Non-lymphoid Hematopoietic Cells Research Articles

Mice with a Unique Mutator Phenotype Allow Detection of Lymphoid Leukemia Tumor Suppressor Genes

Mice that are homozygous for a hypomorphic allele of the DNA replication factor minichromosome maintenance protein 2 (designated Mcm2hypo) on a C57Bl/6 background are born viable and are healthy for the first 2 months of life. Despite the fact that this is a germline mutation, all mice develop a specific malignancy– precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL), beginning at 3 months of age. Copy number aberration (CNA) analysis showed that these pre-T LBL samples had 8-14 small (100-1000 kb) interstitial deletions per sample. Remarkably, all mice had two or more deletions, often bi-allelic, that encompassed genes known to be relevant for human pre-T LBL, including Pten, Cdkn1a, Tcf3, and Tcf12.Mice that express a NUP98-HOXD13 (NHD13) transgene develop myelodysplastic syndrome, followed by leukemic transformation in the majority of mice. The acute leukemias that develop in the NHD13 mice are most commonly myeloid, less commonly T-cell, and, rarely, B-lineage.In an effort to identify myeloid tumor suppressor genes, we crossed NHD13 transgene with Mcm2hypo mice (both on C57Bl/6 backgorund), reasoning that bi-allelic mutations of tumor suppressor genes in myeloid cells that collaborated with the NHD13 transgene could be selected due to a growth advantage in vivo. However, all Mcm2hypo:NHD13+ mice developed a pre-T LBL by 3 months of age, reflecting the highly penetrant nature of the Mcm2hypo phenotype, and none of the Mcm2hypo:NHD13+ mice developed myeloid leukemia. Surprisingly, approximately 30% of the Mcm2hypo:NHD13+ mice developed concurrent B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and pre-T LBL. In these animals, the thymus was typically infiltrated with pre-T LBL cells, whereas the bone marrow and spleen were infiltrated with BCP-ALL cells, characterized by Igh clonal VDJ rearrangement and CD19 staining. Parenchymal organs (lung, kidney, liver) were variably infiltrated with pre-T LBL, BCP-ALL, or both. CNA analysis showed that the pre-T LBL were characterized by short (100-1000 kb) deletions including Pten, CDkn1a, Tcf3, and Tcf12 deletions, similar to the Mcm2hypo pre-T LBL, whereas the BCP-ALL were characterized by homozygous or heterozygous deletions including Pax5 and a 400 kb region encompassing Cebpb and Ptpn1. There were no shared deletions present in both BCP-ALL and pre-T LBL from the same mouse, indicating that the BCP-ALL and pre-T LBL arose independently, and not from a common precursor. In addition to the Pax5 and Cebpb/Ptpn1 deletions, most of the BCP-ALL displayed a focal copy number gain of 400kb bounded by Abl1 and Nup214. We hypothesized that this copy number gain may lead to a Nup214-Abl1 fusion gene, via generation of an extrachromosomal episome or tandem duplication, as has been reported for BCP-ALL and pre-T LBL patients. RT-PCR confirmed that these mice expressed a Nup214-Abl1 fusion gene. Finally, in an attempt to determine if Mcm2hypo mice would develop non-T cell malignancy if they were protected from pre-T LBL, we crossed the Mcm2hypo allele onto an athymic nude (nu/nu; NU/J) background. Although this study is still in progress, Mcm2hypo:nu/nu mice have not developed pre-T LBL as of 5 months. However, crossing the Mcm2hypo:nu/nu with a NUP98-PHF23 (NP23) transgene generated Mcm2hypo:nu/nu:NP23+ mice; these mice have developed BCP-ALL similar to the Mcm2hypo:NHD13+ mice. In sum, these studies show that an Mcm2 “deletor” phenotype can induce gross chromosomal rearrangements, including both deletions and amplifications, that delete tumor suppressor genes (e.g. Pten) and activate proto-oncogenes (Abl1), in both T and B lymphocytes. We find it intriguing that both types of malignancy characterized in Mcm2hypo mice occur in cells (pre-B or pre-T) that are programmed to undergo DNA rearrangements, and tolerate DNA double strand breaks (DSBs). Ongoing studies are focused on determining the mechanism(s) that lead to these deletions, and whether the Mcm2 “deletor” phenotype can produce malignancy in non-lymphoid hematopoietic cells. DisclosuresAplan:NIH Office of Technolgy Transfer: Employment, Patents & Royalties: NUP98-HOXD13 mice.

