Expression of v-rel induces mature B-cell lines that reflect the diversity of avian immunoglobulin heavy- and light-chain rearrangements.

The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte.     

Transformation of avian lymphoid cells by reticuloendotheliosis virus

Avian reticuloendotheliosis virus (REV-T) is the most virulent of all retroviruses, inducing an invariably fatal leukemia in chickens with a latent period of 7-10 days. Unlike avian cells transformed by other acutely transforming viruses, lymphoid cells transformed by REV-T are immortalized. Furthermore, in vitro derived, REV-T transformed cells which do not produce virus are tumorigenic and induce lethal reticuloendotheliosis when injected into histocompatible birds. Thus REV-T transforms its target cell both in vitro and in vivo. In addition this transformation is independent of any helper virus functions. Like other acute leukemia viruses, REV-T is replication-defective and must co-replicate with a reticuloendotheliosis associated virus (REV-A). During evolution, a substantial portion of its genome has been deleted and replaced with a host-derived genetic sequence, designated v-rel. Presumably, the v-rel oncogene was transduced from a normal turkey DNA locus, c-rel. There are 9 regions of homology between c-rel and v-rel, however, several differences exist between these genes, suggesting that transformation by REV-T results from the production of an altered v-rel protein. The v-rel sequence is distinct from other known oncogenes and encodes a 57-kDa phosphoprotein. In REV-T transformed cells, this pp57v-rel protein is localized in the cytoplasm. The product of the v-rel oncogene is present at a low level, representing only about 0.003% of total methionine-labelled protein. In addition, pp57v-rel is relatively stable, having an estimated half-life of 4-10 h. The v-rel protein when purified close to homogeneity is complexed with a 40-kDa cellular phosphoprotein in transformed lymphoid cells and possesses serine kinase activity. This review discusses the molecular aspects of transformation by REV-T in the context of other oncogene-encoded proteins.     

Avian reticuloendotheliosis virus: identification of the hematopoietic target cell for transformation

Non-virus-producing hematopoietic cells transformed in vitro by reticuloendotheliosis virus (REV-T) induce lethal "reticuloendotheliosis" when inoculated into histocompatible chickens. This is the first direct demonstration that an in vivo target cell of an avian acute leukemia virus can be transformed in vitro. The tumorigenic, REV-T-transformed non-virus-producing cells fail to express helper-virus-coded proteins. REV-T transformed tumorigenic cells therefore do not require helper-virus functions. Cells transformed in vivo or in vitro by REV-T have lymphoblastoid morphology and express low levels of terminal-deoxynucleotidyl-transferase activity and bursal-cell determinants. One clone synthesized Ig mu. The preferred target cells for REV-T transformation are therefore immature lymphoid cells that express B-cell determinants. We propose that the unique transforming sequence of REV-T be designated rel (lymphoid).     

Lymphoid precursor cells adhere to two different sites on fibronectin

Several precursor lymphoid cell lines, blocked at specific stages of differentiation, adhere specifically to fibronectin in vitro. Whereas the Ba F3 cell line, which has both immunoglobulin heavy- and light-chain genes in germline configuration, interacts with the arg-gly-asp-containing cell-binding domain of fibronectin, the B-committed line PD 31, which is undergoing rearrangement of immunoglobulin light-chain genes, does not. Accordingly the Ba F3, but not the putative PD 31 surface fibronectin receptor, binds to an affinity matrix containing the 115-kD cell-binding domain of fibronectin. PD 31 cells recognize a different domain of the fibronectin molecule, which is contained within the carboxy terminal segment possessing a high-affinity binding site for heparin. A polyclonal antibody raised against the fibronectin receptor of mouse erythroleukemic cells inhibits adhesion of these lymphoid lines to fibronectin. It precipitates two major species of 140 and 70 kD from surface-radioiodinated Ba F3 cells and species of 140 and 120 kD from PD 31 cells. We propose that the two types of cells express different fibronectin receptors mediating substrate adhesion, and suggest that receptor(s) with different specificity might be expressed in the course of B cell maturation. Because we show that these adhesion properties are shared by normal bone marrow lymphoid precursors, we infer that these receptors may play a role in normal lymphopoiesis.     

Characterization of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning.

