Deficient expression of adhesion molecules by human CD5- B lymphocytes both after bone marrow transplantation and during normal ontogeny

Despite the relatively early reconstitution of blood B-lymphocyte counts observed in patients treated with bone marrow transplantation (BMT), these patients undergo a prolonged phase of humoral immunodeficiency. Adhesion molecules perform relevant functions in many cell types. The present study examines the expression of several adhesion molecules on human B lymphocytes newly formed after BMT. Blood B cells from 38 patients were studied by flow cytometry and three-color analysis. Blood CD5- B lymphocytes obtained at an early stage after BMT (2 to 4 months) showed a markedly low expression of the adhesion molecules CD54, CD44, CD11a, and CD62L. However, these cells exhibited a normal expression of other molecules including CD29, CD19, CD20, and DR. This deficiency was progressively corrected, reaching normal levels in the late post-BMT period (12 to 15 months). In contrast, CD54, CD44, CD11a, and CD62L expression on the patients' CD5+ B lymphocytes was found to be consistently normal. Deficient adhesion molecule expression on CD5- B cells in the early post-BMT period was similarly observed in patients treated with either an allo-BMT (n = 24) or an auto-BMT (n = 14). Because the post-BMT period mimics normal ontogeny, adhesion molecule expression was also investigated in cord-blood B lymphocytes. Cord-blood CD5- B lymphocytes, in contrast to CD5+, also expressed CD54, CD44, CD11a, and CD62L at levels much lower than those found in normal adults. Present data suggest that progressive expression of CD54, CD44, CD11a, and CD62L seems to be a part of the maturational program of CD5- B lymphocytes during both post-BMT and normal development periods. This observation may help to explain the humoral immunodeficiency observed in both conditions.     

Antioxidant enzymes in lymphocytes from normal subjects and patients with chronic lymphocytic leukaemia: increased glutathione peroxidase activity in CLL B lymphocytes

The activities of several enzymes that protect against oxidative injury were determined in blood lymphocytes from patients with B chronic lymphocytic leukaemia (CLL) and from normal subjects. Similar glutathione reductase (GR), catalase and glucose-6-phosphate dehydrogenase (G6PD) activities were found in normal and CLL lymphocytes. Higher glutathione peroxidase (GP) activity was found in CLL lymphocytes. This activity in CLL B lymphocytes was 2-fold higher than that of normal B lymphocytes, and 3-fold higher than that of T lymphocytes from either source. Several disease processes have been associated with decreased glutathione peroxidase activity. Our finding with CLL B lymphocytes is believed to be the first example of an increased GP activity in a disease. It may reflect either the expansion of a rare type of B cell population or be an expression of the malignant process.     

Ontogeny of mouse B lymphocytes and inactivation by antigen of early B lymphocytes.

Taking advantage of recent findings about membrane fluidity, we have studied and compared the biosynthetic capacities of fetal or neonatal mouse B (bone-marrow derived) lymphocytes (until 10 days after birth) and adult B lymphocytes. Although both early and adult lymphocytes can synthesize surface immunoglobulins, they have a different physiological behavior after interaction with a ligand (anti-immunoglobulin sera or antigen), either in vivo or in vitro. Fetal and neonatal lymphocytes bearing surface immunoglobulins do not reexpress their membrane receptors after capping and endocytosis promoted by anti-immunoglobulin sera. On the other hand, adult lymphocytes resynthesize completely their receptors after the same treatment. Furthermore, intrafetal injections of hemocyanin in pregnant mice lead to a striking decrease in the number of hemocyanin-binding cells. It seems plausible that this non-reexpression of surface immunoglobulins could be the first step in tolerance establishment.     

The membrane receptor complex regulating induction and clonal expansion of normal murine B lymphocytes

Induction of resting B lymphocytes results from the interaction of competent ligands or helper cells with "triggering receptors." Subsequent clonal expansion and performance are thought to be regulated by the interaction of selective growth or maturation factors with specific receptors on induced B cells. A set of membrane molecules of B lymphocytes, including IgM, IgD, IA, IE, lipopolysaccharide receptors, receptors for Fc and C3b, and other non-immunoglobulin structures recognized by some antiidiotypic antibodies, display ligand-induced relationships. Functional studies also indicate that this group of molecules functions as a complex that regulates delivery of inductive signals, the expression of growth and maturation receptors, and/or the outcome of the interactions of these receptors with the corresponding factors.     

Visualization of tubulin in lymphocytes. I. Comparison of normal and chronic lymphocytic leukemia (CLL) lymphocytes.