Lymphtoxin β Receptor-Ig Protects from T-Cell-Mediated Liver Injury in Mice through Blocking LIGHT/HVEM Signaling

LIGHT is a member of the TNF superfamily, which is transiently expressed on the surface of activated T lymphocytes and immature dendritic cells. Its known receptors are herpesvirus entry mediator (HVEM) prominently in T lymphocytes, and lymphtoxin beta receptor (LTbetaR) in stromal cells or nonlymphoid hematopoietic cells. Previous studies have shown that overexpression of LIGHT on T cells could lead to autoimmune reaction including lymphocytes activation, inflammation, and tissue destruction. To address the role of LIGHT/HVEM signaling in autoimmune hepatitis, an experimental colitis model induced by intravenous administration of concanavalin A (ConA) was given a soluble LTbetaR-Ig fusion protein as a competitive inhibitor of LIGHT/HVEM pathway. Marked elevation of LIGHT expression was detected in isolate intrahepatic leukocytes (IHLs) of the experimental animal. Treatment with LTbetaR-Ig significantly attenuated the progression and histological manifestations of the hepatic inflammation and reduced the production of inflammatory cytokines including TNF-alpha, IFN-gamma. Moreover, LTbetaR-Ig treatment significantly down-regulated LIGHT expression, leading to reduced lymphocytes (particularly CD4+ T cells), infiltrating into the hepatic inflammation and inhibited NF-kappaB activation and expression. We postulated that blockade of LIGHT/HVEM signaling by LTbetaR-Ig may ameliorate hepatitis by down-regulating LIGHT expression, and therefore we envision that LTbetaR-Ig would prove to a promising strategy for the clinical treatment of human autoimmune hepatitis.

Lymphtoxin β receptor-Ig ameliorates TNBS-induced colitis via blocking LIGHT/HVEM signaling

LIGHT is a member of the TNF superfamily, which is transiently expressed on the surface of activated T lymphocytes and immature dendritic cells. Its known receptors are herpesvirus entry mediator (HVEM) prominently in T lymphocytes, and lymphtoxin β receptor (LTβR) in stromal cells or nonlymphoid hematopoietic cells. Previous studies have shown that overexpression of LIGHT on T cells could lead to lymphocytes activation, inflammation, and tissue destruction focused on intestinal mucosal tissues. To address the role of LIGHT/HVEM signaling in colonic inflammation, an experimental colitis model induced by rectal administration of trinitrobenzene sulfonic acid (TNBS) was given a soluble LTβR-Ig fusion protein as a competitive inhibitor of LIGHT/HVEM pathway. Marked elevation of LIGHT expression was detected in colonic tissue of the experimental colitis. Treatment with LTβR-Ig significantly attenuated the progression and histological manifestations of the colonic inflammation and reduced the production of inflammatory cytokines including TNF-α, IL-1β and IL-8. Moreover, LTβR-Ig treatment significantly down-regulated LIGHT expression, leading to reduced lymphocytes, particularly CD4 + T cells, infiltrating into the colonic inflammation tissue as shown by histological analysis. In addition, comparison of the therapeutic effects on TNBS-induced colitis between LTβR-Ig and mesalazine showed that both treatments were equally efficacious. We postulated that blockade of LIGHT/HVEM signaling by LTβR-Ig may ameliorate TNBS-induced colitis by down-regulating LIGHT expression, and therefore we envision that LTβR-Ig would prove to a promising strategy for the clinical treatment of inflammatory bowel disease.

Vav promotes differentiation of human tumoral myeloid precursors

Vav is one of the genetic markers that correlate with the differentiation of hematopoietic cells. In T and B cells, it appears crucial for both development and functions, while, in non-lymphoid hematopoietic cells, Vav seems not involved in cell maturation, but rather in the response of mature cells to agonist-dependent proliferation and phagocytosis. We have previously demonstrated that the amount and the tyrosine phosphorylation of Vav are up-regulated in both whole cells and nuclei of tumoral promyelocytes induced to granulocytic maturation by ATRA and that tyrosine-phosphorylated Vav does not display any ATRA-induced GEF activity but contributes to the regulation of PI 3-K activity. In this study, we report that Vav accumulates in nuclei of ATRA-treated APL-derived cells and that the down-modulation of Vav prevents differentiation of tumoral promyelocytes, indicating that it is a key molecule in ATRA-dependent myeloid maturation. On the other hand, the overexpression of Vav induces an increased expression of surface markers of granulocytic differentiation without affecting the maturation-related changes of the nuclear morphology. Consistent with an effect of Vav on the transcriptional machinery, array profiling shows that the inhibition of the Syk-dependent tyrosine phosphorylation of Vav reduces the number of ATRA-induced genes. Our data support the unprecedented notion that Vav plays crucial functions in the maturation process of myeloid cells, and suggest that Vav can be regarded as a potential target for the therapeutic treatment of myeloproliferative disorders.

