a prospective, double-blind, placebo-controlled randomized trial of clodronate in multiple myeloma.

Oral clodronate (1600 mg/d) has been shown to significantly reduce the incidence of skeletal complications in multiple myeloma. Preliminary analysis of a double-blind placebo-controlled trial of this treatment indicated that clodronate might prolong survival in patients without vertebral fractures at presentation. This issue was re-examined after further follow-up of the patients recruited into the Medical Research Council (MRC) VIth Myeloma Study. The trial examined the effects of clodronate on the natural history of skeletal disease in multiple myeloma; 619 patients were randomized between June 1986 and May 1992 commencing 15 d after the start of ABCM [adriamycin, BCNU (carmustine), cyclophosphamide, melphalan] chemotherapy or 43 d after ABCMP (ABCM + prenisolone); 535 patients who received clodronate or placebo were included in the analysis. The presence or absence of spinal fractures was assessed centrally from spinal X-rays; long-bone fractures were assessed locally. With a median follow-up of 8.6 years, there was no overall significant difference in survival between the two treatment groups (O/E, chi2 = 0.78, P = 0.38). Among the subgroup of 153 patients with no skeletal fractures at presentation there was a significant survival advantage (O/E, chi2 = 7.52, P = 0.006) in favour of the 73 patients receiving clodronate, with median survivals being, respectively, 59 months (95% CI 43-71 months) and 37 months (95% CI 31-52 months), and 5-year survivals being 46% and 35%. The original analysis of this study shows that there is a benefit in taking 1600 mg clodronate daily for patients with myelomatosis to prevent the development of new skeletal disease. Bearing in mind the limitations of subgroup analysis, the present study indicates that treatment may prolong survival in patients without overt skeletal disease at diagnosis. These observations, however, require confirmation in prospective clinical trials.

Tracking the response of Xid B cells in vivo: TI-2 antigen induces migration and proliferation but Btk is essential for terminal differentiation.

X-linked immunodeficient (Xid) mice carry a Bruton's tyrosine kinase (Btk) mutation and exhibit a selective failure to produce antibodies against bacterial capsular polysaccharides. Studies in vitro point to a fundamental survival defect of Xid B cells after receptor cross-linking by thymus-independent type-2 (TI-2) antigen because B cells undergo apoptosis without proliferating. We describe results from a novel model, which we have used to investigate the impact of the Xid mutation on migration, proliferation and differentiation of B cells after polysaccharide immunization in vivo. Immunoglobulin knock-in mice, in which a large proportion of B cells express transgene-encoded receptors specific for (4-hydroxy-3-nitrophenyl)-acetyl (NP), were crossed with CBA/N mice. The male progeny contain NP-specific Xid B cells, while the female progeny contain NP-specific B cells with normal Btk. After immunization with the TI-2 antigen NP-Ficoll, NP-specific Xid B cells migrate to the T zones and proliferate. Despite transient up-regulation of blimp-1 and survival beyond the time when terminal differentiation is normally underway, Btk-defective B cells fail to differentiate to plasmablasts or germinal center cells. CD40 ligation partially restores their ability to form plasma cells in response to TI-2 antigen.

Changing responsiveness to chemokines allows medullary plasmablasts to leave lymph nodes.

During T cell-dependent antibody responses lymph node B cells differentiate either to plasmablasts that grow in the medullary cords, or to blasts that proliferate in follicles forming germinal centers. Many plasmablasts differentiate to plasma cells locally, but some leave the medullary cords and migrate to downstream lymph nodes. To assess the basis for this migration, changes in the responsiveness of B cells to a range of chemokines have been studied as they differentiate. Naive B cells express high levels of CCR6, CCR7, CXCR4 and CXCR5. When activated B cells grow in follicles the expression of these chemokine receptors and the responsiveness to the respective chemokines is retained. During the extrafollicular response, plasmablast expression of CXCR5 and responsiveness to B-lymphocyte chemoattractant (CXCR5) as well as to secondary lymphoid tissue chemokine (CCR7) and stromal cell-derived factor (SDF)-1 (CXCR4) are lost while a weak response towards the CCR6 chemokine LARC is maintained. Despite losing responsiveness to SDF-1, extrafollicular plasmablasts still express high levels of CXCR4 on the cell surface. These results suggest that the combined loss of chemokine receptor expression and of chemokine responsiveness may be a necessary prerequisite for cells to migrate to the medullary cords and subsequently enter the efferent lymph.

Intrinsic constraint on plasmablast growth and extrinsic limits of plasma cell survival.

