Stromal cells from murine embryonic aorta-gonad-mesonephros region, liver and gut mesentery expand human umbilical cord blood-derived CAFC(week6) in extended long-term cultures.

The first definitive long-term repopulating hematopoietic stem cells (HSCs) emerge from and undergo rapid expansion in the embryonic aorta-gonad-mesonephros (AGM) region. To investigate the presumptive unique characteristics of the embryonic hematopoietic microenvironment and its surrounding tissues, we have generated stromal clones from subdissected day 10 and day 11 AGMs, embryonic livers (ELs) and gut mesentery. We here examine the ability of 19 of these clones to sustain extended long-term cultures (LTCs) of human CD34(+) umbilical cord blood (UCB) cells in vitro. The presence of in vitro repopulating cells was assessed by sustained production of progenitor cells (extended LTC-CFC) and cobblestone area-forming cells (CAFC). The embryonic stromal clones differed greatly in their support for human HSCs. Out of eight clones tested in the absence of exogenous cytokines, only one (EL-derived) clone was able to provide maintenance of HSCs. Addition of either Tpo or Flt3-L + Tpo improved the long-term support of about 50% of the tested clones. Cultures on four out of 19 clones, ie the EL-derived clone mentioned, two urogenital-ridge (UG)-derived clones and one gastrointestinal (GI)-derived clone, allowed a continuous expansion of primitive CAFC and CFU-GM with over several hundred-fold more CAFC(week6) produced in the 12th week of culture. This expansion was considerably higher than that found with the FBMD-1 cell line, which is appreciated by many investigators for its support of human HSCs, under comparable conditions. This stromal cell panel derived from the embryonic regions may be a powerful tool in dissecting the factors mediating stromal support for maintenance and expansion of HSCs.

Inhibition of Tat-mediated transactivation and HIV replication with Tat mutant and repressor domain fusion proteins.

Strategies to inhibit the spread of HIV infection consist of a number of specific molecular approaches. Since viral production is dependent upon Tat-mediated transactivation of the HIV promoter through the Tat activating region (TAR), tat antisense RNA, anti-tat ribozymes, TAR decoys and dominant negative Tat mutant proteins have been suggested as therapeutic inhibitors. We produced and tested several Tat mutant proteins, including a newly generated form Tat delta 58, for the ability to inhibit Tat-mediated transactivation and HIV production. In addition, we generated a new Tat fusion mutant between a C-terminus truncated form of Tat (Tat delta 53) and the Drosophila Engrailed (Eng) transcription repressor domain to test the hypothesis that transcriptional repression can be targeted to the HIV promoter. This fusion mutant was also examined for its capacity to block both Tat-mediated transactivation and HIV replication. We show that three mutants Tat delta 53. Tat delta 58 and Tat delta 53/Eng result in a transdominant phenotype inhibiting wild-type Tat-mediated transactivation, and that the inhibiting potential is increased by the presence of the entire basic domain or the fusion of a repressor domain. However, only the transdominant mutants Tat delta 58 and Tat delta 53/Eng significantly inhibit HIV-1 replication after infection of transfected T cell lines. These results demonstrate the potent inhibiting activity of Tat mutants on HIV replication, and suggest a synergistic effect of Tat transdominant mutant fusion with the Drosophila Engrailed transcription repressor domain.

Development of the definitive hematopoietic hierarchy in the mouse.

Recent research on the ontogeny of the hematopoietic system in mammals has shown that a simple textbook steady-state hematopoietic hierarchy can not be strictly applied to the hematopoietic cells found within the embryo. During embryonic development, hematopoietic cells originate, migrate and differentiate in a number of distinct anatomical sites such as the yolk sac AGM region and liver and thus represent various classes of cells within diverse microenvironments. In this manuscript we review both cellular and molecular aspects of developmental hematopoiesis and present our current views on the numerous complex mechanisms underlying the establishment of definitive hematopoiesis.

HIV- I Nef severely impairs thymocyte development and peripheral T-cell function by a CD4-independent mechanism.

