A novel lymphocyte function-associated antigen (LFA-1): cellular distribution, quantitative expression, and structure

The derivation of a new human pre-B cell line from the leukocytes of a girl in the leukemic phase of lymphoblastic lymphoma is reported. Cells of this line, termed SMS-SB, synthesized mu but not light chains. These mu-chains were found only in the cytoplasm and were not secreted. SMS-SB cells bore HLA-DR determinants. They did not express the common ALL antigen or terminal deoxynucleotidyl transferase and, therefore, differed from previously described human pre-B leukemia cell lines. SMS-SB cells resembled certain Abelson leukemia virus-transformed mouse bone marrow cells. Except for the lack of formed mouse bone marrow cells. Except for the lack of mu secretion, the phenotype of SMS-SB cells was the same as the major subpopulation of marrow pre-B cells.     

Normal thymic cortical epithelial cells developmentally regulate the expression of a B-lineage transformation-associated antigen

Nonlymphoid, stromal cells in the mouse thymus are believed to be important in T cell maturation and have been proposed to play a central role in the acquisition of major histocompatibility complex (MHC) restriction and self-tolerance by maturing thymocytes. Both cortical and medullary epithelial cells in the thymus express high levels of class II (A) major histocompatibility antigens (MHC Ags). We show here that a specific subset of these A^– epithelial cells express a transformation-associated antigen (6C3Ag) found previously on the surfaces of Abelson murine leukemia virus-transformed pre-B cells and on those bone marrow-derived stromal cell clones which support normal and preneoplastic pre-B cell proliferation. Among solid lymphoid organs, only the thymus contains 6C3Ag¹ cells and within the thymus, this antigen is found exclusively on A^– epithelial cells in cortical regions. It is striking that the expression of the 6C3Ag on thymic epithelium is developmentally regulated, suggesting a role for this lymphostromal antigen in the maturation of the thymic microenvironment.     

Abnormal recombination of Igh D and J gene segments in transformed pre-B cells of scid mice

Studies of Ig and TCR genes in transformed lymphocytes of scid mice have revealed aberrant DNA rearrangements. Here we present a more detailed analysis of the Igh gene recombination in nine scid pre-B cell lines transformed by Abelson murine leukemia virus. We found 85% of the rearranged Igh alleles to contain abnormal Dh-Jh deletions of varying size. All of these deletions encompassed Jh elements and extended into the Igh enhancer region, occasionally involving the switch (S) region of the C mu gene. Some of these rearrangements removed most of the Dh elements, but none appeared to extend to the Vh genes. DNA sequence analysis of the two abnormally rearranged Igh alleles in one pre-B cell line showed that no Dh or Jh coding sequences were retained at the recombination sites though heptamer-like (CACTGTG) recognition signal sequences were present in the absence of nonamer (GGTTTTTGT) recognition signal sequences. These results imply that a deregulated recombinase activity may be responsible for the abnormal Dh-Jh deletions and the absence of Vh-Dh joining in established lines of Abelson murine leukemia virus-transformed scid pre-B cells.     

Abelson virus-infected cells can exhibit restricted in vitro growth and low oncogenic potential.

We have designed a method for growing bone marrow cells infected with Abelson murine leukemia virus which permits examination of target cell growth early after infection. This culture system increases the efficiency of target cell growth by favoring rapid growth of a mixed population of adherent cells in the primary culture. The nonadherent Abelson virus-infected cell populations expressed pre-B-cell differentiation markers characteristic of Abelson virus-transformed cells (mu-heavy chains of immunoglobulin M and terminal deoxynucleotidyltransferase). Early after infection, these cell populations exhibited restricted in vitro and in vivo growth properties which differed from those of an established Abelson virus-transformed cell line, 2M3. These included a marked dependency upon the adherent cell layer for growth and viability, a lower efficiency of agar colony formation, and a lower capacity for tumor production in syngeneic animals. Growth of the early populations could be maintained in the absence of the adherent cell layer by using conditioned medium from long-term adherent cell cultures established in the absence of viral infection. After passage of the populations for several weeks, the in vitro growth properties gradually shifted toward that of the 2M3 cell line. Twelve-week-old populations grew independently of the adherent cell layer and showed an increased efficiency of agar colony formation. These data indicate that many lymphoid target cells exhibit an intermediate transformed phenotype when infected with Abelson virus. Growth of these cells in culture is mediated via a synergistic interaction between intracellular expression of the viral transforming gene and an exogenous growth-promoting activity which can be provided by cultures of adherent bone marrow cells.     

