Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule

Bone marrow stromal cell lines have been isolated that directly support B lymphopoiesis in vitro. Single B-lineage precursors proliferate and differentiate on certain of these stromal cell lines to establish long-term B-lineage cultures. These lymphopoietic stromal cells produce novel soluble factors that support proliferation of in vitro established pre-B cell populations. Lymphoid populations established on lymphopoietic stromal cell lines lack surface Ig-bearing cells, but give rise to surface Ig+ cells when transferred to mixed bone marrow feeder layers. Several stromal lines expressed a B-lineage neoplasia marker detected by the monoclonal antibody MAb6C3. Remarkably, only the 6C3Aghi stromal lines supported long-term proliferation of B-lineage cells. We propose that the 6C3 antigen-bearing molecule may play a role in stromal cell-dependent, pre-B cell proliferation, as well as in neoplastic proliferation of pre-B leukemias.     

Bone marrow stromal cell regulation of B-lymphopoiesis. I. The role of macrophages, IL-1, and IL-4 in pre-B cell maturation

Our experiments have addressed regulation of B lymphocyte formation by bone marrow stromal cells. Stromal cells appear to produce a regulatory factor that acts at the pre-B cell stage to induce the expression of Ig L chains and surface Ig. Bone marrow stromal cell conditioned medium was found to contain this factor and the active component was partially purified by HPLC. This stromal cell-derived factor had a m.w. between 16,000 and 20,000, was specifically neutralized by anti-IL-4 mAb, 11B11, and enhanced the proliferation of anti-mu-stimulated B cells. We also found that rIL-4 induced B cell formation in culture. In our studies, IL-1 had no direct effect on pre-B cell maturation, however, IL-1 was found to stimulate the production of IL-4 by both heterogeneous bone marrow stromal cells and a cloned stromal cell line, SCL-160. These effects of IL-1 on factor production by stromal cells were duplicated by the addition of bone marrow-derived macrophages to SCL-160 cells. We conclude that stromal cell-derived IL-4 is a physiologic stimulator for B cell generation. In addition, macrophages appear to play a role in B cell formation by regulating the production of IL-4 by stromal cells via the secretion of IL-1.     

Pre-B cell generation potentiated by soluble factors from a bone marrow stromal cell line

Two bone marrow stromal cell lines isolated from the adherent layer of a Dexter-type long term bone marrow culture differ markedly in their hemopoietic support capacity. S17 supports myelopoiesis and the differentiation of early B cell precursors into B lymphocytes while S10 supports myeloid cell differentiation and not B lymphopoiesis. The identification of a stromal cell line with B cell support capacity prompted an investigation of whether the effects of S17 were mediated via soluble factors. Results presented herein indicate that medium conditioned by S17 but not S10 contains an activity that can induce the expression of the 220,000 m.w. 14.8 antigen and cytoplasmic mu H chain of Ig in B lymphocyte progenitors that have not yet expressed these markers. Bone marrow cells were depleted of 14.8+, cytoplasmic mu+ pre-B cells on antibody-coated petri dishes. After 24-h liquid culture newly generated pre-B cells were enumerated as cells that expressed cytoplasmic mu H chain of Ig but not Ig L chains by immunofluorescence. Expression of Ly5(220) was monitored by 14.8 antibody binding. This pre-B cell differentiation activity was abrogated by digestion with pronase, aminopeptidase, or carboxypeptidase. Isoelectric focusing data revealed the activity to have isoelectric point of 5.9 to 6.2. S17-conditioned medium was fractionated using HPLC and each fraction tested for pre-B cell-generating activity. Fractions collected from a Superose 12 gel filtration column were found to have two peaks of activity associated with molecules of apparent m.w. of approximately 60,000 and 10,000. Virtually identical peaks of activity were observed when medium conditioned by heterogeneous stromal cell cultures was fractionated. Separation of S10-conditioned medium revealed no cryptic activity. S17-conditioned medium was further characterized by anion exchange chromatography and the majority of the pre-B cell generating activity shown to be associated with the void volume that eluted from a MonoQ column. These fractions were rechromatographed on Superose and the activity again found to be associated with two fractions corresponding to apparent m.w. of 60,000 and 10,000. The S17 pre-B cell differentiation activity appears to result from the presence of a novel molecule because other well characterized mediators had no activity in this short-term liquid culture system. No pre-B cell-generating activity was observed when IL-1 or conditioned medium containing IL-2, IL-3, or IL-4 (B cell stimulatory factor 1) were added to cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Association of Ig L chain-like protein lambda 5 with a 16-kilodalton protein in mouse pre-B cell lines is not dependent on the presence of Ig H chain protein

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.     

