Interleukin-10 suppression enhances T-cell antitumor immunity and responses to checkpoint blockade in chronic lymphocytic leukemia.

T-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL), where CLL cells downregulate T-cell responses through regulatory molecules including programmed death ligand-1 (PD-L1) and Interleukin-10 (IL-10). Immune checkpoint blockade (ICB) aims to restore T-cell function by preventing the ligation of inhibitory receptors like PD-1. However, most CLL patients do not respond well to this therapy. Thus, we investigated whether IL-10 suppression could enhance antitumor T-cell activity and responses to ICB. Since CLL IL-10 expression depends on Sp1, we utilized a novel, better tolerated analogue of the Sp1 inhibitor mithramycin (MTMox32E) to suppress CLL IL-10. MTMox32E treatment inhibited mouse and human CLL IL-10 production and maintained T-cell effector function in vitro. In the Eµ-Tcl1 mouse model, treatment reduced plasma IL-10 and CLL burden and increased CD8+ T-cell proliferation, effector and memory cell prevalence, and interferon-γ production. When combined with ICB, suppression of IL-10 improved responses to anti-PD-L1 as shown by a 4.5-fold decrease in CLL cell burden compared to anti-PD-L1 alone. Combination therapy also produced more interferon-γ+, cytotoxic effector KLRG1+, and memory CD8+ T-cells, and fewer exhausted T-cells. Since current therapies for CLL do not target IL-10, this provides a novel strategy to improve immunotherapies.

Mutations in the ligand-binding domain of the kit receptor: an uncommon site in human piebaldism.

Heterozygous mutations in the gene for the Kit transmembrane receptor have been identified recently in human piebaldism and mouse "dominant spotting." Interestingly, all of the 14 known missense mutations that cause depigmentation in these species map to the tyrosine kinase domain of the receptor, whereas none have involved the extracellular ligand-binding domain. In an attempt to detect these uncommon mutations, we screened the nine exons encoding the extracellular portion of Kit for single-strand conformation polymorphisms (SSCP) in eight piebald subjects previously reported to be negative for kinase mutations. Four of these eight kindreds proved to carry novel mutations. The first mutation, found in two apparently unrelated probands with mild piebaldism and English ancestry, substitutes an arginine for a highly conserved cysteine at codon 136. This substitution disrupts a putative disulfide bond required for formation of the second Ig-like (D2) loop of the Kit ligand-binding domain. The second mutation, detected in a piebald kindred characterized by unusually limited depigmentation, substitutes a threonine for an alanine at codon 178, a site just proximal to conserved cysteines at codons 183 and 186. The third mutation, occurring in a kindred with more extensive depigmentation, is a novel four-base insertion in exon 2 that results in a proximal frameshift and premature termination. The data strongly suggest that piebaldism can result from missense mutations in the Kit ligand-binding domain, although the resulting phenotype may be milder than that observed for null or kinase mutations. The apparent clustering of these uncommon mutations at or near the conserved cysteines for the D2 Ig-like loop further suggests a critical role for this region in Kit receptor function.

Isolation of endothelial-like stromal cells that express Kit ligand and support in vitro hematopoiesis.

Although macrophages account for 70-90% of the adherent cells in mouse long-term bone marrow cultures (LTBMC) and CFU-F colonies, the predominant nonhematopoietic stromal cell is endothelial-like (EL), expressing cytoplasmic collagen IV, laminin, and an antigen recognized by the monoclonal antibody MECA-10. We report the isolation of this stromal cell lineage from primary LTBMC by immunomagnetic cell selection using MECA-10. More than 95% of the cells in the MECA-10-positive fraction are EL cells as judged by morphology, surface staining for MECA-10, cytoplasmic staining for collagen IV, and electrophoretic analysis of MECA-10-positive cells isolated from radiation chimeras. When plated under LTBMC conditions, EL cell monolayers recharged with either wild-type or Sl/Sld marrow support an increased density and number of clonogenic and mature hematopoietic cells in short-term cultures. In accord with this finding, Northern blots of mRNA from unstimulated EL cells demonstrate constitutive expression of Kit ligand (KL). Moreover, in situ two-color immunofluorescence staining for cytoplasmic collagen IV and surface KL suggests that EL cells are the exclusive source of membrane-bound KL in mouse cultures. The ability to isolate EL cells from primary cultures without the need for repeated cell passage or immortalization provides a novel approach to dissecting the molecular basis of stem cell-stromal cell interactions.

