Bernard A. Ruether

Severe deficiency of B lymphocytes in peripheral blood from multiple myeloma patients.

A major problem in the assessment of circulating B lymphocytes in multiple myeloma is the extent to which cells with passively absorbed Ig contribute to the assay. We have analyzed peripheral blood B cell numbers in 51 patients in various treatment categories by using an assay that is not subject to artifacts involving cytophilic Ig. We have defined a B lymphocyte by three different criteria (a) expression of a high surface density of Ig (b) expression of a high density of HLA.DR and (c) expression of a marker exclusive to surface Ig+ B cells. By these criteria, normal individuals have an average of 6% B cells. In multiple myeloma patients, B cell levels in purified mononuclear cell preparations are severely reduced. Untreated patients and the majority of patients on intermittent chemotherapy have 20-600-fold fewer B cells than do normal donors (average = 0.3%). This decrease was even greater in whole blood of patients as compared with normal donors (100-1,000-fold fewer B cells). The number of B cells did not correlate with disease status or paraprotein concentration. We found no evidence to support the idea that B lymphocytes in patients include a substantial monoclonal subset.

Restricted expression of immunoglobulin light chain mRNA and of the adhesion molecule CD11b on circulating monoclonal B lineage cells in peripheral blood of myeloma patients.

Circulating monoclonal B cells in peripheral blood from patients with multiple myeloma or with monoclonal gammopathy of undetertmined siginificance (MGUS) have previously been shown to express CD19, CD20, and PCA‐1 and are predominantly CD45R0+, characterizing them as very late stage B cells. This work shows that the abnormal B cells are monoclonal as defined by their exclusive expression of either K or λ light chain mRNA, and that the same type of light chain mRNA is expressed in both bone marrow plasma cells and blood B cells. These abnormal tumour related circulating B cells express high densities of CD11b, a β2‐integrin, which is expressed in a conformationally active state as defined by reactivity with monoclonal antibody 7E3. Normal peripheral blood B cells which do not bear CD11b acquire a high density after stimutation with pokeweed mitogen (PWM). At day 4 of culture, the expression of CD11b on normal CD19+ B cells was nearly comparable to that of the circulating myeloma late stage B cells. After PWM stimulation of circulating myeloma B cells the expression of CD11b was gradually lost during 4 days of culture, suggesting that its expression is dynamically regulated. Two patients with no phenotypically abnormal B cells in their blood at diagnosis acquired a large subset of CD11b+ B cells 4 weeks after initiation of chemotherapy. In most patients, a subset of the circulating myeloma B cells express a low density of CD5. The proportion of CD19+ B cells in the bone marrow expressing CD11b was much reduced compared with peripheral blood B cells, and CD11b was not detectable on plasma cells in the bone marrow, suggesting a sequential relationship of the B‐cell subsets detected in our population of patients, involving gradual loss of CD11b concurrent with the loss of CD19 during B lineage differentiation. These cells appear to represent a continuously differentiating monoclonal B lineage culminating in the CD11b plasma cell entrenched in the bone marrow. We speculate that the expression of conformationally active CD11b on the abnormal B cells in peripheral blood mononuclear cells of myeloma patients facilitates transendothelial migration of circulaling myeloma B cells to the bone marrow.

Abnormal function of B lymphocytes from peripheral blood of multiple myeloma patients. Lack of correlation between the number of cells potentially able to secrete immunoglobulin M and serum immunoglobulin M levels.

Multiple myeloma patients are deficient in normal polyclonal serum immunoglobulins. To determine the reasons for this decrease, we quantitated and compared the number of surface IgM+ B lymphocytes, and the number of B cells susceptible to transformation by Epstein-Barr virus (EBV) with the concentration of IgM in serum. Serum IgM levels varied considerably in individual patients, temporally shifting from undetectable to normal amounts and then dropping again to undetectable levels. A transient rise to normal serum IgM concentrations was seen in 42% of patients assessed at two or more time points. Of 44 patients, 52% showed a lack of correlation between the number of surface IgM+ (sIgM+) B cells and serum IgM concentration. One subset of patients (25%) had detectable to normal numbers of sIgM+ B cells in blood but undetectable levels of serum IgM. Transformation of B cells from these patients indicated a block in IgM secretion that was extrinsic to the B cells that were fully able to transcribe, translate, and secrete IgM after EBV transformation. A second subset of patients (27%) had undetectable numbers of sIgM+ B cells but near normal levels of serum IgM, suggesting abundant secretion by few clones of B cells. Of 18 patients with monoclonal gammopathy of undetermined significance (MGUS), 26% showed a lack of correlation between the numbers of sIgM+ B cells and serum IgM concentration. We suggest that in patients with multiple myeloma, and in some with MGUS, there exists a mechanism(s) extrinsic to the B cell that mediates an arrest in terminal B lymphocyte maturation.

