In multiple myeloma, clonotypic B lymphocytes are detectable among CD19+ peripheral blood cells expressing CD38, CD56, and monotypic Ig light chain [published erratum appears in Blood 1995 Jun 1;85(11):3365]

Multiple myeloma (MM) is characterized by a plasma cell infiltrate of the bone marrow (BM). However, late-stage monotypic B cells have been detected in the blood. This work analyzes the effects of clinical treatment on late stage CD19+ B cells present in 752 blood samples from 152 MM patients. MM patients have 2 to 8 times as many circulating CD19+ cells as do normal donors. Analysis of the Ig heavy chain (IgH) gene rearrangements using polymerase chain reaction indicates that the CD19+ population includes cells sharing the same clonotypic CDR3 region as is detected in the BM plasma cells, for patients analyzed during chemotherapy or in relapse. They are also monotypic as defined by their cytoplasmic or surface expression of Ig kappa or lambda light chain. The light chain restriction is the same as that of the BM plasma cells. Individual patients observed over 1- to 2-year periods exhibit considerable variation in the number of B cells present in blood; this number does not correlate with the concentration of serum monoclonal Ig. The monoclonal blood CD19+ cells are not eliminated by any of the chemotherapy regimens analyzed and remain at high levels during transient remissions. Patients in the progressive phase of disease or in relapse have significantly higher numbers of B cells than do patients in transient remission or untreated patients. During periods when the quantity of blood B cells approaches normal, phenotypically their quality is highly abnormal, with physical and phenotypic heterogeneity. Most B cells express CD45R0, a high density of CD38, and CD56 characteristic of late-stage B or pre-plasma cells. CD38hi blood B cells had a cyclical presence. We conclude that monoclonal B cells in the blood of myeloma patient populations include drug-resistant reservoirs of clonotypic cells that may underlie relapse.     

Ontogeny of cell-mediated immunity. I. Early development of alloantigen- specific cytotoxic T-cell precursors in postnatal mice

Cytotoxic T-cell precursors have been shown to occur in spleens of 2-3-day-old mice. By 12 days after birth, the cytotoxic T-cell response of spleen cells to alloantigens has reached 23-32% of adult levels. Addition of extra T-helper cells did not permit cytotoxic T-cell development in spleen cells from newborn to 2-day-old mice suggesting either a lack of precursors or suppression of precursors. The ontogeny of cell-mediated immune functions has thus been shown to correlate well with other work on the development of humoral immunity, accessory cells, and graft versus host reactivity.     

Intrinsic Expression of the Multidrug Transporter, P-Glycoprotein 170, in Multiple Myeloma: Implications for Treatment

Multidrug resistance, mediated by the P-glycoprotein 170 transport pump, is a serious problem in multiple myeloma. In this review we discuss the expression of P-gp as a differentiation antigen on normal T and B lymphocytes. In myeloma, circulating presumptively malignant B cells express P-gp prior to chemotherapy. A variety of evidence characterizes these circulating B cells as members of the malignant clone in myeloma, including the demonstration that they share immunoglobulin heavy chain (IgH) rearrangements with bone marrow plasma cells, and their extensive DNA aneuploidy. In some patients the only components of the clonal populations that express P-gp are the circulating B cells suggesting that they represent a reservoir of multidrug resistant cells that maintain malignant growth and spread in myeloma. We speculate that exposure to chemotherapy alters clonal homeostasis and exerts positive selection pressure on generative components of the myeloma clone. Thus the possibility exists that chemotherapy perpetuates rather than eradicates myeloma stem cells. P-gp is detectable on bone marrow plasma cells in myeloma but appears to be in an inactive form that is unable to mediate efflux of marker dyes. A similar phenomenon is seen for normal human monocytes which have surface P-gp but lack any functional export of P-gp substrates. P-gp appears to vary depending in a cell-type specific manner suggesting that it may be feasible to design inhibitors of P-gp which selectively block P-gp export by malignant cells and spare the function of P-gp on normal tissue, including lymphocytes and normal hematopoietic stem cells.     