Cell structures containing tubulin were studied in peripheral blood lymphocytes from 8 normal donors and 11 patients with CLL using specific antitubulin antibodies revealed by immunoperoxidase assay. The centriole and microtubules were clearly visible in both groups. A "nucleus-associated tubulin-containing structure" was revealed by antitubulin antibodies and was found in virtually all lymphocytes of normal subjects but in a considerably lower number of CLL lymphocytes. The nature of this structure and its relationship to other cell structures are discussed.     

Production of colony-stimulating activity by normal and neoplastic human B lymphocytes

Normal human B cells were purified from peripheral blood or tonsils and tested for their ability to release colony-stimulating activity (CSA) in short-term cultures. The target cells used in the CSA assays were from peripheral blood or bone marrow. Unstimulated B cells produced CSA in amounts similar to those present in the GCT-conditioned medium used as a positive control. The B cell-derived CSA predominantly promoted the growth of colonies that contained macrophages alone or macrophages and granulocytes. CSA eluted in a single peak from a G-75 Sephadex column with an approximate molecular weight (mw) of 65 to 70 kilodaltons (kd). Fractionation of tonsil B lymphocytes on Percoll density gradients showed that large B cells, probably already activated in vivo, were the main source of CSA. By contrast, small, resting B cells recovered from a different fraction of the Percoll gradient released minimum amounts or no CSA. However, these B cells became CSA producers following stimulation with Staphylococcus aureus Cowan (SAC) in vitro. B cells purified from the peripheral blood of nine out of 12 patients with B-cell chronic lymphocytic leukemia (B-CLL) also released CSA in vitro in the absence of stimuli. These findings suggest that by releasing CSA, B cells may have a role in the regulation of hematopoiesis and in the control of the inflammatory process.     

Cell electrophoretic characterization of peripheral blood lymphocyte subpopulations enriched by rosette formation, from normal individuals and CLL patients

Peripheral blood lymphocytes from healthy subjects and patients with chronic lymphatic leukemia (CLL) were isolated and their subpopulations enriched through formation of spontaneous rosettes with sheep or mouse red blood cells, respectively. Electrophoretic measurements were performed in unseparated as well as in fractionated cell populations. Normal blood lymphocytes showed two clearly distinguishable populations of different electrophoretic mobilities. After separation by SRBC rosette formation the rosette-forming cells could be identified as high mobility cells. CLL lymphocytes showed in most cases an unimodally distributed cytopherogram, the mean electrophoretic mobility being intermediate between the low and high mobility cells of control persons. After separation through mouse erythrocytes rosette formation these cells contained two cell fractions differing in their electrophoretic mobility: a fraction of slower mouse rosette-forming cells and a fraction of the non-MRFC which contained mainly cells of higher mobility that could be identified as enriched T cells. These both fractions showed unimodal distributions. This study shows that CLL lymphocyte subpopulations can be further characterized by surface charge density.     

病毒性心肌炎患者外周血T淋巴细胞亚群及B淋巴细胞的研究

目的: 测定不同时期病毒性心肌炎(VMC)患者T淋巴细胞亚群及B淋巴细胞的数量,并同时检测患者血清心肌损害标志物心肌肌钙蛋白I(cTnI)的水平,探讨其在VMC发病机制中的作用。方法: 采用流式细胞术检测不同时期VMC患者(n=126)外周血CD4+CD45RA+和CD4+CD45RO+以及CD19+的表达,并设正常人群作为对照组(n=50)。结果: 急性期（<3个月）患者CD4+CD45RA+和CD4+CD45RO+以及CD19+与对照组相比差异显著。<1个月以CD4+CD45RA+和CD4+CD45RO+为著（P<0.01;P<0.01）。病程<7 d的患者与对照组相比,CD4+CD45RA+值的降低具有显著性(P<0.01);CD4+CD45RO+值下降明显(P<0.01)。而病程7~14 d、14~21 d及21~30 d的患者CD4+CD45RA+值、CD4+CD45RO+值及CD19+值与对照组相比差异均有统计学意义,CD4+CD45RO+值恢复较快。CD4+CD45RA+/CD4+CD45RO+在发病14 d内的患者与对照组差异无统计学意义;而14~30 d的患者与对照组相比差异有显著性。发病1~3个月患者以CD19+为著（P<0.01）。急性期CD4+CD45RA+/CD4+CD45RO+细胞的比例失衡。恢复期（3~12个月）患者CD4+CD45RA+和CD4+CD45RO+以及CD19+、CD4+CD45RA+/CD4+CD45RO+细胞的比例与对照组相比差异无显著性。结论: VMC患者感染初期（<1个月）以T淋巴细胞亚群异常为主,感染后期（1~3个月）以B淋巴细胞异常较为显著,以上两时期均存在CD4+CD45RA+/ CD4+CD45RO+细胞比例失衡。恢复期（3~12个月）T淋巴细胞及B淋巴细胞逐渐恢复至正常。     

