The pathology and homing of a transplantable murine B cell leukemia (BCL1).

The pathology and homing characteristics of a murine B cell leukemia are described. Experiments utilizing autoradiography to determine the early homing pattern of the leukemic cells revealed a pronounced localization of the labeled cells to the spleen. The cells that were seen in the white pulp showed preferential localization to the follicles or B cell domains. Tissue section immunofluorescence with antibodies to kappa- and lambda-light chains was used to study the initial mouse with this disease as well as to study the mice that were injected with in vivo passaged cells. These mice also showed predominant involvement of the spleen. Although the initial mouse with this disease had 200,000 lambda-bearing B lymphocytes per mm3 in the peripheral blood and closely resembled a human chronic lymphocytic leukemia patient, the studies described suggest that this murine B cell neoplasm is a lymphoma with a striking predilection for splenic involvement. The other organs including the bone marrow as well as the peripheral blood appeared to be involved secondarily. This unusual spontaneously occurring murine B cell disease provides a useful model for the investigation of certain commonly occurring human lymphomas and leukemias.     

Murine B cell leukemia (BCL1): organ distribution and kinetics of growth as determined by fluorescence analysis with an anti-idiotypic antibody.

The life history of a transplantable B cell leukemia (BCL1) that arose spontaneously in a BALB/c mouse is described. Animals bearing this tumor live from 2 to 4 months in apparently good health despite massive splenomegaly and leukemia. Antibody to the idiotype or gamma light chain of the tumor IgM was used in conjunction with the fluorescence-activated cell sorter to identify tumor cells in the BCL1-bearing mice. The results suggest that these cells multiply and differentiate in the spleen and subsequently emigrate to the blood. Tumor cells do not recirculate as evidenced by their failure to enter the thoracic duct or to infiltrate lymph nodes to a significant extent. During tumor growth, a population of T cell blasts appears that may be involved with an immune response against the tumor.     

Immunosuppression in a murine B cell leukemia (BCL1): role of an adherent cell in the suppression of primary in vitro antibody responses

The ability of lymph node cells from mice bearing the BCL1 tumor to respond in vitro to mitogens, allogeneic cells, and both TI and TD antigens was investigated. The lymph nodes of such mice are not invaded with tumor cells and contain normal numbers of T and B cells. Nevertheless, at the peak of tumor burden in the spleen and blood (approximately 8 to 12 wk after injection with tumor cells), the lymph node cells from the tumor-bearing mice display markedly decreased responsiveness both to allogeneic cells and to antigens. In addition, small numbers of lymph node cells from the tumor-bearing mice suppress primary antibody responses of normal lymph node cells. This nonspecific suppression of antibody responses is mediated by a G-10 Sephadex adherent, non-T, non-B cell present in the nodes of the tumor-bearing mice. Since the BCL1 tumor model is in many respects similar to the prolymphocytic type of human chronic lymphocytic leukemia, the present results may be helpful in elucidating the mechanisms underlying the in vivo immunosuppression associated with lymphocytic neoplasms in humans.     

Proliferative response of murine B-cell leukemia (BCL1) to poly(L-lysine)

Peripheral blood lymphocytes (PBL) isolated from BALB/c mice bearing a B-cell leukemia (BCL1) showed a marked proliferative response upon two days culturing with poly(L-lysine) (PLL) of various molecular weights. An inverse relationship was noted between the molecular weight of the PLL and the dose required for optimal proliferative response. PLL showed no proliferative activity when cultured with normal PBL or with lymphocytes isolated from the spleen or other lymphoid organs of BCL1-bearing mice. Double exposure to PLL and lipopolysaccharide (LPS) had a marked synergistic effect on BCL1 PBL stimulation but not on PBL isolated from normal mice. The data suggest that PLL, in contrast to LPS, may cause a selective proliferation of a subpopulation(s) of B-tumor cells at a particular stage(s) of differentiation.     

Characterization of a spontaneous murine B cell leukemia (BCL1). II. Tumor cell proliferation and IgM secretion after stimulation by LPS.