BP-1/6C3 expression defines a differentiation stage of transformed pre-B cells and is not related to malignant potential

BP-1 antibody recognizes a cell surface molecule related to the zinc-dependent metallopeptidases that is expressed during a narrow window early in B cell differentiation. Expression of the same molecule, as originally recognized by the mAb 6C3, is widely accepted to be associated with the complete malignant transformation of pre-B lymphoid cells. We have examined BP-1/6C3 expression in a panel of established Abelson virus-transformed cells that includes both cells analogous to pre-B cells and to less differentiated B lineage cells that have not yet completed Ig H chain gene rearrangement. This analysis reveals that many of the less differentiated transformants do not express BP-1/6C3 for an extended culture period. In contrast, virtually all transformants that are analogous to normal pre-B cells express the determinant early in their culture history. The BP-1/6C3 negative transformants are fully tumorigenic in syngeneic mice, demonstrating that BP-1/6C3 expression is not required for complete malignant transformation. Our data thus suggest that the pattern of BP-1/6C3 expression in Abelson virus-transformed cells mimics that observed in normal cells and is indicative of a differentiation event unrelated to the malignant potential of the cells.     

The role of recombinant stem cell factor in early B cell development. Synergistic interaction with IL-7.

The cDNA for stem cell factor was recently isolated from Buffalo rat liver cells (BRL-3A) and recombinant rat stem cell factor produced from Escherichia coli (rrSCF164). rrSCF164 synergizes with rhIL-7 to stimulate pre-B clonal growth in agar culture of mouse bone marrow cells, and in this study we have characterized the role of rrSCF164 in B cell development. The combination of rrSCF164 plus rhIL-7 stimulated increased colony numbers compared with the sum of colonies stimulated by rrSCF164 and rhIL-7 alone. Also, increased cell proliferation per colony was stimulated by the combination of rrSCF164 plus rhIL-7 compared with rhIL-7 or rrSCF164 alone. The colonies formed with rrSCF164 plus rhIL-7 and rhIL-7 alone contained exclusively pre-B cells, which expressed B220 Ag and cytoplasmic mu-chain, but were negative for surface Ig expression. Morphological examination of the cells in the colonies showed blast-like characteristics. rrSCF164 alone and in combination with rhIL-7 stimulated generation of B220+ cells in liquid culture of B220- cells, whereas rhIL-7 alone had no stimulatory effect on B220- cells. Both stem cell factor mRNA and bioactivity were detected in a mouse bone marrow-derived stromal cell line, termed OZ-11. We propose that stem cell factor is a stromal-derived factor that synergizes with IL-7 to stimulate the proliferation and differentiation of pro-B cells to pre-B cells, which become responsive to IL-7 alone.     

Class-switching from mu to gamma 3 or gamma 2b production at pre-B cell stage

An Abelson virus-transformed murine pre-B cell line class-switched from mu to gamma 3 or gamma 2b at the pre-B stage during culture. A class-switch was mediated by the deletion mechanism of the intervening CH genes on the active chromosome. This study showed for the first time that class-switching at the pre-B cell stage was not always confined to gamma 2b production.     

Differentiation of cloned populations of immature B cells after transformation with Abelson murine leukemia virus

The nature of the target cell for Abelson virus transformation and the effect of transformation on B cell differentiation were studied with six cloned lines of nontransformed immature B lymphocytes. Three clones were at the pre-B cell stage of maturation prior to A-MuLV infection; two were at the B cell stage, and one appeared to represent a stage prior to rearrangement of the mu heavy chain gene. All six cloned lines could be transformed by Abelson virus. Many of the transformants of the pre-B cell clones underwent kappa light chain gene rearrangement and expression following viral infection. Distinct light chain gene rearrangements were segregated by further subcloning these transformed lines. Abelson virus infection of one cloned cell line believed to represent a stage of maturation prior to the pre-B cell stage produced pre-B cell transformants with a variety of heavy chain gene rearrangements. Thus B lymphoid target cells for Abelson virus are not restricted to a single developmental stage, and some transformed subclones can undergo extensive immunoglobulin gene rearrangements shortly after viral infection.     

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.     

Interleukin-7 retroviruses transform pre-B cells by an autocrine mechanism not evident in Abelson murine.