From white spots to stem cells: the role of the Kit receptor in mammalian development.

The Kit tyrosine kinase membrane receptor is essential for melanogenesis, gametogenesis and hematopoiesis during embryonic development and postnatal life. This review summarizes the genetic evidence implicating Kit and its ligand, Steel factor, in the control of stem cell proliferation, migration and survival, with emphasis on mutations in the human and mouse genes.

Southwestern Internal Medicine Conference: clinical use of hematopoietic growth factors.

The hematopoietic growth factors, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), have been cloned, produced in bacteria and yeast, and approved for clinical use in the treatment of neutropenia. Both factors stimulate the proliferation and maturation of neutrophil progenitors and enhance the effector functions of mature cells by interaction with specific receptors on the cell surface. Serum levels of G-CSF correlate inversely with the neutrophil count, suggesting that G-CSF may be the normal homeostatic regulator of the neutrophil count, while GM-CSF is generally undetectable in the serum and appears under normal physiologic conditions to act locally at inflammatory sites. Phase I and II clinical trials with these factors demonstrated minimal toxicity for G-CSF and mild to moderate dose-dependent toxicity for GM-CSF. Recent clinical trials, including double-blind, randomized studies, support a role for these growth factors in the treatment of chronic neutropenias, such as Kostmann's syndrome, acquired immune deficiency syndrome (AIDS), aplastic anemia, and myelodysplasia, as well as in acute neutropenias, such as cyclic neutropenia, chemotherapy-induced neutropenia, and bone marrow transplantation.

Human piebald trait resulting from a dominant negative mutant allele of the c-kit membrane receptor gene.

Human piebald trait is an autosomal dominant defect in melanocyte development characterized by patches of hypopigmented skin and hair. Although the molecular basis of piebaldism has been unclear, a phenotypically similar "dominant spotting" of mice is caused by mutations in the murine c-kit protooncogene. In this regard, one piebald case with a point mutation and another with a deletion of c-kit have been reported, although a polymorphism or the involvement of a closely linked gene could not be excluded. To confirm the hypothesis that piebaldism results from mutations in the human gene, c-kit exons were amplified by polymerase chain reaction from the DNA of 10 affected subjects and screened for nucleotide changes by single-stranded conformation polymorphism analysis. In one subject with a variant single-stranded conformation polymorphism pattern for the first exon encoding the kinase domain, DNA sequencing demonstrated a missense mutation (Glu583----Lys). This mutation is identical to the mouse W37 mutation which abolishes autophosphorylation of the protein product and causes more extensive depigmentation than "null" mutations. In accord with this "dominant negative" effect, the identical mutation in this human kindred is associated with unusually extensive depigmentation. Thus, the finding of a piebald subject with a mutation that impairs receptor activity strongly implicates the c-kit gene in the molecular pathogenesis of this human developmental defect.

Stem cell quiescence/activation is reversible by serial transplantation and is independent of stromal cell genotype in mouse aggregation chimeras.