A spontaneous BALB/c B lymphocyte leukemia could be stimulated in vitro by the polyclonal B cell activator lipopolysaccharide (LPS) and the conditions for activation were studied. Spleen cells or peripheral blood lymphocytes from tumor-bearing animals responded by increased DNA synthesis and the peak of activation occurred earlier than with normal mouse spleen cells. Tumor cells harvested from the spleen, but not from the peripheral blood, could be induced by LPS to secrete IgM. Direct demonstration that the response was due to tumor cell activation and not that of contaminating normal B lymphocytes was provided by karyotype analysis and by immunoprecipitation, which showed the restriction of light chains on secreted IgM molecules to the lambda isotype.     

Characterization of the spontaneous murine B cell leukemia (BCL1). III. Evidence for monoclonality by using an anti-idiotype antibody.

We have raised an anti-idiotypic antibody against the cell surface IgM of the murine BCL1 tumor cells. This antiserum reacts exclusively with the IgM expressed on the tumor cells and detects a unique population of cells in the spleen and blood of the tumor-bearing mice. When these cells are stimulated in vitro with LPS, they secrete an IgM bearing the same idiotype as the cell surface Ig. These results are discussed in terms of a model for the immunotherapy of a chronic lymphocytic leukemia-like syndrome in mice.     

IgH V-region sequence does not predict the survival fate of human germinal center B cells

Germinal center (GC) B cell survival fate is governed in part by the outcome of successful/failed BCR-mediated interactions with accessory cells. However, the extent to which the BCR primary sequence influences such interactions is not fully understood. Over 1000 IgV(H)4 family cDNAs were sequenced from living (annexin V(-)) and apoptotic (annexin V(+) or from within tingible body macrophages) GC B cell fractions from seven tonsils. Results surprisingly demonstrate that living and dying GC B cells do not significantly differ in IgV(H), D, or J(H) gene segment use; HCDR3 length or positive charge; or mutation frequency. Additionally, equivalent IgH cDNA sequences were identified in both fractions, suggesting that BCR sequence alone is an unreliable predictor of GC B cell survival.     

STAT3 regulates the growth and immunoglobulin production of BCL(1) B cell lymphoma through control of cell cycle progression

STAT3 is constitutively phosphorylated on tyrosine(705) in self-renewing, CD5(+) murine B-1 lymphocytes. Nuclear extracts from untreated primary B-1 or CD5(+) BCL(1) B lymphoma cells were found to contain immunoreactive STAT3 protein that binds to a sis-inducible element present in the promoter of the p21(waf1/cip1) tumor suppressor gene and is constitutively phosphorylated on serine(727). To determine the functional significance of constitutive STAT3 activation in B lymphoma cells, a specific STAT3 antisense oligonucleotide was developed and used to examine basal BCL(1) cell growth and IgM production. Abrogating STAT3 expression in BCL(1) cells inhibited their proliferative capacity and induced a corresponding decrease in secretion of IgM. Cell cycle analysis showed a block in progression through G1 in BCL(1) cells treated with the STAT3 antisense oligonucleotide. These results indicate that STAT3 controls cell growth and immunoglobulin secretion by enhancing progression through the G1 phase of the cell cycle in BCL(1) B cell lymphoma.     

Characterization of a spontaneous murine B cell leukemia (BCL1). I. Cell surface expression of IgM, IgD, Ia, and FcR.

The surface marker expression of a spontaneous B lymphocyte leukemia discovered in a BALB/c mouse (BCL1) was examined and found to include a subset of markers known to occur on normal B lymphocytes. The tumor cells bore surface Ig that included both mu- and delta-chains associated with the lambda light chain. Alloantigens coded for within the murine MHC, including H-2D, H-2K, and I-region products, were identified on the tumor cells. Although normal B lymphocytes are thought to express products coded for within both the I-A and I-E subregions, the BCL1 expressed only normal amounts of I-E subregion products. In addition, the H-2 and Ia antigens revealed by 2-dimensional gel electrophoresis exhibited an abnormal pattern of post-translational modifications. The Fc, but not the complement-receptor, was present on the surface of tumor cells. The presence of IgD, Ia antigens, and the responsiveness to lipopolysaccharide (see subsequent paper) have led us to postulate that the BCL1 tumor represents a later differentiative stage than murine B lymphocyte tumors previously described.     

