Cell surface antigens expressed by subsets of pre-B cells and B cells

A large number of monoclonal antibodies, produced by immunizing rats with mouse pre-B cell lines, have been analyzed for their ability to define cell surface antigens expressed by B cells at early stages of differentiation. Whereas many antibodies recognized antigens on pre-B cell lines, only two clones detected cell surface antigens that were distinguished by their restricted distribution among a panel of continuous cell lines and cells from various tissues. Monoclonal antibody clone AA4.1 recognized a cell surface antigen found on all pre-B lymphomas and on one of three B lymphomas tested. This antigen was found on cells at highest frequency in the bone marrow. Adult spleen and fetal liver also have detectable numbers of AA4.1+ cells. Cells that did not express this antigen include plasmacytomas, two of three B lymphomas, T lymphomas, a stem cell line, adult liver, brain, thymus, and lymph node cells. Clone GF1.2 detected an antigen on some pre-B cell lines, one of three B lymphomas tested, and a small fraction of cells from adult bone marrow, spleen, lymph node, and fetal liver. Plasmacytomas, some pre-B lymphomas, two B lymphomas, T lymphomas, adult liver, brain, and thymus cells were negative. In adult bone marrow, AA4.1 bound to all cytoplasmic IgM+ pre-B cells, whereas GF1.2 detected one-half of these cells. Both antibodies recognized approximately 50% of surface IgM+ (sIgM+) bone marrow cells. A small population of bone marrow cells lacking any detectable Ig (surface or cytoplasmic) also reacted with these antibodies. Depletion of AA4.1 or GF1.2 antigen-bearing cells from bone marrow reduced the ability of bone marrow B cells to respond to LPS by 50 to 65%. Experiments with a cloned pre-B lymphoma demonstrate that AA4.1+ pre-B cells become sIgM+ GF1.2+ B cells after activation with LPS. These antibodies recognize cell surface determinants with restricted distribution among the B lymphocyte lineage because they detect antigens displayed by normal and transformed immature B lymphocytes.     

Constitutive versus cell cycle regulation of c-myb mRNA expression correlates with developmental stages in murine B lymphoid tumors.

The expression of c-myb mRNA is differentially regulated in murine B lymphoid tumors such that B cell lymphomas and plasmacytomas contain significantly less c-myb mRNA than pre-B cell lymphomas. To examine the low level of c-myb mRNA expression in the murine B cell lymphoma cell line BCL1, nonessential amino acid starvation was used to block these cells in a G1 state. When BCL1 cells were released from this block, a 7- to 10-fold increase in c-myb mRNA was detected in late G1 and S phase cells relative to that detected in exponentially growing BCL1 cells. This increase was not inhibited by aphidicolin. To determine whether cell cycle regulation of c-myb mRNA expression occurred during exponential growth in both murine pre-B cell lymphoma and B cell lymphoma cell lines, elutriation was used to separate exponentially growing cell populations. An increase in c-myb mRNA expression was seen in late G1 and S phase fractions from B cell lymphoma cell lines. In contrast, c-myb mRNA levels remained constant in elutriation fractions isolated from pre-B cell lymphoma cell lines. Expression of c-myb mRNA was not detected in exponentially growing or in Go serum-stimulated murine fibroblasts. These results indicate that constitutive vs cell cycle regulation of c-myb mRNA expression is related to the state of differentiation in murine B lymphoid tumors and suggest that a switch in regulation may occur during normal B cell development.     

Antibody-induced redistribution of normal and tumor associated surface antigens

Redistribution (capping) of normal and tumor-associated surface antigens was studied on murine and human cells by the indirect membrane immunofluorescence (MIF) technique. The capping of H-2 isoantigens was compared on normal mouse T-lymphocytes and on YAC cells, a Moloney leukemia virus (MLV) induced lymphoma. H-2 and Moloney virus induced cell surface antigen (MCSA) capping was compared on three YAC lines with different MCSA concentrations. H-2 and tumor-associated surface antigen capping was compared on two polyoma induced sarcoma lines and five methylcholanthrene induced sarcoma lines. In the human system, IgM-capping was compared on normal lymphocytes and on the Burkitt lymphoma derived Daudi line. Capping of HL-A and the Epstein-Barr virus (EBV) determined membrane antigen (MA) was compared on the Burkitt lymphoma derived line Maku and on EBV-superinfected Daudi cells. H-2 antigens on normal murine cells capped more promptly and on a larger fraction of the cell population on the various tumor cells. Surface associated IgM showed a better capping on normal lymphocytes than on Daudi cells. All tumor associated antigens except MCSA, showed good capping. MCSA was almost completely refractory to capping. Increasing concentrations of MCSA appeared to inhibit the capping of H-2 on the YAC sublines with different concentrations of MCSA. The polyoma induced ascites sarcoma (SEWA) did not cap either with regard to H-2 or the polyoma determined surface antigen.     