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus which causes a rapid neoplastic disease of the lymphoreticular system. Upon infection, this virus gives rise to two species of unintegrated linear viral DNA, which are 8.3 and 5.5 kilobase pairs long and represent the helper virus (REV-A) and the oncogenic component (REV-T), respectively. Restriction endonuclease cleavage maps of these two DNA components indicate that REV-T DNA has a large portion of the genome deleted with respect to REV-A DNA and a substitution about 0.8 to 1.5 kilobase pairs long that is unrelated to REV-A DNA. These additional sequences comprise the putative transforming region of REV-T (rel). A chicken spleen cell line transformed by REV-T produced virus which upon infection gives rise to three species of unintegrated linear viral DNA (8.3, 5.5, and 3,3 kilobase pairs). We isolated the proviruses of the 8.3- and 3.3-kilobase pair species from this cell line by cloning in the phage vector Charon 4A. Restriction enzyme mapping showed that the two proviral clones are proviruses of REV-A and a variant of REV-T, respectively. A subclone of the variant REV-T provirus specific for the rel sequences of REV-T was used as a hybridization probe to demonstrate that the rel sequences are different from the putative transforming sequences of Schmidt-Ruppin Rous sarcoma virus strain A, avain myelocytomatosis virus, avian myeloblastosis virus, avian erythroblastosis virus, Abelson murine leukemia virus, and Friend erythroleukemia virus. In addition, the rel-specific hybridization probe was used to identify a specific set of sequences which are present in uninfected avian DNAs digested with several restriction enzymes. The corresponding cell sequences are not arranged like rel in REV-T.     

Administration of IL-7 to mice with cyclophosphamide-induced lymphopenia accelerates lymphocyte repopulation

Lymphopenia was induced in mice by a single injection of cyclophosphamide. IL-7 or a control protein were administered to the mice twice daily and the cellularity and composition of the spleen, lymph node, bone marrow, and thymus were determined at various time points thereafter. In comparison to the control cyclophosphamide-treated mice, animals receiving cyclophosphamide and IL-7 had an accelerated regeneration of splenic and lymph node cellularity. There was no significant difference in the rate of recovery of the bone marrow and thymus of the control and IL-7-treated mice. Assessment of the pre-B cell compartment revealed a dramatic increase in total pre-B cell numbers in the spleen and bone marrow of the IL-7-treated mice as measured by both flow microfluorimetry and a pre-B cell colony-forming assay. This was followed in a few days by a significant increase in surface IgM+B cell numbers to levels above normal values in both the spleen and lymph node. IL-7 administration to cyclophosphamide-treated mice also resulted in an accelerated recovery of peripheral CD4+ and CD8+ cell numbers in the spleen and lymph node. The numbers of CD8+ cells were increased by twofold over normal levels in cyclophosphamide-treated mice receiving IL-7. Myeloid recovery was determined in cyclophosphamide treated mice by assessing the numbers of CFU-granulocyte-macrophage and Mac 1+ cells. There was no significant difference in myeloid recovery between cyclophosphamide-treated mice receiving IL-7 or control protein. These results suggest that administration of IL-7 after chemical-induced lymphopenia may have therapeutic benefits in shortening the period required to achieve normal lymphoid cellularity.     

Serine phosphorylation of the v-rel oncogene product/pp40 complex

The transforming protein encoded by the v-rel oncogene of reticuloendotheliosis virus has been purified from REV-T transformed lymphoid cells by sequential DEAE-Sepharose and immunoaffinity chromatography. The purified preparation consisted of pp59v-rel and the 40 kDa cellular protein which is complexed with the v-rel oncogene product in transformed cells as well as some minor proteins. Incubation of this purified preparation in the presence of Mg2+ and (gamma-32P)ATP resulted in phosphorylation of both pp59v-rel and the 40 kDa protein. This preparation was also able to phosphorylate casein on serine residues. Immunoprecipitates obtained from extracts of REV-T transformed lymphoid cells labeled with 32P-orthophosphate contained 59 and 40 kDa phosphoproteins. Both pp59v-rel and the 40 kDa protein were phosphorylated on serine residues in transformed cells.     

Substitution of 5' helper virus sequences into non-rel portion of reticuloendotheliosis virus strain T suppresses transformation of chicken spleen cells

The genome of the highly oncogenic avian retrovirus reticuloendotheliosis virus strain T (REV-T) differs from that of the helper virus reticuloendotheliosis virus strain A by a substitution (rel and a large deletion. Further deletions, constructed in vitro, within the helper-virus-related sequences of REV-T have little effect on the ability of the virus to transform chicken spleen cells in vitro. However, deletions that extend into rel abolish transformation. Substitution of helper-virus-related sequences for the deleted region in the non-rel portion of REV-T also abolishes transformation. Viruses with revertant phenotype were obtained both spontaneously and by construction in vitro from these substituted recombinants. The revertant viruses have various mutations, including deletions and insertions, in the helper-virus-related sequences. Thus the additional helper-virus-related sequences suppress expression of transformation in cis, and the deletion in REV-T seems necessary for expression of the transforming properties of the virus.     