We made use of a previously described in vivo model of chimeric mice created by embryo aggregation that allows the study of contributions to both lymphohematopoiesis and marrow stroma by two genotypically distinct cell populations. Day-2 embryos from C57BL/6 and DBA/2 strains were fused to produce allophenic chimeric mice that proved to have contributions from each strain in all the tissues of the body and that permitted study of competitive contributions to blood formation. Although the contribution of DBA/2 stem cells to hematopoiesis gradually ceased in an age-related manner so that all blood cells in aged chimeras were C57BL/6 in origin, here we show that, in contrast, the extent of stromal chimerism, determined by the fibroblast colony-forming unit (CFU-F) assay, was maintained and was remarkably uniform from one marrow site to another. This result is consistent with a polyclonal nonhematopoietic origin of the CFU-F and suggests that the decline in DBA/2 blood cells was not dependent on similar changes in genotype-matched stroma, but was instead an intrinsic property of this stem cell population. These intrinsic stem cell properties were further examined by serial bone marrow transplantation. When marrow from a chimera with no detectable DBA/2 blood cells was transplanted into irradiated recipients, cells of DBA/2 genotype significantly contributed to early hematopoietic engraftment, demonstrating that the DBA/2 stem cell population was not extinguished in the chimeric donor, but rather had entered a reversible state of quiescence. Reactivation of the DBA/2 stem cell population, however, was short-lived, and long-term engraftment of recipients was accomplished by donor cells of the partner strain (C57BL/6). However, transient reactivation of the quiescent (DBA/2) stem cell pool again occurred with a second round of transplantation. These surprising results demonstrate, for the first time, selective and reiterated inactivation and reactivation of a stem cell population depending on hematopoietic needs. Moreover, the results suggest that genetic differences in the stem cell populations of coexistent strains account for the selective responses described.

Deletion of the c-kit protooncogene in the human developmental defect piebald trait.

The protooncogene c-kit is critical for development of hematopoietic stem cells, germ cells, and melanoblasts in the mouse. Homozygous mutations of this gene in the mouse cause anemia, infertility, and albinism, whereas heterozygous mutant mice usually exhibit only a white forehead blaze and depigmentation of the ventral body, tail, and feet. The heterozygous mouse phenotype is very similar to human piebald trait, which is characterized by a congenital white hair forelock and ventral and extremity depigmentation. To investigate the possibility that alterations in the human c-kit gene may be a cause of piebald trait, DNA from seven unrelated affected individuals was examined by Southern blot analysis. One subject, although cytogenetically normal, has a heterozygous deletion of the c-kit protooncogene. This deletion encompasses the entire coding region for c-kit and also involves the closely linked gene for platelet-derived growth factor receptor alpha. Fluorescence in situ hybridization of genomic c-kit probes to metaphase chromosomes independently confirmed the deletion in this case. These findings provide molecular evidence mapping piebald trait to the c-kit locus on chromosome 4. Although we cannot exclude the involvement of other closely linked genes, the demonstration of a genomic c-kit deletion in one subject with piebald trait and the marked concordance of the human and mouse phenotypes provide strong evidence for the role of c-kit in the development of human melanocytes and in the pathogenesis of piebald trait.

Human gene therapy.

Recent advances in molecular genetics have made possible the use of retroviral "vectors" to transfer cloned human genes into somatic cells. With this new technology, the genetic defect underlying many recessive inherited disorders can probably be corrected by inserting a normal gene into the patient's hematopoietic stem cells. This article reviews the design and safety of the viral vectors and the results of in vivo studies in mice and large animals that have led to the first human trials. Other target cells for gene transfer, such as endothelial cells, fibroblasts, keratinocytes, and hepatocytes, are also discussed. The use of recombinant retroviruses for gene transfer in vivo is still a new area of research, but the feasibility of "gene therapy" for genetic disorders is rapidly gaining medical and scientific acceptance.

Stromal cell progeny of murine bone marrow fibroblast colony-forming units are clonal endothelial-like cells that express collagen IV and laminin.