A monoclonal antibody defining a lymphoma-associated antigen in man

A monoclonal antibody (Ab 89) was developed against a lymphoma-associated antigen on the tumor cells of a patient (N.B.) with a B cell, poorly differentiated lymphocytic lymphoma (D-PDL). By indirect immunofluorescence, Ab 89 was shown to react only with N.B. lymphoma cells and was unreactive with normal N.B. lymphoid cells, normal fractionated peripheral blood cells, other normal lymphoid tissues, fetal tissues, and non-lymphoid malignant cells. In addition, Ab 89 was unreactive with conventional immunoglobulins, private and public HLA antigens, or Ia-like antigens. More importantly, Ab 89 was reactive with some B cell lymphoid malignancies. Of the 57 B cell lymphomas tested, it was found that Ab 89 reacted with approximately 10% of B cell D-PDL and B cell chronic lymphocytic leukemia (CLL). Of interest was the finding that N.B. serum contained a circulating antigen which could specifically block the reactivity of Ab 89. The data obtained suggests that Ab 89 defines a tumor-associated antigen shared by a unique subgroup of B cell lymphomas. The group of patients reactive with Ab 89 did not fall into any histopathologic classification system. These data support the notion that there is greater heterogeneity of B cell lymphomas than may have been previously recognized.     

Phylogeny of lymphocyte heterogeneity: the cellular requirements for in vitro antibody responses of channel catfish leukocytes

Three functionally distinct leukocyte subpopulations were isolated from the peripheral blood of channel catfish. Surface immunoglobulin-positive (sIg+) and sIg- lymphocytes were isolated by an indirect "planning" procedure employing monoclonal antibodies specific for channel catfish Ig. A third population composed of macrophages was isolated by adherence to baby hamster kidney cell microexudate-coated surfaces. Functional features of these three cell types were established by assessing their role(s) in primary in vitro anti-hapten PFC responses to known murine thymus-dependent (TD) and thymus-independent (TI) antigens. The results indicated that anti-hapten PFC responses to a TI antigen required the presence of sIg+ lymphocytes and macrophages. In contrast, all three cell types were required for responses to TD antigens. Furthermore, the results of studies involving the depletion of antigen-reactive lymphocytes demonstrated that both hapten-specific sIg+ cells and carrier-specific sIg- cells were required for anti-hapten responses to TD antigens. These studies provide direct evidence that catfish have separable B cells (sIg+ lymphocytes), T helper cells (sIg- lymphocytes), and accessory cells (macrophages) quite similar to those seen in higher animals.     

A human alloantiserum precipitates Ia-like molecules from chronic lymphocytic leukemia cells.

Alloantigens specific for human B lymphocytes can be identified with selected antisera. These antigens have similarities to murine Ia antigens in that they are found on human B lymphocytes and are controlled by genes linked to genes controlling HLA. Chronic lymphocytic leukemia cells bearing B cell antigens were labeled with 3H leucine and the membrane components reacting with the B cell antisera isolated by immunoprecipitation. These membrane components had m.w. of 33,000 and 24,000 daltons similar to the murine Ia antigens. The results complete the homology of murine Ia and human B cell alloantigens.     

Rabbit antiserum against Daudi-cell membrane fractions detects cell surface antigens present on subpopulations of human lymphocytes.

An antiserum was produced by immunization of rabbits with membrane preparations of a human lymphoblastoid B cell line, Daudi. After absorption with a human endometrial carcinoma cell line, this antiserum appeared to be specific for antigen(s) present on adult and fetal thymocytes as well as on tonsillar lymphocytes but absent, or present in very small amounts, on normal or phytohemagglutinin- (PHA) stimulated peripheral blood lymphocytes (PBL). When T and B cell-enriched fractions from tonsillar lymphocytes were tested with the anti-Daudi serum, the reactivity was equally distributed in each population. Among 13 human lymphoblastoid cell lines tested, reactivity was demonstrated on three out of four T cell lines, and on four out of nine B cell lines. The positive reacting B cell lines were derived from two African and two American Burkitt lymphomas. The antigen(s) described does not seem to be related either to human Ia-type antigens or to Epstein-Barr virus-associated antigens because these antigens are not present on fetal or adult thymocytes. Reciprocal absorption experiments indicate that this anti-Daudi serum detects the same antigenic structures present on certain subpopulations of T and B lymphocytes.     