Analysis of immunodeficiency in multiple myeloma: Observations and hypothesis

Patients with multiple myeloma suffer from often profound immunodeficiency that seriously compromises both longevity and quality of life. The cellular basis for the immunodeficiency is unknown and the mechanism(s) giving rise to and possibly maintaining it is also unknown. In this review, evidence defining cellular defects in the nonmalignant B and T peripheral blood lymphocyte pool is summarized and discussed in terms of possible mechanisms that could give rise to the abberrant immune phenotype characteristic of many patients. The abnormalities we have defined include a deficiency of mature B lymphocytes, presence of often large numbers of pre-B cells in the blood, arrested differentiation of B cells into immunoglobulin (lg)-secreting cells, and abnormal progenitorlike T cells in blood. The specificity repertoire of the remaining B lymphocyte population is heavily skewed toward recognition of idiotypic and shared epitopes on the variable region of Ig, perhaps reflecting immune reaction with the monoclonal myeloma Ig. This may begin as a response to tumor antigen that gradually progresses to autoimmunity. A second subset of B cells present at abnormally high frequency in myeloma patients is that of antigen-experienced memory B cells, which probably encountered antigen prior to the events leading to frank myeloma. In this instance, the high frequency reflects a decrease in aggregate numbers of B cells, although it seems likely that expansion of memory clones also occurs. This is exemplified by the set of memory B cells specific for tetanus toxoid, which are present at normal number but highly enriched frequency in myeloma PBL. Both the antitumor B cells (anti-idiotype, and anti-Ig) and the memory cells specific for environmental pathogens appear to be arrested in their differentiation to Ig-secretors as evidenced by the lack of serum antibody in patients. The above observations are interpreted in terms of the hypothesis that immunoregulatory events triggered by the monoclonal myeloma Ig establish and maintain the immunodeficiency. We speculate that autoimmune suppressor T cells specific for conserved determinants of Ig prevent pre-B cell differentiation to slg + B cells, and B cell terminal differentiation to Ig-secretors. If correct, this analysis could lead to therapeutic approaches for the reversal of the immunodeficiency and perhaps some degree of control over the malignant growth.     

Transitions in CD45 isoform expression indicate continuous differentiation of a monoclonal CD5+ CD11b+ B lineage in Waldenstrom's macroglobulinemia.

Waldenstrom's macroglobulinemia (WM) has been hypothesized to be a pleomorphic B-cell malignancy with persistent maturation towards plasma cells in all lymphoid tissue. This proposal is based on detection of a heterogeneous density of monoclonal Ig on peripheral blood B-cells in patients with WM. We now present data derived from 2- and 3-color immunofluorescence and flow cytometric analysis that strongly supports this hypothesis. Abnormally high numbers of B lineage cells, defined by expression of CD19, CD20, and CD24, were found among peripheral blood mononuclear cells (PBMC). These B-cells are monoclonal as defined by light chain expression and by the existence of rearranged Ig genes (Southern blot analysis), although they exhibit heterogeneity in the density of surface light chain. Unlike normal PBMC B-cells, the monoclonal B-cells bear CD5 and CD10 (CALLA), express adhesion and adhesion-related molecules (CD11b, CD9), and appear to be actively differentiating during the course of the disease, based on the pattern of CD45 isoform expression. At any given point in time, the population of monoclonal B-cells is heterogeneous in differentiation stage based on transitions in the expression of CD45 isoforms from expression of CD45RA, the high molecular mass isoforms of CD45, to the low molecular mass isoform CD45R0 which appears only on very late stage B-cells and early plasma cells. For one patient, analysis of CD45 isoform expression over 2 years showed that the monoclonal B-cell population as a whole progressed towards terminal differentiation as defined by loss of CD45RA and acquisition of CD45R0. This indicates a continuously differentiating lineage of an unusual B-cell phenotype, and/or malignant transformation of a distinct lineage of B-cells in WM.     

The generation of antibody diversity. II. Plaque morphology as a simple marker for antibody specificity at the single-cell level

This paper investigates the validity of plaque morphology as a simple marker for distinguishing single cells producing antibody with different specificities. Plaques on an indicator mixture of related erythrocytes may be clear, with both red cell types lysed, or partial with only one lysed, or “sombreros” where both target erythrocytes are lysed but to different extents. The technique can be adapted for use with defined antigens by testing cells against indicator erythrocytes half of which are coupled at high density with the antigen and half at low density: again the population is split into clear and partial plaques. Various arguments and control experiments have been put forward to support the claim that plaque morphology depends on antibody specificity, and hence V region structure, and not on amount of antibody or C region changes: morphology remains the same at different temperatures of incubation, and as a plaque grows at 37 °C, and is not affected by different concentrations of complement in the medium.     