Antigen presentation by the BCL1 murine B cell line: in vitro stimulation by LPS

We examined the antigen-presenting capacity of BCL1 tumor cells, which are capable of differentiating in vitro with respect to immunoglobulin synthesis/secretion under the influence of LPS. In vivo passaged BCL1 cells depleted of host cell contamination either by positive selection employing panning with anti-lambda reagents, or by elimination of latex-ingesting adherent cells, are capable of MHC-restricted antigen presentation to a GAT-immune T cell line. The BCL1 cells act as antigen-presenting cells when freshly explanted, but gradual loss of this function occurs, and cells cultured for 3.5 days cannot present antigen unless LPS is included during the culture period. BCL1 cells are equivalently Ia+ after the culture period with or without LPS stimulation. Other B cell lines capable of antigen presentation appear to express this trait constitutively, and the in vivo passaged BCL1 line is therefore unique among B cell lines in having antigen-presenting cell function that can be modulated. The data suggest that freshly explanted or LPS-cultured BCL1 cells are heterogeneous with respect to antigen-presenting capacity, and the basis for this heterogeneity is being sought. BCL1 offers an opportunity to study requirements for antigen presentation by B cells.     

Molecular cloning of interferon-gamma inducible genes from a murine pre-B cell leukemia.

In the present study, a pre-B cell leukemia L1210-C7, representing a very early stage of the B lineage, was used to characterize the molecular mechanisms exploited by IFN-gamma to modulate B cell activity. A cDNA library was prepared with poly (A)+ RNA from cells stimulated with IFN-gamma and three cDNAs clones complementary to IFN-gamma inducible mRNAs were isolated by differential screening. Of these, the 9.5 cDNA hybridized to a 2.4 kb mRNA not homologous with previously cloned IFN-gamma inducible mRNAs. Furthermore, when compared with RNAs obtained from cells of different origins (fibroblasts and T cells) the 9.5 mRNA appeared to be increased only in cells belonging to the B lineage. Taken as a whole, these results demonstrate that in leukemic pre-B cells IFN-gamma induces the expression of a gene that could be employed as specific cell activation marker.     

Partial purification of murine B cell stimulatory factor (BSF)-1

BSF-1 was partially purified from serum-free culture supernatants of cells of the EL-4 thymoma line, which had been induced 48 hr earlier with 4 beta-phorbol-12 beta-myristate-12 alpha-acetate (PMA). BSF-1 in 10-liter batches was adsorbed onto and eluted from trimethylsilyl-controlled pore glass beads (TMS-CpG) and then subjected to reverse-phase high-performance liquid chromatography (RP-HPLC). The recovery of BSF-1 activity by TMS-CpG and RP-HPLC ranged from 52 to 55% and 187 to 227%, respectively. The specific activity in units per milligram of protein of partially purified BSF-1 was approximately 2600 times higher than that of the culture supernatant protein. The partially purified BSF-1 had a single isoelectric point of 6.3 and an apparent m.w. between 18,000 and 21,700 when analyzed by isoelectric focusing and gel filtration-HPLC, respectively. The ability to prepare large amounts of partially purified BSF-1 by a rapid and efficient procedure should be of great help in both biochemical and immunologic studies of this lymphokine.     

Influence of a fibroblastoid cell line derived from murine bone marrow (H-1 cells) on stem cell proliferation

A murine fibroblastoid cell line (H-1) with properties similar to those of adventitial reticular cells can support granulopoiesis and the development of mononuclear phagocytes in vitro. In the current study the effect of these cells on stem cell maintenance in vitro was assessed. The H-1 cells were unable to support CFUs replication in liquid culture, while treatment of some stem cells with H-1 conditioned medium appeared to inhibit their proliferation.     