A murine cDNA encodes a pan-epithelial glycoprotein that is also expressed on plasma cells.

Using a subtractive cDNA approach, we have identified a number of genes expressed in murine plasmacytomas, but not B or pre-B lymphomas. One of these genes, 289A, expresses a 1.8-kb microsomally localized mRNA that encodes a 314-amino-acid protein containing a signal sequence and a hydrophobic transmembrane domain. Sequence comparison suggests that the predicted protein is the murine homologue of a human cell surface pan-epithelial glycoprotein known variously as EGP, GA733-2, KSA, and KS1/4, recognized by mAb HEA125, GA733, KS1/4, CO17-1A, M74, and 323/A3. The 289A mRNA is highly expressed in normal murine tissues containing epithelial cells, and at a low level in plasma cells induced by LPS stimulation of spleen B lymphocytes. It is expressed in 15 of 16 plasmacytomas, but at a much lower level, if at all, in pre-B or B lymphomas. In human B cell lines, 289A detects a 1.5-kb mRNA in the myeloma cell line 8226, but not in Burkitt's lymphoma or lymphoblastoid cell lines. Subsequent FACS analysis of human cell lines with the mAb GA733 and KS1/4 demonstrated concordant expression of the mRNA and the protein. We conclude that 289A is the murine homologue of EGP, GA733-2, KSA, and KS1/4 Ag. Although its expression was previously thought to be restricted to epithelial cells, it is also expressed in plasma cells and is a B lymphocyte differentiation Ag. Because of the multiplicity of names, we propose calling the human gene hEGP314, and the murine gene mEGP314.     

Proteoglycan-targeted antibodies as markers on non-Hodgkin lymphoma xenografts

Summary A family of mono- and polyclonal antibodies raised against proteoglycans or their subcomponents served as novel markers to characterize the phenotypes of three non-Hodgkin lymphoma xenograft lines (HT 58 lymphoblastic, HT 117 centroblastic, HT 130 centrocytic) together with normal, human peripheral blood B lymphocytes. These xenografted NHL lines, maintained by serial transplantations on artificially immunosuppressed mice, expressed very similar B-cell-related antigens and differences on the cell surface (HT 58 > HT 117 > HT 130 > B cells) when they were exposed to monoclonal antibodies (mAb) to cartilage proteoglycans. Anti-proteoglycan antibodies used in this study recognize complex epitopes of core protein segment associated with carbohydrate, shared by human cartilage proteoglycans and certain lymphoma cells. The binding of these antibodies was independent of cell-cycle phase. The results suggest that the anti-proteoglycan mAbs could be used as new phenotypic markers to individualize non-Hodgkin lymphomas.     

Cutting edge: constitutive B cell receptor signaling is critical for basal growth of B lymphoma

B lymphomas account for the majority of the lymphoma cases. BCR expression appears to be important for B lymphoma because most oncogenes are translocated to nonrearranged Ig loci and because all of the variants that arise in anti-idiotypic Ab-treated lymphoma patients remain BCR positive. Based on this and the fact that BCR is required for mature B cell survival, we tested the requirement for continued expression of BCR for the growth and survival of B lymphoma cells. Using Igalpha or Igbeta-specific small interfering RNA (siRNA) to inhibit BCR expression, we demonstrate for the first time that constitutive signaling by BCR is critical for survival and proliferation of both murine and human B lymphoma cells. The BCR signals in lymphoma appear to be mediated by Syk, as it is constitutively active in a variety of B lymphoma cells. Blocking Syk activity by selective inhibitors suppresses growth of several murine and human B lymphomas.     

Cloning of a complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homologous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19

The human B1 (CD20) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In order to determine whether this structure is also expressed by murine B cells, cDNA clones that encode the mouse equivalent of the B1 molecule were isolated. The longest murine cDNA clone isolated, pmB1-1, contained a 1.4-kb insert with an 873 base pair open reading frame that encodes a protein of 32 kDa. The predicted mouse B1 protein contains three hydrophobic domains that may span the membrane four times and shares a 73% amino acid sequence homology with the human B1 protein. The pmB1-1 cDNA probe was used to examine mB1 mRNA expression. Northern blot analysis indicated that pmB1-1 hybridized with two mRNA species of 2.3 and 3.0 kb that were expressed only in murine spleen lymphocytes, in B lineage cell lines representing mature B cells, and were weakly expressed in one of two plasmacytoma cell lines. pmB1-1 failed to hybridize with RNA isolated from murine T cell lines, thymus, and nonlymphoid tissues. Southern blot analysis indicated that mB1 was encoded by a single copy gene. In situ hybridization localized the mB1 gene to chromosome 19 band B, a region that also contains the genes that encode the Ly-1, Ly-10, and Ly-12 Ag. These results suggest that only B cells express this heretofore undescribed murine cell-surface protein that is structurally homologous with the membrane-embedded human B1 Ag.     

