Regulation of hematopoietic and leukemic stem cells by the immune system.

Hematopoietic stem cells (HSCs) are rare, multipotent cells that generate via progenitor and precursor cells of all blood lineages. Similar to normal hematopoiesis, leukemia is also hierarchically organized and a subpopulation of leukemic cells, the leukemic stem cells (LSCs), is responsible for disease initiation and maintenance and gives rise to more differentiated malignant cells. Although genetically abnormal, LSCs share many characteristics with normal HSCs, including quiescence, multipotency and self-renewal. Normal HSCs reside in a specialized microenvironment in the bone marrow (BM), the so-called HSC niche that crucially regulates HSC survival and function. Many cell types including osteoblastic, perivascular, endothelial and mesenchymal cells contribute to the HSC niche. In addition, the BM functions as primary and secondary lymphoid organ and hosts various mature immune cell types, including T and B cells, dendritic cells and macrophages that contribute to the HSC niche. Signals derived from the HSC niche are necessary to regulate demand-adapted responses of HSCs and progenitor cells after BM stress or during infection. LSCs occupy similar niches and depend on signals from the BM microenvironment. However, in addition to the cell types that constitute the HSC niche during homeostasis, in leukemia the BM is infiltrated by activated leukemia-specific immune cells. Leukemic cells express different antigens that are able to activate CD4(+) and CD8(+) T cells. It is well documented that activated T cells can contribute to the control of leukemic cells and it was hoped that these cells may be able to target and eliminate the therapy-resistant LSCs. However, the actual interaction of leukemia-specific T cells with LSCs remains ill-defined. Paradoxically, many immune mechanisms that evolved to activate emergency hematopoiesis during infection may actually contribute to the expansion and differentiation of LSCs, promoting leukemia progression. In this review, we summarize mechanisms by which the immune system regulates HSCs and LSCs.

Activating enhancer binding protein 2 epsilon (AP-2ε)-deficient mice exhibit increased matrix metalloproteinase 13 expression and progressive osteoarthritis development.

INTRODUCTION The transcription factor activating enhancer binding protein 2 epsilon (AP-2ε) was recently shown to be expressed during chondrogenesis as well as in articular chondrocytes of humans and mice. Furthermore, expression of AP-2ε was found to be upregulated in affected cartilage of patients with osteoarthritis (OA). Despite these findings, adult mice deficient for AP-2ε (Tfap2e(-/-)) do not exhibit an obviously abnormal cartilaginous phenotype. We therefore analyzed embryogenesis of Tfap2e(-/-) mice to elucidate potential transient abnormalities that provide information on the influence of AP-2ε on skeletal development. In a second part, we aimed to define potential influences of AP-2ε on articular cartilage function and gene expression, as well as on OA progression, in adult mice. METHODS Murine embryonic development was accessed via in situ hybridization, measurement of skeletal parameters and micromass differentiation of mesenchymal cells. To reveal discrepancies in articular cartilage of adult wild-type (WT) and Tfap2e(-/-) mice, light and electron microscopy, in vitro culture of cartilage explants, and quantification of gene expression via real-time PCR were performed. OA was induced via surgical destabilization of the medial meniscus in both genotypes, and disease progression was monitored on histological and molecular levels. RESULTS Only minor differences between WT and embryos deficient for AP-2ε were observed, suggesting that redundancy mechanisms effectively compensate for the loss of AP-2ε during skeletal development. Surprisingly, though, we found matrix metalloproteinase 13 (Mmp13), a major mediator of cartilage destruction, to be significantly upregulated in articular cartilage of adult Tfap2e(-/-) mice. This finding was further confirmed by increased Mmp13 activity and extracellular matrix degradation in Tfap2e(-/-) cartilage explants. OA progression was significantly enhanced in the Tfap2e(-/-) mice, which provided evidence for in vivo relevance. This finding is most likely attributable to the increased basal Mmp13 expression level in Tfap2e(-/-) articular chondrocytes that results in a significantly higher total Mmp13 expression rate during OA as compared with the WT. CONCLUSIONS We reveal a novel role of AP-2ε in the regulation of gene expression in articular chondrocytes, as well as in OA development, through modulation of Mmp13 expression and activity.