Studies of human and murine bone marrow explants have demonstrated the existence of stromal cell precursors that give rise to colonies of adherent cells in short-term cultures. Because previous data suggested that these colonies were composed of fibroblasts, the precursor cells were termed fibroblast colony-forming units (CFU-F). However, we have recently shown that the stromal cells which support hematopoiesis in murine long-term bone marrow cultures (LTBC) express collagen IV and laminin, markers associated with an endothelial cell lineage, but are negative for collagen I and III, markers associated with a fibroblast cell lineage. Because these conflicting results suggest major functional differences between the stromal cells observed in long-term cultures and the short-term assay, we re-examined the lineage of CFU-F-derived stromal cells. Using two-color immunofluorescence, we characterized virtually all of the cells comprising individual "CFU-F" colonies derived from mouse radiation chimeras. Identification of donor (hematopoietic) or host (stromal) origin was based on surface staining for strain-specific H-2 surface antigens, and, for endothelial or fibroblast properties, on cytoplasmic staining for laminin and collagen IV, or collagens I and III, respectively. The results demonstrate that a large proportion of the cells in CFU-F colonies are donor-derived and fail to stain with any of the antisera specific for nonhematopoietic cells. In addition, these donor-derived cells exhibit marked phagocytic capacity and stain positively with monoclonal antibodies characteristic of the monocyte-macrophage hematopoietic cell lineage (anti-T200, anti-Mac-1, F4/80). However, the remainder of the cells are host-derived cells that stain positively with antisera to collagen IV and laminin. In contrast, stains for collagen types I and III were negative under conditions that allowed for strong staining of control skin fibroblasts. In separate studies, using mixtures of two genetically distinct bone marrows, the cells expressing collagen IV were further shown to be clonal in origin within individual colonies, directly demonstrating that the CFU-F assay provides a quantitative measure of the numbers of marrow stromal cell precursors. Thus, the current studies establish a remarkable similarity between the hematopoietic microenvironment in the short-term CFU-F assay and the long-term culture system: the majority of adherent cells are hematopoietic cells of the monocyte-macrophage lineage, while the remainder are stromal cells whose precise lineage remains uncertain, but whose pattern of collagen expression is more consistent with an endothelial rather than a fibroblast cell origin.

Hematopoietic microenvironment. Origin, lineage, and transplantability of the stromal cells in long-term bone marrow cultures from chimeric mice.

Studies of bone marrow transplant patients have suggested that the stromal cells of the in vitro hematopoietic microenvironment are transplantable into conditioned recipients. Moreover, in patients with myeloproliferative disorders, all of the stromal cells, which include presumptive endothelial cells, appear to be derived from hematopoietic precursors. To confirm these findings, we have constructed two chimeric mouse models: (a) traditional radiation chimeras, and (b) fetal chimeras, produced by placental injection of bone marrow into genetically anemic Wx/Wv fetuses, a technique that essentially precludes engraftment of nonhematopoietic cells. Using two-color indirect immunofluorescence, the stromal cells in long-term bone marrow culture derived from these chimeras were analyzed for donor or host origin by strain-specific H-2 antigens, and for cell lineage by a variety of other specific markers. 75-95% of the stromal cells were shown to be hematopoietic cells of the monocyte-macrophage lineage, based upon donor origin, phagocytosis, and expression of specific hematopoietic surface antigens. The remaining 5-25% of the stromal cells were exclusively host in origin. Apart from occasional fat cells, these cells uniformly expressed collagen type IV, laminin, and a surface antigen associated with endothelial cells. Since these endothelial-like cells are not transplantable into radiation or fetal chimeras, they are not derived from hematopoietic stem cells. The contrast between our findings and human studies suggests either unexpected species differences in the origin of stromal lineages or limitations in the previous methodology used to detect nonhematopoietic stromal cells.

Successful transplantation of Friend virus-induced preleukemia into stem cell-deficient fetal mice.