EBV Latent Membrane Protein 1 Activates Akt,NFκB,and Stat3 in B Cell Lymphomas

Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice. The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFκB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro. Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling. These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. These results suggest that Akt, NFκB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.     

Production of tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) by murine pre-B and B cell lymphomas

Media from murine pre-B and B lymphoma cell cultures, but not from myeloma cell cultures, was cytotoxic to WEHI 164 cells, causing these TNF-sensitive targets to release 51Cr. The cytotoxic activity in the culture medium reached maximum levels approximately 4 days after the cell culture was initiated. The constitutive production of the factors was not influenced by depletion of serum from the medium or by the addition of either phorbol ester or bacterial endotoxin. The factor has a Mr greater than 10 kDa, and its cytotoxicity was abolished by anti-serum against murine TNF. Northern blot analysis with the use of cDNA probes to murine tumor necrosis factor (TNF-alpha) and lymphotoxin (LT, TNF-beta) showed high levels of TNF-mRNA in the pre-B cell lines, lower levels in the mature B cell lines and no TNF-mRNA in the myeloma cell lines. LT mRNA was present in pre-B cell lines, at a much lower concentration in only one of the B cell lines, and was not present in three other B lymphomas or in the myelomas tested. The results show a positive correlation between the presence of TNF and/or LT mRNA and the 51Cr-releasing activity present in the cell culture medium. Our data indicate that TNF and LT can be produced by murine B cells and that the synthesis of these cytokines may be restricted to certain differentiation stages of the B cell lineage.     

B lymphoblast antigen (BB-1) expressed on Epstein-Barr virus-activated B cell blasts, B lymphoblastoid cell lines, and Burkitt's lymphomas

Two new cell surface antigens expressed on B lymphoblastoid cell lines (B-LCL) were defined with cytotoxic mouse monoclonal antibodies. One marker, BB-1 (for B lymphoblast antigen-1), was detected on human and nonhuman primate B-LCL, Epstein-Barr virus (EBV)-activated B cell blasts, most Burkitt's lymphomas, and Ia+ B lymphoblast-like myelomas. Polyclonal B cell activators such as pokeweed mitogen (PWM) and lipopolysaccharide (LPS) also induced the expression of BB-1 on immunoglobulin (Ig)-positive cells. In contrast, BB-1 could not be detected on normal lymphoid tissues by complement-dependent cytotoxicity and immunofluorescence (IF) assays or by analysis with a fluorescence-activated cell sorter (FACS). T cell blasts, T cell leukemias, and pre-B cell or erythroblastic leukemia cell lines were also BB-1 negative. Of particular interest was the finding that BB-1 was expressed on the Jijoye lymphoma but only marginally on a subline of Jijoye, P3HR-1, that lacks receptors for EBV and produces a defective virus incapable of transforming lymphocytes. A second lymphoblast antigen (LB-1) unlike BB-1, was present on both T and B cell blasts and virus-transformed T- and B-LCL but not on normal lymphoid tissues.     

EBV latent membrane protein 1 activates Akt, NFkappaB, and Stat3 in B cell lymphomas

Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice. The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFkappaB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro. Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling. These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. These results suggest that Akt, NFkappaB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.     

Interleukin-21 Enhances Rituximab Activity in a Cynomolgus Monkey Model of B Cell Depletion and in Mouse B Cell Lymphoma Models

Rituximab, a monoclonal antibody targeting CD20 on B cells, is currently used to treat many subtypes of B cell lymphomas. However, treatment is not curative and response rates are variable. Recombinant interleukin-21 (rIL-21) is a cytokine that enhances immune effector function and affects both primary and transformed B cell differentiation. We hypothesized that the combination of rIL-21 plus rituximab would be a more efficacious treatment for B cell malignancies than rituximab alone. We cultured human and cynomolgus monkey NK cells with rIL-21 and found that their activity was increased and proteins associated with antibody dependent cytotoxicity were up-regulated. Studies in cynomolgus monkeys modeled the effects of rIL-21 on rituximab activity against CD20 B cells. In these studies, rIL-21 activated innate immune effectors, increased ADCC and mobilized B cells into peripheral blood. When rIL-21 was combined with rituximab, deeper and more durable B cell depletion was observed. In another series of experiments, IL-21 was shown to have direct antiproliferative activity against a subset of human lymphoma cell lines, and combination of murine IL-21 with rituximab yielded significant survival benefits over either agent alone in xenogeneic mouse tumor models of disseminated lymphoma. Therefore, our results do suggest that the therapeutic efficacy of rituximab may be improved when used in combination with rIL-21.     