Characterization by immunoperoxidase of the lymphocytes with bundle- shaped tubular (BST) inclusions in human normal peripheral blood

In six normal subjects, no surface immunoglobulins were detected on blood lymphocytes containing bundle-shaped tubular (BST) inclusions, after incubation with anti-human IgM peroxidase-labeled Fab'2 fragments. These Fab'2 fragments from pepsin-digested sheep anti-human immunoglobulins revealed human mu kappa, and lambda chains. These results suggest that the BST inclusions do not belong to B lymphocytes but belong to T cells and/or a third lymphocyte population, including K cells and precursors of T and B cells.     

Identification and characterization of a new surface membrane antigen found predominantly on malignant B lymphocytes.

A monoclonal antibody, 1D10, was derived that identifies a new antigenic epitope on the surface of malignant B lymphocytes. Normal resting and stimulated lymphocytes do not express the antigen. The majority of individuals with acute Epstein-Barr virus infection express the antigen on their lymphocytes, and in these patients, the T lymphocyte may also be antigen positive. The antigen was found on B-lymphoid neoplasia from the early pre-B cell stage through terminally differentiated plasma cells, a characteristic not reported for other B cell-associated antigens. Studies on homozygous typing cells and cells from individuals with known HLA phenotypes indicate that the antigen does not segregate in a pattern characteristic for major histocompatibility antigens. The molecule is a heterodimeric polypeptide with the molecular weight and isoelectric points of the alpha and beta chains being 32,000 d/4 and 28,000 d/6, respectively. Evidence is presented that the 1D10 molecule is not HLA-DR, -DP, or -DQ. By extrapolation, we suggest that this novel molecule may represent HLA D-region gene expression of a gene(s) not normally expressed. Potential candidates are D-region pseudogenes. We conclude that the antigenic epitope identified by the 1D10 monoclonal antibody is unique among previously described B-lymphocyte antigens. Further studies of the factors controlling the expression of this molecule, as well as studies designed to look at the possible cellular function, may provide insights for understanding crucial events in the malignant transformation of lymphocytes.     

Differential effects of red cells on the formation of normal and neoplastic mouse B lymphocyte colonies in vitro.

Intact sheep red cells potentiated mouse B lymphocyte colony growth in agar but red cell membranes or lysates exhibited no comparable ability. For maximum colony formation red cells had to be present throughout the entire culture period. Red cells added to cultures late in the culture period did not potentiate growth. Intimate contact between colony-forming cells and red cells was not essential for potentiation to occur. Red cell lysates inhibited normal B lymphocyte colony formation in cultures containing intact red cells, but did not inhibit colony formation by cells of the B lymphoid leukemia ABE-8. This differential effect may provide a means of differentiating normal from neoplastic colony-forming B lymphocytes in the mouse. Eight different tumors were also examined. Intact red cells potentiated colony formation by all of them. Lysed red cells did not potentiate the growth of any of the tumor lines. The mastocytoma P815 was the only tumor whose colony formation was inhibited by the addition of intact and lysed red cells.     

Tyrosine phosphorylation of phospholipase C induced by membrane immunoglobulin in B lymphocytes.

Ligation of membrane IgM on B lymphocytes causes activation of a protein-tyrosine kinase(s) (PTK) and of phospholipase C (PLC). To determine whether these are elements of a common signal-transduction pathway, the effect of three PTK inhibitors on the rise in intracellular free Ca2+ concentration [( Ca2+]i) in human B-lymphoblastoid cell lines was assessed. Tyrphostin completely suppressed the increase in [Ca2+]i and the generation of inositol phosphates induced by ligation of membrane immunoglobulin (mIg) M. Herbimycin and genistein reduced by 30% and 50%, respectively, the rise in [Ca2+]i caused by optimal ligation of mIgM, and they abolished it in cells activated by suboptimal ligation of mIgM. Tyrphostin had no effect on the capacity of aluminum fluoride to increase [Ca2+]i. To determine whether a function of PTK is the phosphorylation of PLC, immunoprecipitates obtained with anti-phosphotyrosine from detergent lysates of B-lymphoblastoid cells were assayed for PLC activity. Ligation of mIgM increased immunoprecipitable PLC activity 2-fold by 90 sec and 4-fold by 30 min. Specific immunoprecipitation and Western blot analysis identified tyrosine phosphorylation of the gamma 1 isoform of PLC after 60 sec of stimulation. Activation of PLC in B cells by mIgM requires PTK function and is associated with tyrosine phosphorylation of PLC-gamma 1, suggesting a mechanism of PLC activation similar to that described for certain receptor PTKs.     