Different localization of the product of the v-rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus that transforms early lymphoid cells in vivo and in vitro, but REV-T does not transform chicken embryo fibroblasts (CEF). Using antisera to p59v-rel, the v-rel oncogene product of REV-T, we show that p59v-rel is expressed at equal levels and is a phosphoprotein in REV-T infected spleen cells and CEF. Biochemical fractionation and immunofluorescence of REV-T infected nontransformed CEF show that p59v-rel is loosely associated with the nucleus. However, in REV-T transformed spleen cells p59v-rel is primarily a cytoplasmic protein. MSB-1 cells, a Marek's disease virus transformed T cell leukemic line, and E26 virus transformed myeloid cells show nuclear staining of p59v-rel when they are infected by REV-T. Our results indicate that there is a correlation between a cytoplasmic localization of p59v-rel and transformation by REV-T, and they suggest that p59v-rel cannot transform cells in which it assumes solely a nuclear location.     

Lipopolysaccharide-unresponsive mutant pre-B-cell lines blocked in NF-kappa B activation.

NF-kappa B activation is a crucial late step in the induction of immunoglobulin kappa light-chain gene expression in pre-B cells by lipopolysaccharide (LPS). We have analyzed NF-kappa B activation in three independent mutant lines of 70Z/3 pre-B cells which are unresponsive to LPS. All three variant cell lines failed to activate NF-kappa B when induced with LPS or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. However, all three cell lines contained functional NF-kappa B, as revealed by detergent treatment of cytoplasmic extracts. Moreover, cycloheximide induced limited activation of NF-kappa B comparable to that in wild-type 70Z/3 pre-B cells in two of the three variant lines. These results indicate that the mutations blocking kappa gene induction in these variant 70Z/3 pre-B-cell lines affect NF-kappa B activation.     

Expression of stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4, on CD34+ human bone marrow cells is a phenotypic alteration for committed lymphoid progenitors.

We found that the stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4 (CXCR4), is expressed on human CD34+ bone marrow (BM) cells. Stringently FACS-sorted CD34+CXCR4+ BM cells completely lack myeloid, erythroid, megakaryocytic, and mixed colony-forming potential (myeloid progenitors), but give rise to B and T lymphoid progenitors, whereas CD34+CXCR4- BM cells can generate colonies formed by myeloid progenitors and can also develop into these lymphoid progenitors. Therefore, expression of CXCR4 on CD34+ BM cells can allow lymphoid progenitors to be discriminated from myeloid progenitors. Because CD34+CXCR4+ cells are differentiated from CD34+CXCR4- cells, multipotential progenitors located in the BM are likely to be negative for CXCR4 expression. CXCR4 seems to be expressed earlier than the IL-7R and terminal deoxynucleotidyl transferase during early lymphohemopoiesis. These results suggest that the expression of CXCR4 on CD34+ BM cells is one of the phenotypic alterations for committed lymphoid progenitors.     

A Monoclonal Antibody Defining Human B Cell Differentiation Antigen (HLB-1 Antigen)

A new monoclonal antibody specific for human B cell differentiation antigen (HLB-1) is produced by a hybridoma established by fusion of splenocytes of mice immunized with the Epstein-Barr virus (EBV)-transformed peripheral B cell line, RPMI-8057. This monoclonal, antibody designated anti-HLB-1 monoclonal antibody (anti-HLB-1), reacted with surface immunoglobulin (sIg)-positive B cells of normal peripheral blood and lymphoid tissues and sIg-positive leukemic cells. The cells of T cell leukemia, non-T non-B acute lymphoblastic leukemia (ALL) and nonlymphoid leukemia were HLB-1 negative. These data were further confirmed by studying a panel of cultured human hematopoietic cell lines. Anti-HLB-1 reacted with B cell lines derived from pre-B, Burkitt's lymphoma, B cell type ALL and EBV-transformed peripheral B cells. Anti-HLB-1 was reactive with only one of three human myeloma cell lines, and with none of the T cell, myeloid and non-T non-B ALL cell lines. This newly defined HLB-1 antigen is different from other conventional human B cell markers such as sIg, Ia antigens, and receptors for the Fc portion of Ig and complement C3.     

T-cell receptor and immunoglobulin genes are rearranged together in Abelson virus-transformed pre-B and pre-T cells.

We have assessed the state of rearrangement and expression of B- and T-cell antigen receptor genes in cells of Abelson murine leukemia virus-transformed thymomas and other tumors. We found that unrearranged TcR gamma genes are expressed, as are unrearranged C mu genes, in pre-T, pre-B, and myeloid cells. We also found TcR gamma genes rearranged and expressed in putative pre-T cells and in cells apparently committed to the B-cell lineage. This is in contrast to the data from more mature T- and B-cell tumors. We conclude that in immature lymphoid cells both immunoglobulin and TcR gamma genes are accessible for rearrangement. We discuss the implications of these observations for an understanding of the B-T lymphoid differentiation event.     

Identification of an octamer-binding site in the human kappa light-chain enhancer.

Octamer motifs contribute to the function and tissue specificity of immunoglobulin heavy- and light-chain gene promoters and the heavy-chain enhancer. A variant octamer-binding site within a conserved region of the human kappa light-chain gene enhancer which contributes to the function of this enhancer has been identified.