In vivo definition of genetic and cellular aspects of mammalian sexual differentiation

The differentiation of the reproductive organs is an essential developmental process required for the proper transmission of the genetic material. Müllerian inhibiting substance (MIS) is produced by testes and is necessary for the regression of the Müllerian ducts: the anlagen of the uterus, fallopian tubes and cervix. In vitro and standard transgenic mouse studies indicate that the nuclear hormone receptor Steroidogenic factor 1 (SF-1) and the transcription factor SOX9 play an essential role in the regulation of Mis. To test this hypothesis, mutations in the endogenous SF-1 and SOX9 binding sites in the mouse Mis promoter were introduced by gene targeting in embryonic stem (ES) cells. In disagreement with cell culture and transgenic mouse studies, male mice homozygous for the mutant SF-1 binding site correctly initiated Mis transcription in the fetal testes, although at significantly reduced levels. Surprisingly, sufficient Mis was produced for complete elimination of the Müllerian duct system. However, when the SF-1 binding site mutation was combined with an Mis -null allele, the further decrease in Mis levels led to a partial retention of uterine tissue, but only at a distance from the testes. In contrast, males homozygous for the mutant SOX9 binding site did not initiate Mis transcription, resulting in pseudohermaphrodites with a uterus and oviducts. These studies suggest an essential role for SOX9 in the initiation of Mis transcription, whereas SF-1 appears to act as a quantitative regulator of Mis transcript levels perhaps for influencing non-Müllerian duct tissues. ^ The Mis type II receptor, a member of the TGF- b superfamily, is also required for the proper regression of the Müllerian ducts. Mis type II receptor-deficient human males and their murine counterparts develop as pseudohermaphrodites. A lacZ reporter cassette was introduced into the mouse Mis type II receptor gene, by homologous recombination in ES cells. Expression studies, based on b -galactosidase activity, show marked expression of the MIS type II receptor in the postnatal Sertoli cells of the testis as well as in the prenatal and postnatal granulosa cells of the ovary. Expression is also seen in the mesenchymal cells surrounding the Müllerian duct and in the longitudinal muscle layer of the uterus. ^

PRKCa: Identification of a Novel Downstream Target of WT1

Wilms tumor is a childhood tumor of the kidney arising from the undifferentiated metanephric mesenchyme. Tumorigenesis is attributed to a number of genetic and epigenetic alterations. In 20% of Wilms tumors, Wilms tumor gene 1 (WT1) undergoes inactivating homozygous mutations causing loss of function of the zinc finger transcription factor it encodes. It is hypothesized that mutations in WT1 result in dysregulation of downstream target genes, leading to aberrant kidney development and/or Wilms tumor. These downstream target genes are largely unknown, and identification is important for further understanding Wilms tumor development. Heatmap data of human Wilms tumor protein expression, generated by reverse phase protein assay analysis (RPPA), show significant correlation between WT1 mutation status and low PRKCα expression (p= 0.00013); additionally, p-PRKCα (S657) also shows decreased expression in these samples (p= 0.00373). These data suggest that the WT1 transcription factor regulates PRKCα expression, and that PRKCα plays a potential role in Wilms tumor tumorigenesis. We hypothesize that the WT1 transcription factor directly/indirectly regulates PRKCα and mutations occurring in WT1 lead to decreased expression of PRKCα. Prkcα and Wt1 have been shown to co-localize in E14.5 mesenchymal cells of the developing kidney. siRNA knockdown, in-vivo ablation, and tet-inducible expression of Wt1 each independently confirm regulation of Prkcα expression by Wt1 at both RNA and protein levels, and investigation into possible WT1 binding sites in PRKCα regulatory regions has identified multiple sites to be confirmed by luciferase reporter constructs. With the goal of identifying WT1 and PRKCα downstream targets, RPPA analysis of protein expression in mesenchymal cell culture, following lentiviral delivered shRNA knockdown of Wt1 and shRNA knockdown of Prkcα, will be carried out. Apart from Wilms tumor, WT1 also plays an important role in Acute Myeloid Leukemia (AML). WT1 mutation status has been implicated, controversially, as an independent poor-prognosis factor in leukemia, leading to decreased probability of overall survival, complete remission, and disease free survival. RPPA analysis of AML patient samples showed significant decreases in PRKCα/p-PRKCα protein expression in a subset of patients (Kornblau, personal communication); therefore, the possible role of WT1 and PRKCα in leukemia disease progression is an additional focus of this study. WT1 mutation analysis of diploid leukemia patient samples revealed two patients with mutations predicted to affect WT1 activity; of these two samples, only one corresponded to the low PRKCα expression cohort. Further characterization of the role of WT1 in AML, and further understanding of WT1 regulated PRKCα expression, will be gained following RPPA analysis of protein expression in HL60 leukemia cell lines with lentiviral delivered shRNA knockdown of WT1 and shRNA knockdown of PRKCα.