Lymphoid-specific Expression of the Id3 Gene in Hematopoietic Cells: SELECTIVE ANTAGONISM OF E2A BASIC HELIX-LOOP-HELIX PROTEIN ASSOCIATED WITH Id3-INDUCED DIFFERENTIATION OF ERYTHROLEUKEMIA CELLS

Accumulating evidence implicates functions of the Id family of helix-loop-helix proteins in the regulation of cell growth and differentiation in metazoa. Within the mammalian hematopoietic organ, expression of the Id3 gene is restricted to the lymphoid cell compartment. We show here that in non-lymphoid hematopoietic cells, repression of transcription is correlated with hypermethylation of sequences in the vicinity of the upstream regulatory region of the Id3 gene, suggestive of a strict developmental control of expression of this gene in lymphoid versus non-lymphoid hematopoietic cells. Enforced ectopic expression of Id3 in K562 erythroid progenitor cells promotes erythroid differentiation and is correlated with a quantitative/qualitative shift in the profile of interacting TAL1 and E protein heterodimers that bind to a consensus E box sequence in in vitro band shift assays, consistent with selective targeting of E2A E protein(s) by Id3 and suggesting a possible mechanism involving TAL1-mediated differentiation. By using a Gal 4-VP16 two-hybrid competition assay and an E box-dependent reporter assay, we demonstrate directly that the E2A protein E47 preferentially associates with Id3 in vivo. These observations provide a paradigm for understanding how overlapping but distinct specificities of individual Id proteins may constitute a developmentally regulated program underlying cell determination in diverse lineages.

CD45 Protein-tyrosine Phosphatase Is Specifically Associated with a 116-kDa Tyrosine-phosphorylated Glycoprotein

CD45 is a protein-tyrosine phosphatase expressed on all cells of hematopoietic origin. In an attempt to further characterize CD45 function, we set out to identify molecule(s) that specifically associate with CD45. A 116-kDa protein was detected in immunoprecipitates from CD45+ cells but not CD45- cells. The association between CD45 and this 116-kDa protein can be reconstituted by mixing lysates from CD45- cell lines with purified CD45. p116 appears to associate with CD45 through the external, transmembrane, or membrane-proximal region of CD45 since p116 is associated with a mutant form of CD45 possessing a truncated cytoplasmic domain. The association of p116 with CD45 is not isoform-specific as p116 associates equally well with various CD45 isoforms. We have determined that p116 is a tyrosine-phosphorylated glycoprotein and that it is associated with CD45 in all hematopoietic cells examined. Because of its broad distribution, it is possible that identification of p116 will provide additional insight into the function of CD45 in lymphoid as well as non-lymphoid hematopoietic cells.

RAG-1 and RAG-2 are not sufficient to direct all phases of immunoglobulin gene rearrangement in pre-B-cell lines.

To probe the factors controlling immunoglobulin heavy-chain gene rearrangement, we analyzed Abelson virus-transformed pre-B-cell lines that fail to undergo VH-to-DJH joining at an appreciable frequency. Despite this feature, some of these cell lines (rechi) rearrange an extrachromosomal recombination substrate at levels normal for transformed pre-B cells. Others (reclo) rearrange these substrates at levels characteristic of nonlymphoid hematopoietic cells. The DJH rearrangements from a representative rechi cell line were aberrant, suggesting that these cells probably fail to complete heavy-chain gene assembly because some of the necessary cis-acting signals are missing. In contrast, both DJH rearrangements from a reclo cell line appeared normal in structure, indicating that trans-acting factors necessary for recombination might be missing. Introduction of the RAG-1 and RAG-2 genes, genes encoding two such factors, failed to confer a rechi phenotype to these cells. However, fusion of the reclo cells to a rechi cell line generated a high frequency of rechi hybrids. In addition, most of the hybrids rearranged the endogenous kappa light-chain locus. Neither the rechi phenotype nor kappa-chain rearrangement correlated with levels of RAG-1 and RAG-2 expression in all of the hybrids. Thus, both gene transfer and cell fusion experiments indicate that RAG-1 and RAG-2 are not sufficient to activate immunoglobulin gene recombination in at least some pre-B-cell lines. In addition, the fusion experiments suggest that two gene products in addition to RAG-1 and RAG-2 may be required for kappa-gene rearrangement.

RAG-1 and RAG-2 are not sufficient to direct all phases of immunoglobulin gene rearrangement in pre-B-cell lines.