Stimulation of B cell growth and differentiation by murine recombinant interleukin 1

Purified splenic B cells from C57BL/6 mice were separated into high-density (resting) and low-density (activated) B cells. Separated B cell populations were cultured at low cell densities (1 X 10(4) cells/well) with recombinant interleukin 1 (r-IL 1) alone or in combination with dextran sulfate (DXS) or anti-IgM monoclonal antibodies (alpha IgM mab), respectively, and proliferative responses were determined. R-IL 1 alone, as well as in synergy with alpha IgM mab or DXS, respectively, stimulated the growth of low-density B cells. Moreover, r-IL 1 and alpha IgM mab costimulated replication of high-density B cells. Separated B cell populations (1 X 10(5) cells/well) were cultured with r-IL 1 alone or in combination with DXS or alpha IgM mab, respectively, and the generation of plaque-forming cells was determined. R-IL 1 alone, as well as in synergy with DXS, stimulated the differentiation of low-density B cells into Ig-secreting cells. These findings suggest that r-IL 1 has B cell growth and differentiation factor activity and is operative on high- and low-density B cells. Thus, IL 1 may play an important role in B cell growth and maturation.     

Liver kinase B1 (LKB1) in the pathogenesis of UVB-induced murine basal cell carcinoma

LKB1, a known tumor suppressor, is mutated in Peutz–Jeghers Syndrome (PJS). It is responsible for the enhanced cancer risk in patients with PJS. Dysregulation of LKB1-dependent signaling also occurs in various epithelial cancers. UVB alters the expression of LKB1, though its role in the pathogenesis of skin cancer is unknown. Here we describe upregulation of LKB1 expression in UVB-induced murine basal cell carcinoma (BCC) and in human skin tumor keratinocytes. AMP-kinase and acetyl Co-A carboxylase, the downstream LKB1 targets, are also enhanced in this neoplasm. In addition, p-Akt, a kinase which inactivates GSK3β by its phosphorylation, is enhanced in BCCs. Consistently, an accumulation of p-GSK3β and an increase in activated nuclear β-catenin are found. mTOR signaling, which is also inhibited by LKB1, remains upregulated in BCCs. However, a marked decrease in the expression of sestrins, which function as potent negative regulators of mTOR is observed. Metformin, a known chemical inducer of this pathway, was found effective in immortalized HaCaT keratinocytes, but failed to activate the LKB1-dependent signaling in human carcinoma A431 cells. Thus, our data show that the LKB1/AMPK axis fails to regulate mTOR pathway, and a complex regulatory mechanism exists for the persistent mTOR activation in murine BCCs.     

Physicochemical and functional properties of murine B cell-derived B cell growth factor II (WEHI-231-BCGF-II)

A murine B lymphoma cell line, WEHI-231, constitutively secreted a kind of B cell stimulatory factor (BSF) that induced proliferation and IgM secretion in splenic B cells as well as BCL1 cells. Growth- and differentiation-promoting activities were not separated by various kinds of chromatographies on the basis of the m.w., isoelectric point, or hydrophobicity, and the degree of both activities in crude supernatants, DEAE-Toyopearl, TSK-3,000SWG, Mono P, and Phenyl-5PW fractions increased in a dose-dependent manner with complete correlation. The partially purified factor (WEHI-231-BGDF) did not show any other activities, such as IL 1, IL 2, interferon, or colony stimulating factor. WEHI-231-BGDF induced proliferation and IgM secretion in activated B cells with low density, but not in small resting B cells. WEHI-231-BGDF showed synergistic effect with dextran sulfate but not with anti-Ig in the induction of proliferation or IgM secretion in small resting B cells. WEHI-231-BGDF did not show any effect on Xid B cells. The relationship with several T cell-derived BSF and the significance of B cell-derived BSF in the B cell responses are discussed.     

Anti-idiotypic mechanisms involved in suppression of a mouse B cell lymphoma, BCL1