Tumor cells expressing a retroviral envelope escape immune rejection in vivo

A model system for the in vivo control of tumor cell proliferation by the immune system has been used to assay for the possible immunosuppressive activity of retroviral proteins. Expression vectors for the entire or the transmembrane subunit of the Moloney murine leukemia virus envelope protein were constructed, as well as control vectors for irrelevant transmembrane proteins—or no protein. They were introduced either into MCA205 murine tumor cells, which do not proliferate upon s.c. injection into an allogeneic host, or into CL8.1 murine tumor cells, which overexpress class I antigens and are rejected in a syngeneic host. In both cases, expression of the complete envelope protein or of the transmembrane subunit resulted in tumor growth in vivo, with no effect of control vectors. Tumor cell growth results from inhibition of the host immune response, as the envelope-dependent effect was no more observed for MCA205 cells in syngeneic mice or for CL8.1 cells in x-irradiated mice. This inhibition is local because it is not observed at the level of control tumor cells injected contralaterally. These results suggest a noncanonical function of retroviral envelopes in the “penetrance” of viral infections, as well as a possible involvement of the envelope proteins of endogenous retroviruses in tumoral processes.

Role of B cells in antigen presentation of the hepatitis B core

The hepatitis B virus (HBV) nucleocapsid or core antigen (HBcAg) is extremely immunogenic during infection and after immunization. For example, during many chronic infections, HBcAg is the only antigen capable of eliciting an immune response, and nanogram amounts of HBcAg elicit antibody production in mice. Recent structural analysis has revealed a number of characteristics that may help explain this potent immunogenicity. Our analysis of how the HBcAg is presented to the immune system revealed that the HBcAg binds to specific membrane Ig (mIg) antigen receptors on a high frequency of resting, murine B cells sufficiently to induce B7.1 and B7.2 costimulatory molecules. This enables HBcAg-specific B cells from unprimed mice to take up, process, and present HBcAg to naive Th cells in vivo and to T cell hybridomas in vitro approximately 105 times more efficiently than classical macrophage or dendritic antigen-presenting cells (APC). These results reveal a structure–function relation for the HBcAg, confirm that B cells can function as primary APC, explain the enhanced immunogenicity of HBcAg, and may have relevance for the induction and/or maintenance of chronic HBV infection.

An autologous oral DNA vaccine protects against murine melanoma

We demonstrated that peripheral T cell tolerance toward murine melanoma self-antigens gp100 and TRP-2 can be broken by an autologous oral DNA vaccine containing the murine ubiquitin gene fused to minigenes encoding peptide epitopes gp10025–33 and TRP-2181–188. These epitopes contain dominant anchor residues for MHC class I antigen alleles H-2Db and H-2Kb, respectively. The DNA vaccine was delivered by oral gavage by using an attenuated strain of Salmonella typhimurium as carrier. Tumor-protective immunity was mediated by MHC class I antigen-restricted CD8+ T cells that secreted TH1 cytokine IFN-γ and induced tumor rejection and growth suppression after a lethal challenge with B16G3.26 murine melanoma cells. Importantly, the protective immunity induced by this autologous DNA vaccine against murine melanoma cells was at least equal to that achieved through xenoimmunization with the human gp10025–33 peptide, which differs in its three NH2-terminal amino acid residues from its murine counterpart and was previously reported to be clearly superior to an autologous vaccine in inducing protective immunity. The presence of ubiquitin upstream of the minigene proved to be essential for achieving this tumor-protective immunity, suggesting that effective antigen processing and presentation may make it possible to break peripheral T cell tolerance to a self-antigen. This vaccine design might prove useful for future rational designs of other recombinant DNA vaccines targeting tissue differentiation antigens expressed by tumors.

Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin

Pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 (PBSF/SDF-1) is a member of the CXC group of chemokines that is initially identified as a bone marrow stromal cell-derived factor and as a pre-B-cell stimulatory factor. Although most chemokines are thought to be inducible inflammatory mediators, PBSF/SDF-1 is essential for perinatal viability, B lymphopoiesis, bone marrow myelopoiesis, and cardiac ventricular septal formation, and it has chemotactic activities on resting lymphocytes and monocytes. In this paper, we have isolated a cDNA that encodes a seven transmembrane-spanning-domain receptor, designated pre-B-cell-derived chemokine receptor (PB-CKR) from a murine pre-B-cell clone, DW34. The deduced amino acid sequence has 90% identity with that of a HUMSTSR/fusin, a human immunodeficiency virus 1 (HIV-1) entry coreceptor. However, the second extracellular region has lower identity (67%) compared with HUMSTSR/fusin. PB-CKR is expressed during embryo genesis and in many organs and T cells of adult mice. Murine PBSF/SDF-1 induced an increase in intracellular free Ca2+ in DW34 cells and PB-CKR-transfected Chinese hamster ovary (CHO) cells, suggesting that PB-CKR is a functional receptor for murine PBSF/SDF-1. Murine PBSF/SDF-1 also induced Ca2+ influx in fusin-transfected CHO cells. On the other hand, considering previous results that HIV-1 does not enter murine T cells that expressed human CD4, PB-CKR may not support HIV-1 infection. Thus, PB-CKR will be an important tool for functional mapping of HIV-1 entry coreceptor fusin and for understanding the function of PBSF/SDF-1 further.