Traffic of T and B lymphocytes in the normal spleen.

The occurrence of C3b and C3d complement receptors and of Fc and E receptors in normal splenic tissue was studied in cryostat sections incubated with specific marker cells. Thereby the topographic distribution of T and B lymphocytes in the splenic tissue was mapped: Te lymphocytes are found especially in the periateriolar sheaths, B lymphocytes in eccentric zones around the arterioles. The cells in the splenic pulp carry especially Fc, but also C3b receptors, whereas C3d receptors appear to be specific of lymphocytes. The basic lymphocyte traffic behind the mapped distribution of the lymphocyte subpopulations in the spleen was elucidated by lymphocyte kinetic studies using autologous 51Cr-labelled reinfused lymphocytes. These studies were performed partly on normal persons and partly on splenectomized persons without immune defects. The results indicate the presence of an exchangeable pool of T as well as of B lymphocytes in the spleen, the T cell pool being larger and making up in normals about 30% of the total exchangeable T lymphocyte mass. The matrix for the T lymphocyte pool in the spleen is the periateriolar sheaths. The difference in structure between the lymph nodes and the lymphoid tissue of the spleen, including differences in the distribution of lymphocyte subpopulations, are explained by the special vascular arrangement in the spleen.     

Comparison of normal and chronic lymphocytic leukemia lymphocyte surface Ig determinants using peroxidase-labeled antibodies. II. quantification of light chain determinants in atypical lymphocytic leukemia.

Five cases of atypical lymphocytic leukemia were investigated with regard to their membrane-associated light chains. Detection and quantitation of antigenic determinants were performed by means of peroxidase-labeled antibodies according to Avrameas et al. The cases studied had clinical and cytologic features in common: an active clinical course, marked splenomegaly, severe anemia and thrombocytopenia, little or no lymph node enlargement, and very high white blood counts with small mature lymphocytes and poorly differentiated lymphoid cells. Blood lymphocytes of all patients carried a single type of light chain, and 90%-100% of the cells were stained. The average number of antigenic sites per cell was 72,500 (range 40,000-97,500). These results differed from those previously found in typical CLL (mean value 9000) and approached the values of normal peripheral blood lymphocytes (90,000). The criteria investigated in this study could be of value for the diagnosis and prognosis of some atypical forms of lymphocytic leukemia.     

B-cell chronic lymphocytic leukemia cells express a surface membrane phenotype of activated,antigen-experienced B lymphocytes

B-cell chronic lymphocytic leukemia (B-CLL) is considered an accumulative disease of antigen-naive CD5(+) B lymphocytes that circulate in the resting state. However, to evaluate the possibility that B-CLL cells resemble antigen-experienced and activated B cells, we analyzed the expression of markers of cellular activation and differentiation on CD5(+)CD19(+) cells from B-CLL patients and from age-matched healthy donors. The leukemic cells from all B-CLL patients, including those that lack significant numbers of V gene mutations, bear the phenotype of activated B cells based on the overexpression of the activation markers CD23, CD25, CD69, and CD71 and the underexpression of CD22, Fcgamma receptor IIb, CD79b, and immunoglobulin D that are down-regulated by cell triggering and activation. Furthermore, these leukemic cells resemble antigen-experienced lymphocytes in the underexpression of molecules that are down-regulated by cell triggering and in the uniform expression of CD27, an identifier of memory B cells. A comparison of the phenotypes of B-CLL patients with and without immunoglobulin V gene mutations suggests that the 2 subgroups differ both in specific marker expression (CD69, CD71, CD62 L, CD40, CD39, and HLA-DR) and in the time since antigenic stimulation, based on the reciprocal relationship of CD69 and CD71 expression. These findings imply that the leukemic cells from all B-CLL cases (irrespective of V gene mutations) exhibit features of activated and of antigen-experienced B lymphocytes and that the B-CLL cells that differ in immunoglobulin V genotype may have different antigen-encounter histories.     

Functional assay of cytotoxic lymphocytes involved in antibody-mediated cytotoxicity in normal and rheumatoid subjects.