BTNL2, a butyrophilin-like molecule that functions to inhibit T cell activation

B7 family members regulate T cell activation and tolerance. Although butyrophilin proteins share sequence homology with the B7 molecules, it is unclear whether they have any function in immune responses. In the present study, we characterize an MHC class II gene-linked butyrophilin family member, butyrophilin-like 2 (BTNL2), the mutation of which has been recently associated with the inflammatory autoimmune diseases sarcoidosis and myositis. Mouse BTNL2 is a type I transmembrane protein with two pairs of Ig-like domains separated by a heptad peptide sequence. BTNL2 mRNA is highly expressed in lymphoid tissues as well as in intestine. To characterize the function of BTNL2, we produced a BTNL2-Ig fusion protein. It recognized a putative receptor whose expression on B and T cells was significantly enhanced after activation. BTNL2-Ig inhibited T cell proliferation and TCR activation of NFAT, NF-kappaB, and AP-1 signaling pathways. BTNL2 is thus the first member of the butyrophilin family that regulates T cell activation, which has implications in immune diseases and immunotherapy.     

Expression of CD28 by bone marrow stromal cells and its involvement in B lymphopoiesis

Young mice lacking CD28 have normal numbers of peripheral B cells; however, abnormalities exist in the humoral immune response that may result from an intrinsic defect in the B cells. The goal of this study was to assess whether CD28 could be involved in the development of B cells. CD28 mRNA was detected preferentially in the fraction of bone marrow enriched for stromal cells. Flow cytometry and RT-PCR analysis demonstrated that CD28 was also expressed by primary-cultured stromal cells that supported B lymphopoiesis. Confocal microscopy revealed that in the presence of B-lineage cells, CD28 was localized at the contact interface between B cell precursors and stromal cells. In addition, CD80 was detected on 2-6% of freshly isolated pro- and pre-B cells, and IL-7 stimulation led to induction of CD86 on 15-20% of pro- and pre-B cells. We also observed that stromal cell-dependent production of B-lineage cells in vitro was greater on stromal cells that lacked CD28. Finally, the frequencies of B-lineage precursors in the marrow from young (4- to 8-wk-old) CD28(-/-) mice were similar to those in wild-type mice; however, older CD28(-/-) mice (15-19 mo old) exhibited a 30% decrease in pro-B cells and a 50% decrease in pre-B cells vs age-matched controls. Our results suggest that CD28 on bone marrow stromal cells participates in stromal-dependent regulation of B-lineage cells in the bone marrow. The localization of CD28 at the stromal cell:B cell precursor interface suggests that molecules important for T cell:B cell interactions in the periphery may also participate in stromal cell:B cell precursor interactions in the bone marrow.     

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.     

A transmembrane trap method for efficient cloning of genes encoding proteins possessing transmembrane domain.

To facilitate searching for genes encoding cell membrane proteins, we developed a method for isolating cDNAs that contain sequences for hydrophobic transmembrane runs. This cloning strategy, termed the "transmembrane (TM) trap method," utilizes a vector that directs the cell surface expression of mouse CD4 fusion protein when an insert encoding hydrophobic transmembrane sequences is cloned in-frame with correct orientation. We applied this novel method to isolation of cytokine receptor cDNAs. Our strategy enabled efficient isolation of relatively rare species encoding receptors such as IL-2Rgamma, IL-3Rbeta, IL-4Ralpha, IL-5Ralpha, and IL-6Ralpha. This method also could be used to isolate cDNAs for intracellular molecules with a transmembrane region, e.g., bcl-2. These results indicate that the TM trap method provides an efficient cloning strategy for identification of various families of genes encoding proteins with one or more transmembrane regions.     

Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.     

Murine pro-B cells require IL-7 and its receptor complex to up-regulate IL-7R alpha, terminal deoxynucleotidyltransferase, and c mu expression