The leukemias induced by the Friend polycythemia virus and other leukemogenic retroviruses have previously not been transplantable until weeks or months after virus inoculation. Because tumor-specific immune mechanisms persist in both irradiated and nude mice, it has not been possible to determine if this result is due to rejection of cells already immortalized by retrovirus infection, or reflects an inherent limitation in the proliferative capacity and malignancy of these "preleukemic" cells. To clarify these issues, we have transplanted virus-infected bone marrow into mouse fetuses that are immunologically immature and thus incapable of graft rejection. We report here that within days of virus inoculation, transplantable cells capable of disease progression in certain fetal hosts can be detected with this technique. These results demonstrate that cells with the capacity for extensive leukemic proliferation arise very early in Friend virus-induced disease. However, successful transplantation was seen only in genetically anemic recipients (Wx/Wv), which are deficient in hematopoietic stem cells, and not in their normal littermates. Thus, in accord with recent in vitro observations, this in vivo data suggests that normal hematopoietic cells, independent of immune mechanisms, can suppress the malignant progression of transformed cells.

Relationships between B-lineage lymphocytes and stromal cells in long-term bone marrow cultures.

In long-term culture of mouse bone marrow, the growth and differentiation of B-lineage lymphocytes depends on interaction with adherent cells or their products. The objectives of these studies were to characterize the types of cells present in the supporting adherent layer as well as the physical relationships of these cells with lymphocytes. With an extensive panel of antibodies against hemopoietic and lymphocyte antigens, two discrete nonlymphoid populations were identified: macrophages and undefined, large cells which we termed "stromal cells". Lymphocyte clusters grew in actual contact with the latter cells only. Stromal cells lacked expression of most hemopoietic antigens, including the common leukocyte antigen, J11d, heat stable antigen (M1/69), Thy-1 and BP 1. Antigens expressed by stromal cells were detected by AA4.1, our 94.2 antibody, and antibody to the Forsmann antigen, but the most distinguishing characteristics of the lymphocyte-binding stromal cells were production of basement membrane components, laminin and collagen IV, and the extremely low uptake of acetylated low density lipoprotein (LDL). Using acetylated LDL uptake as a sorting criterion, the lymphocyte-binding stromal cells were separated from the macrophages, recultured and shown to support lymphocyte proliferation. We found the binding between stromal cells and lymphocytes to be highly selective and dependent on divalent cations; hence, specialized adhesion mechanisms may have a role in B cell development. Moreover, our studies suggest that phosphatidylinositol-anchored cell surface molecules may be involved in this adhesion. Our findings demonstrate the possibility that a single cell type provides physical support and proliferation stimuli for early B-lineage cells. This accessory cell is not a macrophage; rather, it has features of an endothelial or epithelial cell.

Development of adult bone marrow stem cells in H-2-compatible and -incompatible mouse fetuses.

Bone marrow of normal adult mice was found, after transplacental inoculation, to contain cells still able to seed the livers of early fetuses. The recipients' own hematopoietic stem cells, with a W-mutant defect, were at a selective disadvantage. Progression of donor strain cells to the bone marrow, long-term self-renewal, and differentiation into myeloid and lymphoid derivatives was consistent with the engraftment of totipotent hematopoietic stem cells (THSC) comparable to precursors previously identified (4) in normal fetal liver. More limited stem cells, specific for the myeloid or lymphoid cell lineages, were not detected in adult bone marrow. The bone marrow THSC, however, had a generally lower capacity for self-renewal than did fetal liver THSC. They had also embarked upon irreversible changes in gene expression, including partial histocompatibility restriction. While completely allogeneic fetal liver THSC were readily accepted by fetuses, H-2 incompatibility only occasionally resulted in engraftment of adult bone marrow cells and, in these cases, was often associated with sudden death at 3-5 mo. On the other hand, H-2 compatibility, even with histocompatibility differences at other loci, was sufficient to ensure long-term success as often as with fetal liver THSC.

Totipotent hematopoietic stem cells: normal self-renewal and differentiation after transplantation between mouse fetuses.