Expression of low levels of peripheral lymph node-associated vascular addressin in mucosal lymphoid tissues: possible relevance to the dissemination of passaged AKR lymphomas

Lymphoid tumors display a wide variety of growth patterns in vivo, from that of a solitary extralymphoid tumor, to a general involvement of all lymphoid organs. Normal lymphocytes are uniquely mobile cells continuously recirculating between blood and lymph throughout much of their life cycle. Therefore, it is reasonable to propose that disseminating malignant lymphocytes may express recirculation characteristics or homing properties consistent with that of their normal lymphoid counterparts. Trafficking of lymphocytes involves the expression and recognition of both lymphocyte homing receptors and their opposing receptors on endothelium, the vascular addressins. These cell surface elements direct the tissue-selective localization of lymphocyte subsets in vivo into organized lymphoid organs and sites of chronic inflammation where specific binding events occur between lymphocytes and the endothelium of specialized high endothelial venules (HEV). In a recent murine study of 13 lymphoma lines, we found that lymphomas that bind well to high endothelial venules, in the Stamper-Woodruff in vitro assay (an assay of lymphocyte binding to venules in frozen sections of peripheral lymph nodes or Peyer's patches), spread hematogenously to all high endothelial venule bearing lymphoid organs, whereas non-binding lymphomas did not. In some cases lymphomas that bound with a high degree of selectivity to peripheral lymph node (PLN) high endothelial venules exhibited only limited organ preference of metastasis, involving the mucosal lymphoid organs Peyer's patches (PP) in addition to the peripheral lymph nodes of adoptive recipients. Here we demonstrate that Peyer's patch high endothelial venules express a low but functional level of peripheral lymph node addressin (MECA-79) that can be recognized by lymphomas expressing the peripheral lymph node homing receptor (MEL-14 antigen).(ABSTRACT TRUNCATED AT 250 WORDS)     

Some lymphoid cell lines transformed by Abelson murine leukemia virus lack a major 36,000-dalton tyrosine protein kinase substrate.

Fibroblasts transformed by Abelson murine leukemia virus differ from normal fibroblasts in that they contain several cellular proteins, including one of 29 and one of 36 kilodaltons, which are phosphorylated at tyrosine residues. Since it has been shown before that these proteins also become phosphorylated at tyrosine after transformation of fibroblasts by a number of other retroviruses, their phosphorylation may play an important role in the transformation of these cells. In contrast, the 36-kilodalton phosphoprotein was not detectable in three of the four lines of Abelson virus-transformed B lymphoma cell lines studied here. These three cell lines, RAW307.1.1, 18-48, and 18-81, and a B lymphoma induced by mineral oil, WEHI 279, were all found to lack both the phosphorylated and unphosphorylated forms of the 36-kilodalton protein. It thus appears that expression of this major cell protein is not essential for the survival of B lymphoma cells in culture and that the phosphorylation of the 36-kilodalton protein at tyrosine is not essential for transformation of pre-B lymphocytes by Abelson virus.     

Tyrosine phosphorylation of CD19 in pre-B and mature B cells.

Cross-linking of B cell surface immunoglobulins (sIg) results in activation of mature B cells and stimulates a molecular signaling mechanism for antigen-specific B cell expansion and differentiation. This signaling pathway is dependent on tyrosine (Tyr) phosphorylation and results in the activation of sIg-associated src family kinases and p72SYK. Rapid Tyr phosphorylation occurs on multiple protein substrates. Here we show that activation of B cells by cross-linking sIg results in an increase in Tyr phosphorylation of the lineage-restricted B cell surface antigen CD19, and show that it is a major substrate of activated Tyr kinase following sIg stimulation. Lower levels of constitutive CD19 Tyr phosphorylation occurred in most sIg+ mature B cell lines examined and in normal dense tonsillar B cells. We also find that when CD19 is Tyr-phosphorylated it becomes competent to interact with SH2 domains suggesting a mechanism whereby, following B cell activation, CD19 could be linked to intracellular signaling pathways. In sIg- pre-B cell lines, CD19 was expressed but was not constitutively phosphorylated on tyrosine. Upon CD19 cross-linking, Tyr phosphorylation of CD19 was induced in sIg- pre-B cell lines. CD19 cross-linking also directly induced Tyr phosphorylation of CD19 and other substrates in mature B cells. The ability of CD19 to signal in the absence of sIg expression may provide important stimulation in pre-B cell development.