Immunization of BALB/c mice with idiotypic IgM rescued by hybridization from the syngeneic BCL1 lymphoma protects specifically against challenge with tumor cells, with 83% surviving greater than 100 days compared with controls (38 +/- 10 days). Spleens from long-term survivors (greater than 6 mo) with no macroscopically visible tumor, when examined with anti-idiotypic antibody, showed a range of apparently dormant tumor with BCL1 cells present at 2 to 50% of total. A spectrum of protection against tumor resulted from immunization, and tumor emerging in the period 53 to 173 days postpassage was investigated for expression of idiotype. It was found that cells from individual mice expressed variable amounts of idiotypic IgM at the cell surface, although it was always detectable in the intracellular compartment. Unlike typical BCL1 cells, tumor cells developing in immune spleens often secreted little idiotypic IgM either in vitro or in vivo. This modulation of expression and secretion of idiotype was detected even in the apparent absence of serum anti-idiotypic antibody. On passage of spleen cells from the long-term survivors into naive animals, BCL1 tumor developed and killed the recipients in a way indistinguishable from routine tumor passage. These tumor cells, however, both expressed and secreted IgM of the same idiotype as the original tumor. It appears therefore that tumor development in immunized mice is suppressed by a process that includes modulation but not selection of the tumor cell idiotypic determinants. Analysis of possible mechanisms of suppression revealed the presence of cytotoxic anti-idiotypic antibody at variable levels in sera of immunized mice, and splenic T cells that proliferated specifically in response to idiotypic IgM. Only low levels of cytotoxic T cells were found. Passive transfer studies demonstrated a major role for antibody in protection against tumor, with no significant enhancement by immune lymphocytes.     

Influence of staphylococcal enterotoxin B (SEB) on the course of murine listeriosis

Abstract Confrontation of the immune system with bacterial superantigens leads to an initial activation of the immune system followed by a state of profound immunosuppression. To investigate the role of a superantigen in an acute infection with a facultatively intracellular bacterium, we have studied the effect of staphylococcal enterotoxin B on the course of murine listeriosis. Intraperitoneal injection of SEB led to a statistically significant growth restriction of Listeria monocytogenes in the organs of mice infected intravenously or intraperitoneally when treatment with SEB and infection with L. monocytogenes were given simultaneously or when the mice were treated two days before infection. No effect of SEB on murine listeriosis was found when SEB was given more than two days before infection or one or more days after infection. We conclude that initial immunostimulation by SEB which is indicated by a massive liberation of all interleukins measured (IL1α, IL6, TNFα, IL2, IFNγ, IL4) is responsible for the growth restriction of L. monocytogenes in the organs of treated mice. Apoptosis of Vβ8 positive T cells which was accompanied by a 30% reduction of these cells at day 7 after treatment seems to be totally compensated.     

Purification and partial sequence analysis of murine B cell growth factor II (interleukin 5)

Murine B cell growth factor II (BCGF-II/interleukin 5) was purified from the conditioned media of the helper T cell line D10 . G4 . 1. The purification scheme consisted of sequential batch adsorption onto trimethylsilyl-controlled pore glass beads, high pressure ion exchange chromatography, and reverse phase high pressure liquid chromatography. The purified BCGF-II had a relative molecular weight of 45,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Identical analysis of BCGF-II under reducing conditions yielded a m.w. of 22,500, suggesting that native BCGF-II exists as a homodimer. The NH2-terminal amino acid sequence of the purified lymphokine was determined by automated Edman degradation. A single amino acid sequence of 24 residues was obtained that, upon comparison, was contained within the cDNA pSP6K-mTRF23 recently described as encoding murine BCGF-II/T cell-replacing factor. The NH2-terminal methionine in mature BCGF-II is found at position 21 of the amino acid sequence predicted from the cDNA pSP6K-mTRF23. This finding supports the contention of Kinashi et al. (Kinashi, T., N. Harada, E. Severinson, T. Tanabe, P. Sideras, M. Konishi, C. Azuma, A. Tominaga, S. Bergstedt-Lindqvist, M. Takahashi, F. Matsuda, Y. Yaoita, K. Takatsu, and T. Honjo. 1986. Nature 324:70) that amino acids 1-20 serve as the signal sequence for the BCGF-II gene. The ability of BCGF-II to stimulate the proliferation of the B cell lymphoma BCL1 was used to assess the potency of the lymphokine. BCGF-II at 13.5 pM induced 50% of the maximal proliferative response in the BCL1 cells; concentrations as low as 2 pM were still effective in stimulating the growth of the cells. Assuming that the amount of BCGF-II necessary to mount a 50% response in the BCL1 assay is defined as one unit of activity, then the purified BCGF-II has a specific activity of 16.5 U/ng of protein.