The normal counterpart of IgD myeloma cells in germinal center displays extensively mutated IgVH gene, Cmu-Cdelta switch, and lambda light chain expression.

Human myeloma are incurable hematologic cancers of immunoglobulin-secreting plasma cells in bone marrow. Although malignant plasma cells can be almost eradicated from the patient's bone marrow by chemotherapy, drug-resistant myeloma precursor cells persist in an apparently cryptic compartment. Controversy exists as to whether myeloma precursor cells are hematopoietic stem cells, pre-B cells, germinal center (GC) B cells, circulating memory cells, or plasma blasts. This situation reflects what has been a general problem in cancer research for years: how to compare a tumor with its normal counterpart. Although several studies have demonstrated somatically mutated immunoglobulin variable region genes in multiple myeloma, it is unclear if myeloma cells are derived from GCs or post-GC memory B cells. Immunoglobulin (Ig)D-secreting myeloma have two unique immunoglobulin features, including a biased lambda light chain expression and a Cmu-Cdelta isotype switch. Using surface markers, we have previously isolated a population of surface IgM-IgD+CD38+ GC B cells that carry the most impressive somatic mutation in their IgV genes. Here we show that this population of GC B cells displays the two molecular features of IgD-secreting myeloma cells: a biased lambda light chain expression and a C&mu-Cdelta isotype switch. The demonstration of these peculiar GC B cells to differentiate into IgD-secreting plasma cells but not memory B cells both in vivo and in vitro suggests that IgD-secreting plasma and myeloma cells are derived from GCs.

The cytokine profile expressed by human dendritic cells is dependent on cell subtype and mode of activation.

In the present study, we have analyzed the pattern of cytokines expressed by two independent dendritic cell (DC) subpopulations generated in vitro from human cord blood CD34+ progenitors cultured with granulocyte-macrophage CSF and TNF-alpha. Molecularly, we confirmed the phenotypic differences discriminating the two subsets: E-cadherin mRNA was only detected in CD1a+-derived DC, whereas CD68 and factor XIIIa mRNAs were observed exclusively in CD14+-derived DC. Semiquantitative reverse-transcriptase PCR analysis revealed that both DC subpopulations spontaneously expressed IL-1alpha, IL-1beta, IL-6, IL-7, IL-12 (p35 and p40), IL-15, IL-18, TNF-alpha, TGF-beta, macrophage CSF, and granulocyte-macrophage CSF, but not IL-2, IL-3, IL-4, IL-5, IL-9, and IFN-gamma transcripts. Both subpopulations were shown to secrete IL-12 after CD40 triggering. Interestingly, only the CD14+-derived DC secreted IL-10 after CD40 activation, strengthening the notion that the two DC subpopulations indeed represent two independent pathways of DC development. Furthermore, both DC subpopulations expressed IL-13 mRNA and protein following activation with PMA-ionomycin, but not with CD40 ligand, in contrast to IL-12 and IL-10, revealing the existence of different pathways for DC activation. Finally, we confirmed the expression of IL-7, IL-10, and IL-13 mRNA by CD4+ CD11c+ CD3- DC isolated ex vivo from tonsillar germinal centers. Thus, CD14+-derived DC expressing IL-10 and factor XIIIa seemed more closely related to germinal center dendritic cellsGCDC than to Langerhans cells.

Measles virus suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and T cells.

Secondary infections due to a marked immunosuppression have long been recognized as a major cause of the high morbidity and mortality rate associated with acute measles. The mechanisms underlying the inhibition of cell-mediated immunity are not clearly understood but dysfunctions of monocytes as antigen-presenting cells (APC) are implicated. In this report, we demonstrate that measles virus (MV) replicates weakly in the resting dendritic cells (DC) as in lipopolysaccharide-activated monocytes, but intensively in CD40-activated DC. The interaction of MV-infected DC with T cells not only induces syncytia formation where MV undergoes massive replication, but also leads to an impairment of DC and T cell function and cell death. CD40-activated DC decrease their capacity to produce interleukin (IL) 12, and T cells are unable to proliferate in response to MV-infected DC stimulation. A massive apoptosis of both DC and T cells is observed in the MV pulsed DC-T cell cocultures. This study suggests that DC represent a major target of MV. The enhanced MV replication during DC-T cell interaction, leading to an IL-12 production decrease and the deletion of DC and T cells, may be the essential mechanism of immunosuppression induced by MV.

Molecular cloning of human RP105.

RP105 is a 105-kDa type I membrane protein of the leucine-rich repeat (LRR) family. Anti-RP105 sensitizes B cells to antigen-receptor-mediated apoptosis, but protects B cells from radiation-induced apoptosis and stimulates B cell proliferation. The sequence of the mouse RP105 has been reported. Here, we report the characterization of the human RP105. The 2.6-kb cDNA encodes a protein of 661 amino acids which displays 78% homology with mouse RP105. The 22 LRR and the 9 potential N-linked glycosylation sites within the extracellular region are conserved. While previous studies have shown that RP105 is expressed on surface IgM+IgD+2 B cells in mice, human RP105 was shown to be expressed on all subsets of mature B cells and dendritic cells. Human RP105 gene was mapped to the long arm of chromosome 5, where numerous cytokines and receptors have been localized.

Immunolocalization of the ICE/Ced-3-family protease, CPP32 (Caspase-3), in non-Hodgkin's lymphomas, chronic lymphocytic leukemias, and reactive lymph nodes.