Nef is a regulatory protein of the human and simian immunodeficiency viruses (HIV and SIV) whose role in infection and the viral life cycle are not fully understood. In T-lymphocytes Nef down-regulates cell-surface CD4, and has been implicated in an increase in infectivity at low primary viral isolate titres. Additionally, the SIV nef gene is necessary for viraemia and AIDS-like pathogenesis in rhesus macaques. We report here in an in vivo murine transgenic model that thymocyte and T-cell-specific nef gene expression results in a marked decrease in thymic cellularity from 16 days post coitus. This reduction in thymocyte cell number is independent of CD4 expression and Nef-induced CD4 down-regulation, but can be restored by expressing a constitutively active p56lckF505 gene. Functional analyses have revealed a severe decrease in thymocyte and T-cell proliferation in response to both T-cell-receptor- and mitogen-mediated stimuli. In addition, a significant proportion of Nef-expressing peripheral T-cells display cell-surface characteristics associated with cellular activation. These results suggest that Nef expression in developing thymocytes can severely reduce the regeneration capacity of the immune system, whereas expression in mature T-cells dramatically decreases their potential to respond to antigen. With the recent recognition of a persistently high viral load in HIV-infected individuals, these findings have important implications for the mechanism of the progressive deterioration of the immune system that leads to AIDS.

Altered cytokine expression in T lymphocytes from human immunodeficiency virus Tat transgenic mice.

Examination of the interaction between human immunodeficiency virus (HIV) regulatory gene products and the host immune system is fundamental to understanding the pathogenesis of HIV and could reveal possible targets for therapeutic intervention in the treatment of AIDS. The HIV Tat gene is a potential candidate for this type of strategy. Transgenic mice can be used to investigate the in vivo effects of Tat on the developing and dynamic immune system and on cellular gene expression. Thus, we have generated transgenic mice that harbor the HIV type 1 Tat gene under the transcriptional control of the human CD2 gene regulatory elements. This expression cassette results in high-level, tissue-specific transcription of the transgene within the T-cell compartment. In this report, we demonstrate the effects of Tat on the in vivo immune system. CD2-Tat transgenic mice show no signs of aberrant thymic development and have normal levels of T-cell subsets in the thymus and peripheral lymphoid organs. However, activated T cells from transgenic mice contain increased levels of tumor necrosis factor beta mRNA as well as biologically active tumor necrosis factor protein and express elevated levels of transforming growth factor beta and interleukin-4 receptor mRNA. These increased cytokine levels do not appear to alter mitogen- or antigen-stimulated responses or induce the formation of dermal lesions in ageing mice. Such investigations should provide insight into the combination of host immune factors mediating pathogenesis in HIV infection.

Specific ablation of human immunodeficiency virus Tat-expressing cells by conditionally toxic retroviruses.

The identification of human immunodeficiency virus (HIV) as the etiologic agent of AIDS has led to the proposal of novel intervention strategies to block HIV infection and viral replication or eliminate HIV-infected cells. We have produced recombinant retroviruses for a molecular ablation system, whereby a toxin gene can be delivered to hematopoietic cells for the specific elimination of HIV Tat-expressing cells. For this cell-specific ablation, we have coupled the conditional toxin herpes simplex virus type 1 thymidine kinase (tk) gene to the HIV-2 promoter and Tat responsive region (TAR) in order that transcriptional activity be under the absolute control of HIV and simian immunodeficiency virus Tat trans-activator proteins. Since the HIV-2 promoter has a considerable level of basal expression in the absence of Tat, we constructed a number of modifications in the HIV-2 promoter to minimize the risk of cytotoxicity to cells not containing HIV Tat. We demonstrate that certain promoter modifications reduce basal transcription while maintaining high trans-activated levels of expression when transfected or transduced by retroviral vectors into several different cell lines. In mouse and human cells infected with HIV-2 tk retroviruses, we show that Tat-induced expression from the HIV-2 promoter results in differential ablation and a massive reduction in Tat-positive cells after ganciclovir treatment. Thus, the retroviruses produced in these studies may be applicable to HIV ablative therapy.

CD4 cell surface downregulation in HIV-1 Nef transgenic mice is a consequence of intracellular sequestration.

The Nef gene product is a regulatory protein of HIV whose biological function is poorly understood. Nef has been thought to have a negative effect on viral replication in vitro but has been shown in studies with SIV to be necessary in the establishment of viraemia in vivo. In vitro studies in various human cell lines have shown that Nef downregulates the expression of cell surface CD4 and thus could have effects on the immune response. We have generated four transgenic mouse lines, with constructs containing two different Nef alleles under the control of CD2 regulatory elements to examine the interaction of Nef with the host immune system in vivo. In adult transgenic mice we have found marked downregulation in the level of CD4 on the surface of double positive thymocytes and a decrease in the number of CD4+ T cells in the thymus. Functional analyses have revealed a decrease in the total activation of transgenic thymocytes by anti-CD3 epsilon antibody. By specific intracellular staining of T cells in such mice we have found CD4 colocalizing with a Golgi-specific marker. These results strongly suggest a Nef mediated effect on developing CD4 thymocytes resulting from interference of Nef in the intracellular trafficking or post-translational modification of CD4.