Phenotypic analysis of bone marrow cells from IL-7 knockout (KO) mice revealed that B cell development is blocked precisely at the transition between pro-B cells and pre-B cells. In contrast, the generation of pre-pro-B cells and pro-B cells appeared to be normal, as judged by total cell numbers, proliferative indexes, D-JH and V-DJH gene rearrangements, and mRNA for recombinase-activating gene-1 (RAG-1), RAG-2, TdT, Ig mu, lambda 5, and VpreB. However, upon closer inspection, several abnormalities in pro-B cell development were identified that could be corrected by injection of rIL-7 in vivo. These included the absence of the subset of late pro-B cells that initiates cmu expression for pre-B cell Ag receptor (BCR) formation, and the failure of pro-B cells to up-regulate TdT and the IL-7R alpha (but not the common gamma-chain) chain. Similar defects were present in common gamma-chain and Jak3 KO mice, but not in lambda 5 or (excluding cytoplasmic Ig mu heavy chain (c mu)) RAG-1 KO mice, all of which also arrest at the late pro-B cell stage. Consequently, up-regulation of TdT and IL-7R alpha expression requires signaling through the high affinity IL-7R, but does not require cmu expression or a functional pre-BCR. Taken together, these results suggest that IL-7 and its receptor complex are essential for 1) up-regulating the expression of TdT and IL-7R alpha, 2) initiating the production of cmu and 3) promoting the formation of a functional pre-BCR in/on pro-B cells. These key events, in turn, appear to be prerequisite both for differentiation of pro-B cells to pre-B cells and for proliferation of these cell subsets upon continued stimulation with IL-7.     

Normal B cell precursors responsive to recombinant murine IL-7 and inhibition of IL-7 activity by transforming growth factor-beta

The ability of stromal cells in bone marrow to support B lymphopoiesis may be partially mediated by secretion of biologically active factors. The first cytokine with lymphopoietic activity to be molecularly cloned from stromal cells, IL-7, was originally identified by its growth-promoting activity on long term cultured lymphocytes. We now report that murine rIL-7 is a potent proliferative stimulus for B cell progenitors isolated from fresh bone marrow. Proliferation was initially most obvious among large precursor cells which bear the B lineage associated Ag, Ly5/220 and BP1. A majority of these also contained cytoplasmic Ig mu H chains. Extended culture with IL-7 resulted in a predominance of immature c mu- lymphocytes. No effect by IL-7 was observed on the proliferation of mature lymphocytes. It also did not induce maturation in a number of early B lineage cell lines, or promote the formation of LPS-responsive, clonable B cells from precursors. When incorporated into semisolid agar medium, IL-7 specifically and rapidly induced the formation of pre-B cell colonies in a linear fashion with respect to numbers of cells cultured from either purified B cell progenitor preparations or unfractionated bone marrow. In both liquid and agar culture conditions, the IL-7 proliferative activity was inhibitable by two related forms of transforming growth factor (TGF) beta, TGF-beta 1 and TGF-beta 2. Taken together, these results indicate that IL-7 is a stimulus for replication of normal B lineage cells at an early stage of differentiation, and its activity can be modulated by other cytokines. IL-7 also provides a means of studying the progeny of a single B cell progenitor, and of enumerating clonable pre-B cells in the absence of colony formation by other cell types in bone marrow.     

Cloning and characterization of CD300d, a novel member of the human CD300 family of immune receptors

Herein we present the cloning and molecular characterization of CD300d, a member of the human CD300 family of immune receptors. CD300d cDNA was cloned from RNA obtained from human peripheral blood mononuclear cells, and RT-PCR revealed the gene to be expressed in cells of myeloid lineage. The cloned cDNA encoded for a type I protein with a single extracellular Ig V-type domain and a predicted molecular mass of 21.5 kDa. The short cytoplasmic tail is lacking in any known signaling motif, but there is a negatively charged residue (glutamic acid) within the transmembrane domain. CD300d forms complexes with the CD300 family members, with the exception of CD300c. Contrary to other activating members of the CD300 family of receptors, surface expression of CD300d in COS-7-transfected cells required the presence of an immunoreceptor tyrosine-based activating motif-bearing adaptor (FcεRγ). Accordingly, we found that CD300d was able to recruit FcεRγ. Unexpectedly, we could not detect CD300d on the surface of cells expressing FcεRγ, suggesting the existence of unknown mechanisms regulating the trafficking of this molecule. The presence of other CD300 molecules also did not modify the intracellular expression of CD300d. In fact, the presence of CD300d decreased the levels of surface expression of CD300f but not CD300c. Our data suggest that the function of CD300d would be related to the regulation of the expression of other CD300 molecules and the composition of CD300 complexes on the cell surface.     

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.     

Activation of V kappa gene rearrangement in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains.

During B cell development V kappa gene rearrangement seems to occur only in mu-positive pre-B cells. To study the role of the mu chain in the activation of the Ig kappa locus, we introduced expression vectors carrying different forms of the mu gene into null pre-B cells. The activation of the Ig kappa locus followed the expression of the membrane form (micron) of the mu chain. The expression of the secreted form (microS) did not result in the activation of the Ig kappa locus. We further show that both forms of the mu chain differ in their intracellular transport in pre-B cells.