In this study, we have constructed retroviral vectors expressing the interleukin-7 (IL-7) cDNA and have used infection with these retroviruses to express this cytokine endogenously in an IL-7-dependent pre-B-cell line. Infection with IL-7 retroviruses, but not with a control retrovirus, resulted in the conversion of the cells to IL-7 independence. The frequency at which this occurred, together with data on vector expression levels, indicated that secondary events were required for factor independence in this system. Southern analysis showed that the IL-7-dependent clones harbored unrearranged copies of the vector proviruses. The factor-independent cells produced variable quantities of IL-7 as measured by an IL-7-specific bioassay, and their proliferation could be substantially inhibited by a neutralizing antibody directed against IL-7, indicating that a classical autocrine-mechanism was responsible for their transformation. These IL-7-independent cells were tumorigenic, in contrast to the parental IL-7-dependent cells or those infected with a control vector. These results showed that IL-7 could participate in the malignant transformation of pre-B cells. However, neither of two Abelson murine leukemia virus (A-MuLV)-transformed pre-B-cell lines expressed detectable IL-7 mRNA, at a level of sensitivity corresponding to less than one molecule of mRNA per cell. Moreover, the proliferation of the A-MuLV transformants was unaffected by addition of the IL-7 antisera under conditions in which parallel experiments with IL-7 virus-infected cells resulted in greater than 70% growth inhibition. Thus, transformation of pre-B cells by A-MuLV was not associated with a demonstrable autocrine loop of IL-7 synthesis. These results show that IL-7 can participate in the malignant transformation of pre-B cells and suggest studies aimed at assessing the role of autocrine production of IL-7 in the generation of human leukemias and lymphomas.     

BP-3 alloantigen. A cell surface glycoprotein that marks early B lineage cells and mature myeloid lineage cells in mice

To explore the cell surface molecules expressed on pre-B cells we have produced a panel of alloantibodies against transformed pre-B cells from BALB/c mice by immunizing a wild mouse, Mus spretus. One of these antibodies, BP-3, recognized glycoproteins of Mr 38,000 to 48,000 on pre-B cells transformed either by the Abelson murine leukemia virus or an erb B oncogene construct. Removal of N-linked oligosaccharides from the BP-3 Ag revealed a single core protein of Mr 32,000. The Ag was expressed by bone marrow cells in all but one (A/J) of the inbred mouse strains tested and in wild mice of biochemical groups Mus-1 and Mus-2. Analysis of the tissue distribution revealed expression of the BP-3 reactive molecule on normal pre-B and B cells in the bone marrow, 35% of B cells in the circulation, 30% of the B cells in the spleen, and less than or equal to 20% of B cells in lymph nodes, peritoneal cavity, and Peyer's patches. The subpopulation of BP-3+ B cells in bone marrow and peripheral tissues displayed an immature phenotype (IgM IgD +/- ). Examination of a panel of transformed B lineage cells confirmed the early stage-specific expression of the BP-3 alloantigen. In addition, a myeloid cell line and normal myeloid cells were found to express the BP-3 alloantigen. In contrast to B lineage cells, the level of BP-3 expression increased as a function of myeloid cell differentiation. Myeloid cells in the bone marrow expressed relatively little Ag, whereas circulating neutrophils and peritoneal macrophages expressed relatively high levels of the BP-3 alloantigen with Mr 38,000, 41,000, and 46,000. The data suggest that this variably glycosylated cell surface protein could play different roles in the differentiation of B lineage and myeloid lineage cells. The BP-3 alloantigen appears to be a useful marker for virgin B cells that have recently migrated from the bone marrow to the periphery.     

Modulated Expression of the Epidermal Growth Factor-Like Homeotic Protein dlk Influences Stromal-Cell–Pre-B-Cell Interactions, Stromal Cell Adipogenesis, and Pre-B-Cell Interleukin-7 Requirements