Immunohistochemical analysis of the apoptosis-effector protease CPP32 (Caspase-3) in normal lymph nodes, tonsils, and nodes affected with reactive hyperplasia (n = 22) showed strong immunoreactivity in the apoptosis-prone germinal center B-lymphocytes of secondary follicles, but little or no reactivity in the surrounding long-lived mantle zone lymphocytes. Immunoblot analysis of fluorescence-activated cell sorted germinal center and mantle zone B cells supported the immunohistochemical results. In 22 of 27 (81%) follicular small cleaved cell non-Hodgkin's B-cell lymphomas, the CPP32-immunopositive germinal center lymphocytes were replaced by CPP32-negative tumor cells. In contrast, the large cell component of follicular mixed cells (FMs) and follicular large cell lymphomas (FLCLs) was strongly CPP32 immunopositive in 12 of 17 (71%) and in 8 of 14 (57%) cases, respectively, whereas the residual small-cleaved cells were poorly stained for CPP32 in all FLCLs and in 12 of 17 (71%) FMs, suggesting that an upregulation of CPP32 immunoreactivity occurred during progression. Similarly, cytosolic immunostaining for CPP32 was present in 10 of 12 (83%) diffuse large cell lymphomas (DLCLs) and 2 of 3 diffuse mixed B-cell lymphomas (DMs). Immunopositivity for CPP32 was also found in the majority of other types of non-Hodgkin's lymphomas studied. Plasmacytomas were CPP32 immunonegative in 4 of 12 (33%) cases, in contrast to normal plasma cells, which uniformly contained intense CPP32 immunoreactivity, implying downregulation of CPP32 in a subset of these malignancies. All 12 peripheral blood B-cell chronic lymphocyte leukemia specimens examined were CPP32 immunopositive, whereas 3 of 3 small lymphocytic lymphomas were CPP32 negative, suggesting that CPP32 expression may vary depending on the tissue compartment in which these neoplastic B cells reside. The results show dynamic regulation of CPP32 expression in normal and malignant lymphocytes.

Mechanisms of selection and differentiation in germinal centers.

The choice between death and survival, a feature of early B cell development, is a choice also faced by mature B cells. If they survive this early developmental decision, mature B cells then face a second choice: either to undergo either terminal differentiation into plasma cells or to differentiate into memory B cells. Antigens, T cell signals (cytokines, CD40 ligand and Fas ligand) as well as the activation state of B cells determine their ultimate fate.

Efficient MHC class II-restricted presentation of measles virus to T cells relies on its targeting to its cellular receptor human CD46 and involves an endosomal pathway.

The role of the measles virus (MV) receptor, human CD46, in the uptake of MV and antigen presentation by Major Histocompatibility Complex (MHC) class II molecules was investigated. Expression of CD46 in murine B cells resulted in cells highly efficient in capturing UV-inactivated MV particles and presenting both envelope hemagglutinin H and nucleoprotein N to specific T cell hybridomas. Although MV fuse with the plasma membrane of its target cells, presentation of both MV-H and -N was sensitive to inhibition by chloroquine but was not affected by a tripeptide which prevents virus-cell fusion. Whereas 50 microM of chloroquine was required to inhibit presentation of MV-H, purified H or soluble N, only a two-fold lower concentration was required to inhibit that of MV-N. This shows that some CD46-mediated captured MV particles are endocytosed, then disrupted and processed in an endosome/lysosome compartment.

Efficient major histocompatibility complex class II-restricted presentation of measles virus relies on hemagglutinin-mediated targeting to its cellular receptor human CD46 expressed by murine B cells.

Measles virus after binding to its cell surface human CD46 receptor fuses with the plasma membrane. This fusion results in envelope hemagglutinin (H) and fusion glycoprotein (F) incorporated into the plasma membrane and injection of the nucleocapsid made of nucleoprotein (NP) into the cytosol. The influence of targeting measles virus (MV) to CD46 in the processing and presentation of MV H and NP to antigen specific MHC class II I-E(d)- and I-A(d)-restricted T cell hybridomas was explored using murine M12-CD46 B cell transfectants. Parent M12 cells, which lack any MV receptor, were unable to present any of these two viral proteins when incubated with MV particles. Incubating M12.CD46 cells with 200 ng and 10 micrograms of MV could strongly stimulate H-specific and NP-specific T cells, respectively. Neosynthesis of MV proteins was not necessary since the efficiency of antigen presentation was similar when using ultraviolet-inactivated MV. Similar enhancing effects (more than 1,000-fold) on antigen presentation were also observed when using purified native H soluble or incorporated into liposomes whereas denaturating H glycoprotein resulted in a poor efficiency in T cell stimulation, M12.CD46 being no more potent than the parental M12 counterpart. MV H and NP presentation efficiency did not depend on MV fusion with plasma membrane as revealed by the lack of effect of specific fusion inhibitors. Both MV H and NP presentations were sensitive to chloroquine inhibition indicating that antigens from CD46-mediated captured MV were likely processed in the endosome/lysosome compartment. Altogether these data indicate that (a) MV targeting via CD46 has a strong effect on the efficiency of antigen presentation by MHC class II, (b) the effect is mediated by the binding of H to CD46, and (c) though MV does fuse with plasma membrane, endocytosis, and processing of virus particles are also occurring. Since, in humans, CD46 is expressed in almost every tissue including professional antigen-presenting cells, such a targeting is likely to play a crucial role in the CD4+ T cell-mediated primary immune response against the pathogen in vivo.