Specificity repertoire of lymphocytes from multiple myeloma patients. I. High frequency of B cells specific for idiotypic and F(ab′)2-region determinants on immunoglobulin

The specificity repertoire of B lymphocytes from 14 multiple myeloma patients has been studied using the technique of Epstein-Barr virus (EBV) transformation of peripheral blood lymphocytes (PBL) coupled with clonal analysis by limiting dilution. We find that up to 100% of the B cells from myeloma patients undergoing EBV transformation secrete IgM specific for determinants on the F(ab′)2 region of autologous and/or heterologous monoclonal immunoglobulin. In normal individuals 0.02–0.73% of the transformed B cells secrete IgM specific for F(ab′)2 determinants. Two patients with monoclonal gammopathy of undetermined significance had only a weak reactivity to F(ab′)2 fragments. The number of anti-F(ab′)2 B cells was up to 145-fold greater in patients than in normal donors. The majority of antibodies from patient clones recognized determinants shared among 3–12 different F(ab′)2 fragments, whereas those originating from normal donor B cells saw determinants expressed on only one or two of the panel of test F(ab′)2 fragments. There was a preference for autologous M components and a high proportion of antiidiotypic reactivity in five of eight patients so analyzed. We speculate that these findings indicate the existence of an anti-F(ab′)2 immunoregulatory network mediating patient immunodeficiency, thereby creating an abnormality that may enable the progression of multiple myeloma.

Life-threatening hemolytic anemia due to an autoanti-Pr cold agglutinin: evidence that glycophorin A antibodies may induce lipid bilayer exposure and cation permeability independent of agglutination

BACKGROUND: The hemoglobin of a 29‐year‐old man fell below 35 g/L over 5 days, despite 14 units of red blood cells (RBCs), due to an anti‐Pr cold agglutinin (CA). His hemolytic anemia necessitated respiratory support in intensive care for 4 weeks.

Expression of Multiple Adhesion Molecules on Circulating Monoclonal B Cells in Myeloma

Multiple myeloma is a tumor of the B cell lineage, with characteristic monoclonal end stage plasma cells in the bone marrow, responsible for producing large quantities of monoclonal immunoglobulin present in serum, and for triggering osteoclasts to produce lesions of the bone, leading to high blood calcium, the possibility of kidney failure, and severe pathological damage. The proliferative potential of these plasma cells is low, and they express very few surface molecules involved in locomotion and migration. Candidates for spreading of the disease are circulating, monoclonal B cells, which have been reported by us (1, 2) and others (3–8). We have described a large subset of circulating cells belonging to the same clone as the bone marrow plasma cells in myeloma, which represent late stages of B cell differentiation towards pre-plasma cells, predominantly expressing the CD45R0 isoform (1), only found on activated late stage B cells (9). This subset of monoclonal B cells in the peripheral blood was observed among newly diagnosed patients, as well as patients undergoing intermittent chemotherapy, or at stable phases of disease (1), and thus seems unaffected by conventional therapy (see also Pilarski et al., this issue). In contrast to the bone marrow plasma cells, the blood B cells comprise a proliferating subpopulation (2). The monoclonal B cells in the blood of myeloma patients are at a late stage of B cell differentiation, and appear to be continuously progressing towards the plasma cell stage. Significant numbers of plasma cells, however, are only found in the bone marrow, except in terminal or highly aggressive myeloma, where they may be present in the blood as well. These observations suggest that if the blood B cells are, or include, a precursor population for the bone marrow plasma cells of the same clonality, the blood B cells must be capable of extravasating and migration to the bone marrow. It further suggests that in myeloma patients, the blood does not provide the micro environmental stimuli to support the terminal differentiation to end stage plasma cells. The myeloma blood B cells express multiple adhesion molecules, including CD11b, L-Selectin, CD44, α2β1, and α6β1, in contrast to the plasma cells in the myeloma bone marrow. This strongly indicates that the circulating subset is indeed potentially more motile and capable of extravasating and homing to the bone marrow.