Cloning of the complete Ly-6E.1 gene and identification of DNase I hypersensitive sites corresponding to expression in hematopoietic cells.

The Sca-1 antibody recognizes antigens encoded by members of the Ly-6 multigene family. These antigens are expressed on fetal and adult hematopoietic stem cells, progenitor cells, mature activated T cells, and some nonhematopoietic cells and are most likely encoded by the Ly-6E.1 and Ly-6A.2 genes. Characterization and isolation of regulatory elements of Ly-6E.1 and A.2 genes that govern tissue-specific and high levels of expression in the cells of the hematopoietic system (particularly stem cells) are of considerable interest. To characterize the control elements of this gene, we have cloned a 30-kb fragment encoding a fully functional Ly-6E.1 gene and 13 kb of 5' and 13 kb of 3' flanking sequence. Transfection studies in murine erythroleukemia (MEL) cells show that a 14-kb BamHI fragment from this clone is sufficient to confer Ly-6E.1 gene expression at levels equivalent to those of the endogenous gene. By mapping regions of chromatin sensitive to DNase I digestion, we have located hypersensitive sites in the 5' and 3' regions of the gene in FDCP-1 cells, MEL cells, and various T-cell lines. The appearance of two 5' hypersensitive sites in hematopoietic cells correlates with Ly-6E.1 expression after gamma-interferon induction. We show that the presence of hypersensitive sites in the 5' and 3' regions corresponds to Sca-1 expression, and we also discuss the localization of putative regulatory control elements.

Studies of HIV-2 promoter activity and cell specific ablation.

We have a developed a retroviral mediated molecular ablation method to specifically eliminate HIV Tat-expressing cells. This approach utilizes the Tat-inducible HIV-2 promoter and a conditional toxin gene. The Herpes Simplex Virus thymidine kinase gene product is toxic to mammalian cells only after treatment with Ganciclovir (GCV) or other nucleoside analogues. We demonstrate here that certain promoter modifications can decrease basal expression while retaining the ability to be transactivated. Furthermore, we show that a HIV-2 promoter thymidine kinase gene cassette transduced via retroviral vectors into tissue culture cells can specifically promote the ablation of HIV-Tat expressing cells in the presence GCV. We also show that there is a large differential in HIV-thymidine kinase gene transcription and lethal drug dose between Tat-expressing cells and Tat-negative cells.

ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients.

While hematopoietic stem cells from adult and fetal stages of murine development are capable of long term reconstitution of all mature blood lineages in vivo, embryonic hematopoietic stem cell repopulation in vivo has proved difficult. It is thought that there are many fewer hematopoietic stem cells in the embryo than in the fetal/adult stages of mouse development and that these cells possess a different developmental potential. One source of such cells are embryonic stem (ES) cells which can differentiate into most mature blood lineages in vitro. We have therefore used transplantation of differentiated ES cells to assess the hematopoietic potential of embryonic hematopoietic cells in vivo. We demonstrate here that precursors obtained from in vitro cultures of normal ES cells can contribute only to restricted and limited hematopoiesis in a mouse without leading to tumour formation. Repopulation occurs for greater than 6.5 months at levels ranging from 0.1% to 6% in B and T cell lineages in peripheral blood. In contrast to in vitro colony data demonstrating the myeloid lineage developmental potential of ES cells, no donor-derived myeloid repopulation was observed in CFU-S assays and no macrophage and mast cells were found in long term repopulated recipients. Thus, the hematopoietic potential of ES cells in vivo is limited to low levels of repopulation and is restricted to the lymphoid lineage.

An early pre-liver intraembryonic source of CFU-S in the developing mouse.

It is widely accepted that during murine embryogenesis, totipotent haematopoietic stem cells first originate in the yolk sac, then migrate to the fetal liver and finally colonize the bone marrow shortly before birth. This view is based on in vitro studies showing that yolk sac cells can differentiate into various haematopoietic lineages and in vivo studies showing that yolk sac contains spleen colony-forming units (CFU-S) beginning at day 8 of gestation. However, some investigators have failed to find statistically significant numbers of CFU-S arising from day 9 yolk sac and, although one group reported that yolk sac could repopulate the haematopoietic system of W mutant mice, others have failed to confirm yolk sac-derived repopulation of adults. In the avian and amphibian systems, the yolk sac gives rise only to early, transitory haematopoiesis whereas the definite adult haematopoietic stem cells in these vertebrates are derived from the mesodermal region containing the dorsal aorta. Because this analogous area of the mouse embryo has not been previously examined for haematopoietic activity, we directly compared the CFU-S activity of the aorta, gonad, mesonephros (AGM) region with the yolk sac and fetal liver during embryogenesis. Here we report that this intra-embryonic AGM region contains CFU-S activity at a higher frequency than that in embryonic yolk sac and that such activity appears in the AGM region before the fetal liver.

Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells.

Recombinant retroviral genomes encoding a chromosomal human beta-globin gene have been used to transduce murine haematopoietic stem cells in vitro. After permanent engraftment of lethally irradiated recipients with the transduced cells, the human beta-globin gene is expressed at significant levels only within the erythroid lineage. These results indicate that it is possible to obtain stable expression of exogenous chromosomal DNA sequences introduced into mature haematopoietic cells in vivo via stem cell infection, and that human disorders of haemoglobin production may be more feasible candidates for somatic cell gene therapy than previously suspected.

Molecular characterization of antibodies bearing Id-460. I. The structure of two highly homologous VH genes used to produce idiotype positive immunoglobulins.

The heavy chain immunoglobulin genes encoding a variety of antibodies specific for DNP or Pasteurella pneumotropica and bearing the dominant idiotype of MOPC 460, Id-460, were cloned and sequenced. The VH genes encoding the M460 and D35 (DNP binding) antibodies were found to be homologous but not identical to the VH gene encoding the LB8 (P. pneumotropica binding) monoclonal antibody. Two of at least eight genes in the VH460 cross-hybridizing gene family can encode Id-460 positive antibodies. The VH460 gene family overlaps with the gene family described by VH36-60 and more completely describes this germ-line VH gene family. We have previously demonstrated a genetic requirement for VK1 expression in order to observe the expression of Id-460 in anti-DNP antibody responses. Southern blot analysis of these monoclonal antibody-producing cells demonstrates, as with the cross-hybridizing VH, that two cross-hybridizing VK1 genes (VK1A and VK1C) can be used to encode Id-460-positive immunoglobulins. We demonstrate that immunoglobulin idiotype determinant expression can be the result of the expression of nonidentical but highly homologous genes in the VH460 cross-hybridizing VH gene family and also in the VK1 cross-hybridizing VL family.

Molecular characterization of antibodies bearing Id-460. II. Molecular basis for Id-460 expression.

Id-460+ immunoglobulins can be induced in vivo by immunization with dinitrophenyl (DNP) or P. pneumotropica and form two nonoverlapping groups of antibodies with respect to antigen binding specificity. In this study, using Id-460+ antibodies of differing antigen binding specificities, we compared on the molecular genetic level the five gene segment combinations (VH, DH, JH, VL, and JL) that encode the variable regions of these idiotype-positive immunoglobulins. The Id-460 determinant appears to be a conformational or combinatorial determinant encoded by VH460 and VK1 crosshybridizing genes. DH, JH, and JK gene segments appear to have no measurable effect upon expression of Id-460. Finally, antigen binding specificity does not appear to simply localize to any particular gene segment but may in part be the result of somatic mutation and/or VDJH junctional sequences, whose length correlates roughly with antigen binding specificity.

Non-dinitrophenyl-binding immunoglobulin that bears a dominant idiotype (Id460) associated with antidinitrophenyl antibody is specific for an antigen on Pasteurella pneumotropica.

We have previously described an idiotype (Id460) that transiently dominates anti-2,4-dinitrophenyl (DNP) antibody responses of mice that possess the appropriate Igh-V and V kappa genotypes. Normal serum has significant levels of Id460 that does not bind DNP, and hybridomas derived from spleen cell fusions that produce monoclonal antibodies with these characteristics have been generated. Many of these monoclonal, Id460-positive antibodies bind the opportunistic mouse pathogen Pasteurella pneumotropica. P. pneumotropica induces a marked increase in serum Id460 titers without significantly increasing serum anti-DNP titers. Both normal serum and P. pneumotropica-induced Ig460-positive immunoglobulin specifically bind to P. pneumotropica. These results suggest that the normal serum Id460-positive immunoglobulin is induced by environmentally encountered antigens on P. pneumotropica. We propose that this naturally occurring Id460 activates antiidiotypic regulatory cells that in turn promote production of Id460-positive anti-DNP antibody following DNP-ovalbumin immunization. These data are compatible with those obtained in several other idiotypic systems that suggest that dominant idiotypes may be associated with antibodies that have been evolutionarily selected for expression because of their specificity for antigens on environmentally encountered pathogens.