A close relationship exists between adipocyte differentiation of stromal cells and their capacity to support hematopoiesis. The molecular basis for this is unknown. We have studied whether dlk, an epidermal growth factor-like molecule that intervenes in adipogenesis and fetal liver hematopoiesis, affects both stromal cell adipogenesis and B-cell lymphopoiesis in an established pre-B-cell culture system. Pre-B-cell cultures require both soluble interleukin-7 (IL-7) and interactions with stromal cells to promote cell growth and prevent B-cell maturation or apoptosis. We found that BALB/c 3T3 fibroblasts express dlk and function as stromal cells. Transfection of these cells with antisense dlk decreased dlk expression and increased insulin-induced adipocytic differentiation. When antisense transfectants were used as stroma, IL-7 was no longer required to support the growth of pre-B cells and prevent maturation or apoptosis. Antisense dlk transfectants of S10 stromal cells also promoted pre-B-cell growth in the absence of IL-7. These results show that modulation of dlk on stromal cells can influence their adipogenesis and the IL-7 requirements of the pre-B cells growing in contact with them. These results indicate that dlk influences differentiation signals directed both to the stromal cells and to the lymphocyte precursors, suggesting that dlk may play an important role in the bone marrow hematopoietic environment.     

Loss of heterozygosity at the Ink4a/Arf locus facilitates Abelson virus transformation of pre-B cells

In many tumor systems, analysis of cells for loss of heterozygosity (LOH) has helped to clarify the role of tumor suppressor genes in oncogenesis. Two important tumor suppressor genes, p53 and the Ink4a/Arf locus, play central roles in the multistep process of Abelson murine leukemia virus (Ab-MLV) transformation. p53 and the p53 regulatory protein, p19Arf, are required for the apoptotic crisis that characterizes the progression of primary transformed pre-B cells to fully malignant cell lines. To search for other tumor suppressor genes which may be involved in the Ab-MLV transformation process, we used endogenous proviral markers and simple-sequence length polymorphism analysis to screen Abelson virus-transformed pre-B cells for evidence of LOH. Our survey reinforces the role of the p53-p19 regulatory pathway in transformation; 6 of 58 cell lines tested had lost sequences on mouse chromosome 4, including the Ink4a/Arf locus. Consistent with this pattern, a high frequency of primary pre-B-cell transformants derived from Ink4a/Arf +/- mice became established cell lines. In addition, half of them retained the single copy of the locus when the transformation process was complete. These data demonstrate that a single copy of the Ink4a/Arf locus is not sufficient to fully mediate the effects of these genes on transformation.     

IL-3 and stromal cell-derived factor synergistically stimulate the growth of pre-B cell lines cloned from long-term lymphoid bone marrow cultures

The addition of IL-3 to modified Whitlock-Witte long-term lymphocyte cultures was found to enhance the growth of a small but significant number of B cell precursors supported by an adherent stromal cell monolayer. Several pre-B cell lines were cloned from IL-3-treated long-term lymphocyte cultures. The growth requirements and physical properties of one representative clone, BL/3, are described. BL/3 cells were shown to be unresponsive to IL-3 except when it is used at very high concentrations. In contrast, significant growth was stimulated by stromal cell conditioned medium previously shown to contain a pre-B cell growth factor. Optimal growth of the pre-B cell clone was stimulated by stromal cell conditioned medium plus IL-3. Synergy between the stromal cell-derived factor and IL-3 occurred when IL-3 was used over a wide range of concentrations including a relatively low amount that was ineffective as a growth stimulus by itself. The finding that more than one factor is required to sustain optimal growth of some pre-B cells parallels the complex growth requirements reported for some primitive myeloid/erythroid progenitors.     

Secondary genomic rearrangement events in pre-B cells: VHDJH replacement by a LINE-1 sequence and directed class switching.

We describe rearrangement events which alter expression from a productive VHDJH rearrangement in an Abelson murine leukemia virus-transformed pre-B cell line. One such rearrangement results in replacement of the initially expressed variable region gene by a site-specific join between the open reading frame of a LINE-1 repetitive element and a remaining JH segment. We discuss this event in the context of the 'accessibility' model of recombinase control, and with respect to similar rearrangements involved in oncogene activation. In another subclone of the same pre-B cell line, altered heavy chain expression resulted from a mu to gamma 2b class switch recombination which occurred by a recombination-deletion mechanism but involved a complex inversion. We provide evidence that the germline gamma 2b region is specifically expressed in pre-B cell lines and early in normal development. We propose that the predisposition of pre-B cell lines to switch to gamma 2b production may reflect a normal physiological phenomenon in which the switch event is directed by an increased 'accessibility' of the germline gamma 2b locus to switch-recombination enzymatic machinery. Our findings support the hypothesis that the apparently distinct recombination systems involved in variable region gene assembly and heavy chain class switching are both directed by the accessibility of their substrate gene segments.