Antibody-mediated lymphocyte-induced cytotoxicity (K cell activity) was measured in a system consisting of Chang human liver cells, rabbit anti-Chang antiserum, and Triosil-Ficoll purified human lymphocytes. It was concluded that one K cell probably inactivates or kills one target cell, and that the number of active K cells is similar in the peripheral blood of normal and rheumatoid subjects and is about 0-5%.     

Complement and virus-specific antibody-dependent infection of normal B lymphocytes by human immunodeficiency virus type 1

We tested the susceptibility of human purified, normal B lymphocytes to human immunodeficiency virus type 1 (HIV-1) infection, in the presence or absence of complement-sufficient serum and of virus-specific antibodies. Virus replication was detected when cells were infected in the presence of both complement and anti-HIV antibodies (C'-ADE conditions), by day 2 postinfection. Similar results were obtained when B lymphocytes were purified either from peripheral blood (three healthy donors) or from tonsils (four individuals with chronic tonsillitis). HIV infection was shown by polymerase chain reaction (PCR) detection of proviral sequences (gag and pol genes), by p24 antigen synthesis, and by cocultivation assay with MT2 cells. The higher p24 production was obtained when B cells were preactivated for 2 days by phorbol 12- myristate 13-acetate (PMA) before infection and then cultured in the presence of low-molecular weight B-cell growth factor (LMW-BCGF). Expression of virus envelope glycoprotein (gp) 120 could also be detected on a subpopulation of B cells (CD19+, CD22+) by flow cytometry. Blocking experiments with monoclonal antibodies (MoAbs) against CD4, CD21 (complement receptor 2 [CR2]), CD35 (CR1), CD19, and CD5 surface molecules indicated that infection of B cells involves CD4, CD21, and CD35 antigens. Indeed, blocking of CD4 receptor inhibited 10% of p24 production, and blocking of both CD21 and CD35 led to extinction of p24 signal. CR-dependent pathway is thus a major route for C'-ADE of HIV infection in normal B cells. Our results emphasize the importance of studying interactions between HIV and the complement system for better understanding infection mechanisms and the major dysfunctions of B cells in HIV-infected individuals.     

Detection of monoclonal B lymphocytes in multiple myeloma by immunofluorescence tests of surface immunoglobulins.

Peripheral blood lymphocytes of 16 patients with secretory type of multiple myeloma and 5 with nonmyelomatous monoclonal gammopathy were investigated for the surface immunoglobulins on the cell by immunofluorescence. A low pH shock of cells before staining was applied to dissociate the passively absorbed immunoglobulins present on the cell surface. Increases of B lymphocytes bearing surface immunoglobulins which have the same light chains as those of monoclonal immunoglobulins produced by the plasma cells were found in 5 of 11 common secretory myeloma patients and in all of 6 Bence-Jones myeloma patients. Ratios of cells bearing light chains of kappa- and lambda-types (kappa/lambda) appeared abnormal in almost all with an exception of only 3 cases of myeloma patients, even in the cases where the number of Ig bearing cells did not increase. Increases of possible monoclonal B cells bearing IgG, in addition to IgA cells, were observed in some patients with IgA myeloma. Increases of B cells bearing certain heavy chains were also observed in all 5 patients with Bence-Jones myeloma during the course of disease. No abnormalities of B cells bearing surface immunoglobulin were found in nonmyelomatous monoclonal gammopathy. These results suggest that proliferation of monoclonal B lymphocytes, which may be progenitors to the malignant plasma cells, occurs in a majority of myeloma patients, but not in nonmyelomatous monoclonal gammopathy.     

Myristoylation and the post-translational acquisition of hydrophobicity by the membrane immunoglobulin heavy-chain polypeptide in B lymphocytes.

Membrane immunoglobulin heavy chain in pre-B and in B cells is initially synthesized as a relatively hydrophilic protein that is nonetheless stably anchored in the endoplasmic reticulum membrane. In B cells, but not in pre-B cells, the membrane immunoglobulin heavy chain is post-translationally converted to a relatively hydrophobic form that partitions into the oil phase when solubilized with the phase-separating detergent Triton X-114. Covalent myristoylation of the membrane and secretory forms of immunoglobulin heavy chains as well as of light chains was observed in B cells. Myristoylation of the membrane immunoglobulin heavy chain correlates with its transport to the cell surface and its post-translational conversion to a relatively hydrophobic form. This post-translational modification is hydroxylamine resistant and may be responsible for the assembly and transport of membrane immunoglobulin to the cell surface in B cells.