B cells recruited into splenic antibody responses grow exponentially, either in extrafollicular foci as plasmablasts, or in follicles where they form germinal centers. Both responses yield plasma cells. Although many splenic plasma cells survive <3 d, some live much longer. This study shows that early plasma cell death relates to a finite capacity of the spleen to sustain plasma cells rather than a life span endowed by the cell's origin or the quality of antibody it produces. Antibody responses were compared where the peak numbers of plasma cells in spleen sections varied between 100 and 5,000 cells/mm(2). In each response, plasmablast clones divided some five times, with the peak numbers of plasma cells produced relating directly to the number of B cells recruited into the response. The spleen seems to have the capacity to sustain between 20 and 100 plasma cells/mm(2). When this number is exceeded, there is a loss of excess cells. Immunoglobulin variable region gene sequencing, and 5-bromo-2'-deoxyuridine pulse-chase studies indicate that long-lived splenic plasma cells are a mixture of cells derived from the extrafollicular and germinal center responses and cells derived from virgin and memory B cells. Only a proportion has switched immunoglobulin class.

B-cell memory and the persistence of antibody responses.

Antigens such as viral envelope proteins and bacterial exotoxins induce responses which result in the production of neutralizing antibody. These responses persist for years and provide highly efficient defence against reinfection. During these antibody responses a proportion of participating B cells mutate the genes that encode their immunoglobulin variable regions. This can increase the affinity of the antibody, but can also induce autoreactive B cells. Selection mechanisms operate which allow the cells with high affinity for the provoking antigen to persist, while other B cells recruited into the response die.

Germinal centers without T cells.

Germinal centers are critical for affinity maturation of antibody (Ab) responses. This process allows the production of high-efficiency neutralizing Ab that protects against virus infection and bacterial exotoxins. In germinal centers, responding B cells selectively mutate the genes that encode their receptors for antigen. This process can change Ab affinity and specificity. The mutated cells that produce high-affinity Ab are selected to become Ab-forming or memory B cells, whereas cells that have lost affinity or acquired autoreactivity are eliminated. Normally, T cells are critical for germinal center formation and subsequent B cell selection. Both processes involve engagement of CD40 on B cells by T cells. This report describes how high-affinity B cells can be induced to form large germinal centers in response to (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll in the absence of T cells or signaling through CD40 or CD28. This requires extensive cross-linking of the B cell receptors, and a frequency of antigen-specific B cells of at least 1 in 1,000. These germinal centers abort dramatically at the time when mutated high-affinity B cells are normally selected by T cells. Thus, there is a fail-safe mechanism against autoreactivity, even in the event of thymus-independent germinal center formation.

Dendritic cells associated with plasmablast survival.

A subset of myeloid dendritic cells is described which is associated with the ability of splenic and lymph node plasmablasts to survive and differentiate into plasma cells. Plasmablast-associated dendritic cells (PDC) are CD11c(high), DEC-205(-) and unlike conventional dendritic cells do not associate with T cells. The following findings suggest a requirement for PDC if plasmablasts are to differentiate to plasma cells. First, when large numbers of B cells are recruited into antibody responses and plasmablasts outgrow the PDC stroma, only those associated with PDC survive and differentiate into plasma cells. Conversely, if the number of PDC is increased by ligating their CD40, more plasmablasts survive on the expanded PDC stroma and differentiate into plasma cells. Finally, in T cell-deficient mice, the plasma cells that develop atypically in the T zones in response to thymus-independent antigens are associated with ectopic PDC.

Anti-CD40 antibody enhances responses to polysaccharide without mimicking T cell help.

Protection against infection with encapsulated bacteria is mediated by IgG antibodies against the capsular polysaccharides. Production of such antibodies is impaired during infancy, when susceptibility to bacterial meningitis is greatest. Recent studies have proposed the use of anti-CD40 antibody to increase responsivenesses to polysaccharide antigens. We show here that the IgG response to a model polysaccharide antigen is greatly increased, but retains thymus-independent characteristics--switching continues to be mainly to IgG3 and neither germinal centers nor memory B cells are formed. Furthermore, anti-CD40 causes striking splenomegaly in mice, which is accompanied by dramatic cellular redistribution and proliferation of dendritic cells, macrophages, T cells and endothelium, as well as B cells. These findings raise the possibility that the anti-CD40 effect is due mainly to increased activity of accessory cells that affect plasmablast growth and differentiation rather than mimicry of T cell help.

Interplays between mouse mammary tumor virus and the cellular and humoral immune response.

Mouse mammary tumor virus has developed strategies to exploit the immune response. It requires vigorous immune stimulation to achieve efficient infection. The infected antigen-presenting cells present a viral superantigen on the cell surface which stimulates strong CD4-mediated T-cell help but CD8 T-cell responses are undetectable. Despite the high frequency of superantigen-reactive T cells, the superantigen-induced immune response is comparable to classical antigen responses in terms of T-cell priming, T-cell-B-cell collaboration as well as follicular and extra-follicular B-cell differentiation. Induction of systemic anergy is observed, similar to classical antigen responses where antigen is administered systemically but does not influence the role of the superantigen-reactive T cells in the maintenance of the chronic germinal center reaction. So far we have been unable to detect a cytotoxic T-cell response to mouse mammary tumor virus peptide antigens or to the superantigen. This might yet represent another step in the viral infection strategy.