Successful engraftment of mouse fetal liver cells in early fetal recipients, after microinjection via the placental circulation, is attributable to seeding of the recipient's liver by a cell type that is ancestral to both the myeloid and lymphoid definitive lineages and is capable of sustained self-renewal and differentiation for more than 2 years. This primitive cell is therefore the normal totipotent hematopoietic stem cell (THSC). The use of a large series of mutant anemic recipients with decreasing severity of an endogenous stem-cell defect (W/W, Wv/Wv, Wf/Wf, Wv/+), and therefore of graded selective advantage to normal donor cells, has revealed that engraftment entails marginal numbers of cells--probably individual ones--in the least afflicted hosts. Thus the observed progressive and coordinate shift toward donor-strain erythrocytes, granulocytes and B and T lymphocytes, over time, indicates THSC expansion to form a larger stem-cell pool and normally regulated differentiation of cells from the pool. This transplant system allows allogeneic combinations with impunity and therefore provides many novel experimental possibilities for investigating THSC normal development, genetic abnormalities or neoplastic potential in relation to the intact developmental succession of hematopoietic tissue environments in vivo.

Prevention of genetic anemias in mice by microinjection of normal hematopoietic stem cells into the fetal placenta.

Mice homozygous for mutant genes at the W locus have a marked macrocytic anemia that is fatal in some genotypes. The defect is believed to originate in the developmentally pluripotent hematopoietic stem cell population. Anemia is first grossly manifest on day 13 of gestation, when the liver is the chief hematopoietic organ. The known paucity of blood-forming foci in livers of homozygotes and the limited formation of their erythrocytes suggested that such fetuses-unlike normal ones-might have conditions favorable for in utero seeding of genetically normal hematopoietic tissue. If this were accomplished before day 13, the anemia might essentially be prevented, or at least substantially mitigated, and normalcy soon achieved by cell selection. This proved to be the case. Allogeneic normal fetal liver cells were microinjected into the blood vessels of the fetal placenta on day 11 of gestation. Of eight mutant homozygotes born from segregating matings, six (four W/W, two W(v)/W(v)) were successfully populated with donor cells. Strain-specific hemoglobin markers demonstrated replacement of the erythroid lineage with the normal type, the rate of substitution being more rapid in the W/W (ordinarily more anemic) recipients. Strain-specific isozyme differences revealed that white blood cells were also replaced. Thus, the initial selective pressure, hence the W-mutant phenotypic lesion, must have occurred at the pluripotent stem cell stage. The animals remained immunologically tolerant of the donor cells and no graft-versus-host reaction occurred. The early introduction of hematopoietic cells differing genetically from all the other tissues of the animal provides possibilities for tracing normal hematopoietic lineages in vivo, for analyzing cell and tissue interactions, such as those between lymphocytes and thymus, and for clarifying the etiology of other blood or immune insufficiencies or malignancies.

Modification and restriction of T-even bacteriophages. In vitro degradation of deoxyribonucleic acid containing 5-hydroxymethylctosine.

Using the single-stranded circular DNA of bacteriophage fd as template, double-stranded circular DNA has been prepared in vitro with either 5-hydroxymethylcytosine ([hmdC]DNA) or cytosine ([dC]DNA) in the product strand. Extracts prepared from Escherichia coli cells restrictive to T-even phage containing nonglucosylated DNA degrade [hmdC]DNA to acid-soluble material in vitro, but do not degrade [dC]dna. In contrast, extracts prepared from E. coli K12 rglA- rglB-, a strain permissive to T-even phage containing nonglucosylated DNA, do not degrade [hmdC]DNA or [dC]DNA. In addition, glucosylation of the [hmdC]DNA renders it resistant to degradation by extracts from restrictive strains. The conversion of [hmdC]DNA to acid-soluble material in vitro consists of an HmCyt-specific endonucleolytic cleavage requiring the presence of the RglB gene product to form a linear molecule, followed by a non-HmCyt-specific hydrolysis of the linear DNA to acid-soluble fragments, catalyzed in part by exonuclease V. The RglB protein present in extracts of E. coli K12 rglA- rglB+ has been purified 200-fold by complementation with extracts from E. coli K12 rglA- rglB-. The purified RglB protein does not contain detectable HmCyt-specific endonuclease or exonuclease activity. In vitro endonucleolytic cleavage of [hmdC]DNA thus requires additional factors present in cell extracts.