Transcriptome of a mouse kidney cortical collecting duct cell line: Effects of aldosterone and vasopressin

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCDcl4) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCDcl4 cell line either by Northern blot hybridization or reverse transcription–PCR. The hepatocyte nuclear transcription factor HNF-3-α (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is crucial for production of mature red cells. Although it is well established that the Epo-bound EpoR is a dimer, it is not clear whether, in the absence of ligand, the intact EpoR is a monomer or oligomer. Using antibody-mediated immunofluorescence copatching (oligomerizing) of epitope-tagged receptors at the surface of live cells, we show herein that a major fraction of the full-length murine EpoR exists as preformed dimers/oligomers in BOSC cells, which are human embryo kidney 293T-derived cells. This observed oligomerization is specific because, under the same conditions, epitope-tagged EpoR did not oligomerize with several other tagged receptors (thrombopoietin receptor, transforming growth factor β receptor type II, or prolactin receptor). Strikingly, the EpoR transmembrane (TM) domain but not the extracellular or intracellular domains enabled the prolactin receptor to copatch with EpoR. Preformed EpoR oligomers are not constitutively active and Epo binding was required to induce signaling. In contrast to tyrosine kinase receptors (e.g., insulin receptor), which cannot signal when their TM domain is replaced by the strongly dimerizing TM domain of glycophorin A, the EpoR could tolerate the replacement of its TM domain with that of glycophorin A and retained signaling. We propose a model in which TM domain-induced dimerization maintains unliganded EpoR in an inactive state that can readily be switched to an active state by physiologic levels of Epo.

Rad51 expression and localization in B cells carrying out class switch recombination.

Rad51 is a highly conserved eukaryotic homolog of the prokaryotic recombination protein RecA, which has been shown to function in both recombinational repair of DNA damage and meiotic recombination in yeast. In primary murine B cells cultured with lipopolysaccharide (LPS) to stimulate heavy chain class switch recombination, Rad51 protein levels are dramatically induced. Immunofluorescent microscopy shows that anti-Rad51 antibodies stain foci that are localized within the nuclei of switching B cells. Immunohistochemical analysis of splenic sections shows that clusters of cells that stain brightly with anti-Rad51 antibodies are evident within several days after primary immunization and that Rad51 staining in vivo is confined to B cells that are switching from expression of IgM to IgG antibodies. Following switch recombination, B cells populate splenic germinal centers, where somatic hypermutation and clonal proliferation occur. Germinal center B cells are not stained by anti-Rad51 antibodies. Rad51 expression is therefore not coincident with somatic hypermutation, nor does Rad51 expression correlate simply with cell proliferation. These data suggest that Rad51, or a highly related member of the conserved RecA family, may function in class switch recombination.

Subcutaneous vaccination with irradiated, cytokine-producing tumor cells stimulates CD8+ cell-mediated immunity against tumors located in the "immunologically privileged" central nervous system.

Vaccination with cytokine-producing tumor cells generates potent immune responses against tumors outside the central nervous system (CNS). The CNS, however, is a barrier to allograft and xenograft rejection, and established tumors within the CNS have failed to respond to other forms of systemic immunotherapy. To determine what barriers the "immunologically privileged" CNS would pose to cytokine-assisted tumor vaccines and what cytokines would be most efficacious against tumors within the CNS, we irradiated B16 murine melanoma cells producing murine interleukin 2 (IL-2), IL-3, IL-4, IL-6, gamma-interferon, or granulocyte-macrophage colony stimulating factor (GM-CSF) and used these cells as subcutaneous vaccines against tumors within the brain. Under conditions where untransfected B16 cells had no effect, cells producing IL-3, IL-6, or GM-CSF increased the survival of mice challenged with viable B16 cells in the brain. Vaccination with B16 cells producing IL-4 or gamma-interferon had no effect, and vaccination with B16 cells producing IL-2 decreased survival time. GM-CSF-producing vaccines were also able to increase survival in mice with pre-established tumors. The response elicited by GM-CSF-producing vaccines was found to be specific to tumor type and to be abrogated by depletion of CD8+ cells. Unlike the immunity generated against subcutaneous tumors by GM-CSF, however, the effector responses generated against tumors in the CNS were not dependent on CD4+ cells. These data suggest that cytokine-producing tumor cells are very potent stimulators of immunity against tumors within the CNS, but effector responses in the CNS may be different from those obtained against subcutaneous tumors.