T-independent type 2 antigens induce B cell proliferation in multiple splenic sites, but exponential growth is confined to extrafollicular foci.

During the primary splenic response to the T-independent type 2 (TI-2) antigen (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll, small numbers of antigen-specific B cells have entered S phase of the cell cycle 24 h after intraperitoneal immunization. These are distributed in all splenic compartments (T zones, marginal zones, follicles, and red pulp), indicating early proliferation induced by NP-Ficoll does not require accessory signals delivered in a particular splenic microenvironment. Subsequently B blasts accumulate selectively in the outer T zone areas, but exponential growth leading to plasma cell production occurs only in extrafollicular foci. This growth peaks after 5 days, but 20% of peak numbers of antibody-containing cells are still present 3 months after immunization and 9% of these cells are proliferating. It is unclear if these late plasmablasts are sustained by self-renewal or continued recruitment of virgin cells into the response. Unlike TD and TI-1 responses NP-specific memory cells do not accumulate in the splenic marginal zones. The level of Cgamma3 switch transcripts increases during the first 24 h of the response, but does not increase further during exponential plasmablast growth.

Defective immunoglobulin class switching in Vav-deficient mice is attributable to compromised T cell help.

Vav, a guanine nucleotide exchange factor for members of the Rho family of small GTPases, is activated through engagement of B and T lymphocyte antigen receptors. It is important for establishing the signaling threshold of the TCR, as mice lacking Vav display defective thymocyte selection. Here, conventional B cells are shown to develop normally in Vav-deficient mice but these mice have few B-1 B cells. The threshold for inducing B cell proliferation through BCR engagement in vitro is greater in Vav-deficient B cells. Nevertheless, in vivo the mutant mice have normal antibody responses to haptenated Ficoll. In contrast, Vav-/- mice show defective class switching to IgG and germinal center formation when immunized with haptenated protein. Interestingly, this defect is reversed in chimeras where normal T cells are present. Antigen-specific proliferation of T cells in the T zone was found to be similar in wild-type and Vav-/- mice but the induction of IL-4 mRNA and switch transcripts was specifically impaired. These results suggest that defective immunoglobulin class switching in Vav-deficient mice is attributable to compromised T cell help.

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 130kD 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.

T helper 1 (Th1) and Th2 characteristics start to develop during T cell priming and are associated with an immediate ability to induce immunoglobulin class switching.

The respective production of specific immunoglobulin (Ig)G2a or IgG1 within 5 d of primary immunization with Swiss type mouse mammary tumor virus [MMTV(SW)] or haptenated protein provides a model for the development of T helper 1 (Th1) and Th2 responses. The antibody-producing cells arise from cognate T cell B cell interaction, revealed by the respective induction of Cgamma2a and Cgamma1 switch transcript production, on the third day after immunization. T cell proliferation and upregulation of mRNA for interferon gamma in response to MMTV(SW) and interleukin 4 in response to haptenated protein also starts during this day. It follows that there is minimal delay in these responses between T cell priming and the onset of cognate interaction between T and B cells leading to class switching and exponential growth. The Th1 or Th2 profile is at least partially established at the time of the first cognate T cell interaction with B cells in the T zone. The addition of killed Bordetella pertussis to the hapten-protein induces nonhapten-specific IgG2a and IgG1 plasma cells, whereas the anti-hapten response continues to be IgG1 dominated. This indicates that a Th2 response to hapten-protein can proceed in a node where there is substantial Th1 activity.

MRC trial of alpha2b-interferon maintenance therapy in first plateau phase of multiple myeloma. MRC Working Party on Leukaemia in Adults.

Plateau phase has been achieved in 64% of all newly diagnosed patients with multiple myeloma treated with the ABCM (adriamycin, BiCNU, cyclophosphamide and melphalan) regimen in the Medical Research Council (MRC) trials; this stable clinical stage of the disease is associated with no more than minimal symptoms. Several studies have found that alpha-interferon (alpha-IFN) maintenance therapy increases the duration of plateau phase, but it is less clear if this translates into prolonged survival. We report the effect of alpha-IFN on the duration of plateau phase and overall survival in a trial with 284 patients who were randomized to receive alpha2b-IFN (Intron-A) or no maintenance therapy during first plateau phase. The minimum follow-up after randomization was 21 months. There was no significant difference in the overall survival between the two treatment groups (X2=0.32, P=0.57). There was a trend towards longer relapse-free survival in the patients allocated alpha-IFN, but this trend to longer plateau phase was not statistically significant (X2 = 1.62, P = 0.2). Disease progression at relapse on alpha-IFN appears to be more severe with greater elevations from plateau levels of serum paraprotein (P = 0.06) and beta2-microglobulin (P= 0.03) levels. Physicians tended to start chemotherapy sooner after diagnosis of relapse when patients had received alpha-IFN (P = 0.16). Although, in common with most other studies, there is a trend for patients treated with alpha-IFN to have a longer plateau phase, this is counteracted by morbidity attributable to the treatment and a somewhat shortened survival post relapse. Meta-analysis of interferon trials is required to assess whether the minor trend for longer survival in patients maintained on alpha-IFN found in some studies is significant and, if so, the extent of this advantage.