Analysis of clonotypic switch junctions reveals multiple myeloma originates from a single class switch event with ongoing mutation in the isotype-switched progeny

Multiple myeloma (MM) is a cancer of plasma cells (PCs) expressing immunoglobulin heavy chain (IgH) postswitch isotypes. The discovery of earlier stage cells related to postswitch PCs, called preswitch clonotypic IgM (cIgM) cells led to the hypothesis that cIgM cells may be MM progenitors, replenishing the tumor throughout malignancy. cIgM cells may do this by undergoing class switch recombination (CSR), a process detectable in postswitch PCs as multiple IgH switch junctions associated with a single clonotypic IgH V/D/J. We addressed this with a specific clonotypic-switch polymerase chain reaction (PCR), informative for 32 of 41 cases. Here we made 2 significant discoveries: (1) in all cases, we detected only a single clonotypic switch fragment that persists over time (1-7.6 years), and (2) we detected ongoing mutation upstream of the switch junction in 5 of 6 patients, often targeting the intronic enhancer, a key control region in IgH expression. The presence of a single, unchanging clonotypic switch junction suggests that cIgM cells are not MM-PC progenitors; rather, postswitch PCs arise from a single cIgM cell, and MM-PC progenitors reside in the postswitch population. Furthermore, mutations revealed here provide a new marker to identify MM-PC progenitors and aggressive clones that evolve throughout malignancy.     

A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma

Although the in vitro expansion of the multiple myeloma (MM) clone has been unsuccessful, in a novel three-dimensional (3-D) culture model of reconstructed bone marrow (BM, n = 48) and mobilized blood autografts (n = 14) presented here, the entire MM clone proliferates and undergoes up to 17-fold expansion of malignant cells harboring the clonotypic IgH VDJ and characteristic chromosomal rearrangements. In this system, MM clone expands in a reconstructed microenvironment that is ideally suited for testing specificity of anti-MM therapeutics. In the 3-D model, melphalan and bortezomib had distinct targets, with melphalan targeting the hematopoietic, but not stromal com-partment. Bortezomib targeted only CD138(+)CD56(+) MM plasma cells. The localization of nonproliferating cells to the reconstructed endosteum, in contact with N-cadherin-positive stroma, suggested the presence of MM-cancer stem cells. These drug-resistant CD20(+) cells were enriched more than 10-fold by melphalan treatment, exhibited self-renewal, and generated clonotypic B and plasma cell progeny in colony forming unit assays. This is the first molecularly verified demonstration of proliferation in vitro by ex vivo MM cells. The 3-D culture provides a novel biologically relevant preclinical model for evaluating therapeutic vulnerabilities of all compartments of the MM clone, including presumptive drug-resistant MM stem cells.     

Developmental nicotine exposure alters neurotransmission and excitability in hypoglossal motoneurons

Hypoglossal motoneurons (XII MNs) control muscles of the mammalian tongue and are rhythmically active during breathing. Acetylcholine (ACh) modulates XII MN activity by promoting the release of glutamate from neurons that express nicotinic ACh receptors (nAChRs). Chronic nicotine exposure alters nAChRs on neurons throughout the brain, including brain stem respiratory neurons. Here we test the hypothesis that developmental nicotine exposure (DNE) reduces excitatory synaptic input to XII MNs. Voltage-clamp experiments in rhythmically active medullary slices showed that the frequency of excitatory postsynaptic currents (EPSCs) onto XII MNs from DNE animals is reduced by 61% (DNE = 1.7 ± 0.4 events/s; control = 4.4 ± 0.6 events/s; P < 0.002). We also examine the intrinsic excitability of XII MNs to test whether cells from DNE animals have altered membrane properties. Current-clamp experiments showed XII MNs from DNE animals had higher intrinsic excitability, as evaluated by measuring their response to injected current. DNE cells had high-input resistances (DNE = 131.9 ± 13.7 MΩ, control = 78.6 ± 9.7 MΩ, P < 0.008), began firing at lower current levels (DNE = 144 ± 22 pA, control = 351 ± 45 pA, P < 0.003), and exhibited higher frequency-current gain values (DNE = 0.087 ± 0.012 Hz/pA, control = 0.050 ± 0.004 Hz/pA, P < 0.02). Taken together, our data show previously unreported effects of DNE on XII MN function and may also help to explain the association between DNE and the incidence of central and obstructive apneas.