Specific induction of cAMP in Langerhans cells by calcitonin gene-related peptide: relevance to functional effects.

Epidermal Langerhans cells (LC) are associated anatomically with epidermal nerves, and a product of these nerves, calcitonin gene-related peptide (CGRP), inhibits the antigen-presenting capacity of LC and macrophages. As the CGRP receptor appears to be coupled to Gs alpha protein, which in turn activates adenylate cyclase, the ability of CGRP to induce cAMP in LC was examined and correlated with functional effects. LC were isolated from murine epidermal cells using antibodies on magnetic microspheres. Exposure to CGRP induced a significant increase in cAMP content, which could be inhibited by coculture with a truncated form of CGRP [CGRP-(8-37)] that is a specific competitive inhibitor of CGRP. Substance P and calcitonin failed to induce cAMP in LC. Although culture in CGRP reduced the ability of murine epidermal cells enriched for LC content to present pigeon cytochrome c to a responsive clone or to present antigen for elicitation of delayed-type hypersensitivity in immune mice, culture in forskolin had little or no effect on antigen presentation despite increased cAMP content of LC as much or more than that induced by CGRP. The effect of CGRP on antigen presentation in these systems could be blocked with CGRP-(8-37). CGRP inhibited the induction of B7-2 by lipopolysaccharide on peritoneal macrophages and a LC line, whereas calcitonin did not. CGRP induces specific accumulation of cAMP in LC and inhibits LC antigen-presenting function by a receptor-mediated event. However, the induction of cAMP by itself does not account for inhibition of antigen presentation. Suppression of the expression of B7-2 may be one mechanism by which CGRP inhibits antigen presentation.

Construction, purification, and functional incorporation on tumor cells of glycolipid-anchored human B7-1 (CD80).

To generate a potent cell-mediated immune response, at least two signals are required by T cells. One is engagement of the T-cell receptor with peptide-bearing major histocompatibility complex molecules. The other signal can be delivered by various molecules on the antigen-presenting cell, such as B7-1 (CD80). Many tumor cells escape immune recognition by failing to express these costimulatory molecules. Transfection of the B7 gene into some murine tumor cells allows for immune recognition and subsequent rejection of the parental tumor. We have studied an alternative approach for the introduction of B7-1 onto the surface of tumor cells. This method involves purified glycosyl-phosphatidylinositol (GPI)-anchored proteins which can spontaneously incorporate their lipid tail into cell membranes. We have created and purified a GPI-anchored B7-1 molecule (called GPI-B7) which is able to bind its cognate ligand, CD28, and incorporate itself into tumor cell membranes after a short incubation. Tumor cells that have been reconstituted with GPI-B7 can provide the costimulatory signal needed to stimulate T cells. These findings suggest an approach for the introduction of new proteins onto cell membranes to create an effective tumor vaccine for potential use in human immunotherapy.

Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis.

Murine endothelial cells are readily transformed in a single step by the polyomavirus oncogene encoding middle-sized tumor antigen. These cells (bEND.3) form tumors (hemangiomas) in mice which are lethal in newborn animals. The bEND.3 cells rapidly proliferate in culture and express little or no thrombospondin 1 (TS1). To determine the role of TS1 in regulation of endothelial cell phenotype, we stably transfected bEND.3 cells with a human TS1 expression vector. The cells expressing human TS1 were readily identified by their altered morphology and exhibited a slower growth rate and lower saturation density than the parental bEND.3 cells. The TS1-expressing cells also formed aligned cords of cells instead of clumps or cysts in Matrigel. Moreover, while the bEND.3 cells formed large tumors in nude mice within 48 hr, the TS1-expressing cells failed to form tumors even after 1 month. The TS1-transfected cells expressed transforming growth factor beta mRNA and bioactivity at levels similar to those of the parental or vector-transfected bEND.3 cells, indicating that the effects of TS1 expression are not due to the activation of transforming growth factor beta by TS1. TS1 expression resulted in a > 100-fold decrease in net fibrinolytic (urokinase-type plasminogen activator, uPA) activity due to more plasminogen-activator inhibitor 1 and less uPA secretion. TS1 thus appears to be an important regulator of endothelial cell phenotype required for maintaining the quiescent, differentiated state.