B-cell receptor regulation of peripheral B cells.

Recent studies indicate that immature B cells compete with recirculating B cells for survival signals. The signals, delivered through the B-cell receptor for antigen, induce immature cells to differentiate into recirculating cells and maintain the survival of recirculating cells. They do not induce proliferation or differentiation to antibody-producing cells.

A randomized trial of the effect of clodronate on skeletal morbidity in multiple myeloma. MRC Working Party on Leukaemia in Adults.

In patients with multiple myeloma, despite a major reduction of bone pain achieved with chemotherapy, skeletal disease continues to progress. The effects of clodronate, an inhibitor of osteoclastic bone resorption, are evaluated on the natural history of skeletal disease in patients with newly diagnosed multiple myeloma. Within the framework of the VIth MRC Multiple Myeloma Trial, 536 patients (218 women, 318 men) with recently diagnosed multiple myeloma were randomized to receive either clodronate 1600 mg daily (n=264) or an outwardly identical placebo (n=272) in addition to chemotherapy. Treatment with clodronate was associated with a 50% decrease in the proportion of patients with severe hypercalcaemia (5.1% v 10.1%, P=0.06) and a similar reduction in reported non-vertebral fractures (6.8% v 13.2%, P=0.04). Fewer patients receiving clodronate sustained vertebral fractures after entry to the trial (38% v 55%, P=0.01) and patients also lost less height over 3 years compared to those receiving placebo (2.0 v 3.4 cm, P=0.01). Biochemical indices of bone turnover were significantly lower in patients receiving concomitant clodronate, both at plateau and at disease relapse. The frequencies of back pain and poor performance status were significantly lower at 24 months in clodronate than in placebo-treated patients (10.9% v 19.9%, P=0.05, and 18.3% v 30.5% P=0.03 respectively.) There was no statistically significant difference in survival between the clodronate and placebo treated patients. The study indicates that long-term oral clodronate slows the progression of skeletal disease in multiple myeloma and decreases the associated morbidity. Patients without overt skeletal disease at diagnosis were also found to benefit from clodronate, indicating that this treatment should be initiated as early in the course of the disease as possible.

Memory B-cell clones and the diversity of their members.

Memory B-cell clones develop from virgin B cells that take up processed antigen, make cognate interaction with primed T cells and then grow in germinal centres. Within the germinal centre the proliferating B cells undergo Ig variable-region mutation and are subsequently selected on their ability to bind antigen held on follicular dendritic cells and then to make cognate interaction with germinal centre T cells. The selected cells emerge as memory B cells or plasmablasts. Although many of the memory B cells and most of the plasma cells emerging from follicles have undergone Ig class switch recombination a substantial minority of the memory B cells have not switched. These non-switched memory cells can be induced to switch on re-exposure to antigen. Affinity maturation following a single immunization ceases as germinal centres wane some 3-4 weeks after immunization - memory cells and antibody production, on the other hand, persist for months and even years.

The changing preference of T and B cells for partners as T-dependent antibody responses develop.

Recirculating virgin CD4+ T cells spend their life migrating between the T zones of secondary lymphoid tissues where they screen the surface of interdigitating dendritic cells. T-cell priming starts when processed peptides or superantigen associated with class II MHC molecules are recognised. Those primed T cells that remain within the lymphoid tissue move to the outer T zone, where they interact with B cells that have taken up and processed antigen. Cognate interaction between these cells initiates immunoglobulin (Ig) class switch-recombination and proliferation of both B and T cells; much of this growth occurs outside the T zones B cells migrate to follicles, where they form germinal centres, and to extrafollicular sites of B-cell growth, where they differentiate into mainly short-lived plasma cells. T cells do not move to the extrafollicular foci, but to the follicles; there they proliferate and are subsequently involved in the selection of B cells that have mutated their Ig variable-region genes. During primary antibody responses T-cell proliferation in follicles produces many times the peak number of T cells found in that site: a substantial proportion of the CD4+ memory T-cell pool may originate from growth in follicles.