Expression of an idiotype (Id-460) during in vivo anti-dinitrophenyl antibody responses. III. Detection of Id-460 in normal serum that does not bind dinitrophenyl.

Using an anti-idiotypic antibody previously characterized as specific for the hapten binding site of the 2,4-dinitrophenyl (DNP)-binding BALB/c myeloma protein MOPC-460, we have detected substantial amounts of this idiotype (Id-460) in the serum of normal mice. Whereas the idiotypic material in DNP-immune serum binds to DNP, the Id-460-positive material in normal mouse serum is not specific for DNP. The material in normal serum appears to be immunoglobulin. Furthermore, Id-460-positive, non-DNP-binding monoclonal immunoglobulins that completely inhibit our assay for Id-460 are repeatedly isolated when hybridomas are prepared from LPS-activated normal spleen cells. These data are interpreted in the context of Jerne's network hypothesis. It is our conclusion that the non-DNP-binding form of Id-460 is the inherited form and that this form establishes an idiotypic network favoring the production of anti-DNP bearing Id-460. Thus, the paradox of finding an inherited idiotype in the antibody response to the nonpathogen DNP may be resolved by proposing that the true form of Id-460 is specific for an environmental pathogen and that Id-460 dominance in the anti-DNP response is simply a consequence of idiotype-specific regulatory events preconditioned by Id-460-bearing immunoglobulin specific for antigenic determinants unrelated to DNP.

Expression of an idiotype (Id-460) during in vivo anti-dinitrophenyl antibody responses. II. Transient idiotypic dominance.

After immunization of mice with 2,4-dinitrophenyl-ovalbumin (DNP-OVA), it was shown previously that strains having Igh-Va genes and able to express light chains of the Vk1 group produce high levels of anti-DNP antibody bearing an idiotype (Id-460) associated with the combining site of the BALB/c DNP-binding myeloma protein MOPC 460. Expression of Id-460 in serum is transient; Id-460 levels peak early in the response and are regulated independently of total anti-DNP antibody. In this paper, the transient dominance of Id-460 expression has been confirmed at the cellular level by inhibition of splenic anti-DNP plaque-forming cells (PFC) with rabbit anti-Id-460 antiserum. Id-460+ PFC can account for 52-91% of anti-DNP PFC early after secondary challenge with DNP-OVA. Furthermore, Id-460 is represented at these high levels in IgM, IgG, and IgG1, and IgG2a, the three isotypes tested in the PFC assay, as well as in IgE, as tested by passive cutaneous anaphylaxis. Thus, there is no preferential association of Id-460 with a given isotype. We conclude from these studies that Id-460 is a dominant idiotype in the anti-DNP antibody response of BALB/c mice to DNP-OVA. This dominance is expressed transiently and is independent of isotype. A further conclusion from these studies is that regulation of isotype expression is independent of the regulation of idiotype expression in this system. We would suggest that regulation of Id-460 expression involves Ig-dependent helper T cells specific for Id-460 that induce Id-460+ B cells and also activate suppressor T cells, both events occurring via idiotype-anti-idiotype interactions.

Expression of an idiotype (Id-460) during in vivo anti-dinitrophenyl antibody responses. I. Mapping of genes for Id-460 expression to the variable region of immunoglobulin heavy-chain locus and to the variable region of immunoglobulin kappa-light-chain locus.

The genetic contro of the expression of an idiotype (Id-460) associated with the 2,4-dinitrophenyl (DNP)-binding BALB/c myeloma protein MOPC 460 was studied using congenic strains of mice. It was shown that the expression of high levels of Id-460 during secondary in vivo anti-DNP-ovalbumin responses was determined by genes governing immunoglobulin heavy-chain variable and kappa-light chain variable regions (V kappa). Appropriate alleles at both loci were required for the expression of Id-460. Genes in the major histocompatability complex and the X-linked immune deficiency gene found in strain CBA/N did not greatly affect Id-460 expression. The V kappa gene controlling Id-460 expression can be differentiated from Lyt-3, and it is the first instance in which expression of an idiotype subdivides the V kappa genes associated with the Lyt-3a allele. Although it is likely that the V kappa gene(s) involved are structural, the involvememt of a regulatory gene linked to the structural gene can not be excluded.