To probe the factors controlling immunoglobulin heavy-chain gene rearrangement, we analyzed Abelson virus-transformed pre-B-cell lines that fail to undergo VH-to-DJH joining at an appreciable frequency. Despite this feature, some of these cell lines (rechi) rearrange an extrachromosomal recombination substrate at levels normal for transformed pre-B cells. Others (reclo) rearrange these substrates at levels characteristic of nonlymphoid hematopoietic cells. The DJH rearrangements from a representative rechi cell line were aberrant, suggesting that these cells probably fail to complete heavy-chain gene assembly because some of the necessary cis-acting signals are missing. In contrast, both DJH rearrangements from a reclo cell line appeared normal in structure, indicating that trans-acting factors necessary for recombination might be missing. Introduction of the RAG-1 and RAG-2 genes, genes encoding two such factors, failed to confer a rechi phenotype to these cells. However, fusion of the reclo cells to a rechi cell line generated a high frequency of rechi hybrids. In addition, most of the hybrids rearranged the endogenous kappa light-chain locus. Neither the rechi phenotype nor kappa-chain rearrangement correlated with levels of RAG-1 and RAG-2 expression in all of the hybrids. Thus, both gene transfer and cell fusion experiments indicate that RAG-1 and RAG-2 are not sufficient to activate immunoglobulin gene recombination in at least some pre-B-cell lines. In addition, the fusion experiments suggest that two gene products in addition to RAG-1 and RAG-2 may be required for kappa-gene rearrangement.

Two distinct mechanisms for the SCL gene activation in the t(1;14) translocation of T‐cell leukemias

Molecular study of a t(1;14)(p32;q11) translocation found in an acute T-cell leukemia (Kd cells) with a relatively mature phenotype is reported. Complex DNA rearrangements were characterized in the TCR alpha/delta locus. Besides a productive V alpha/J alpha assembly found on the normal allele, two deletions within the J alpha cluster were identified in the translocated allele. The translocation breakpoints involved the TCR delta gene on chromosome 14 and the SCL locus on chromosome band Ip32 that was recently shown to be activated by the t(1;14) translocation of the DU 528 leukemic cell line. Significantly, both Kd and DU 528 translocation breakpoints were located at the boundaries of D delta or J delta segments and were clustered in a 10 kb genomic fragment of the SCL gene. The presence of recombination signal motifs (heptamer-12/23 bp spacer-nonamer) on both normal chromosome partners, and N nucleotide addition on both derivative chromosomes involved the recombinase system in the translocation event. The SCL locus was highly expressed as a 5 kb transcript in Kd cells and, as already reported, as a 2 kb transcript in DU 528 cells. Importantly, a 5 kb SCL transcript was also detected in immature nonlymphoid hematopoietic cells but not in normal mature T cells, suggesting that it might correspond to the normal SCL transcript. Taken together, our data support the notion that the involvement of the SCL gene in the leukemogenic process may occur through overexpression of an apparently normal transcript (Kd cells) or expression of a truncated RNA (DU 528 cells).

Glycoproteins of 210,000 and 130,000 m.w. on activated T cells: cell distribution and antigenic relation to components on resting cells and T cell lines.

A glycoprotein complex of 210,000 and 130,000 m.w., found on mitogen or alloantigen-stimulated human T cells and not on other hematopoietic cells, has been defined by a monoclonal antibody (Mab). The components of this complex are a subset of a larger family of proteins (210,000, 165,000 and 130,000 m.w.) defined by a second Mab. In a panel of hematopoietic cell lines and cell types, only activated T cells (including the cell line HUT-102) express the 210,000/130,000 complex and these cells also express the IL 2 receptor, a characteristic marker for activated T cells. The 210,000/130,000 m.w. complex (reactive with the Mab TS2/7) is present on all long-term activated T cells, including both the OKT4 and OKT8 subsets. The 210,000 m.w. subunit is expressed only on activated T cells. Other lymphoid cells express either the 130,000 m.w. subunit alone (unactivated lymphocytes, thymocytes, HUT-78) or the 130,000 subunit together with a 165,000 subunit (MOLT-4, HSB, and other leukemic T cell lines). The 210,000/130,000 m.w., 165,000/130,000 m.w. and 130,000 m.w. complexes are antigenically related in that all share reactivity with the Mab A- 1A5 . Among non-lymphoid hematopoietic cells and cell lines, none express the 210,000 m.w. chain; adherent cells (monocytes) and myeloid cell lines each express single proteins of 130,000 to 155,000 m.w. Granulocytes and red blood cells are negative and platelets express multiple bands (165,000 and 140,000 m.w.). Immunoperoxidase staining of tissue sections showed that a broad range of tissues and cell types had material cross-reactive with the lymphoid 130,000 m.w. protein. However, only a discrete subset of those tissues and cells including blood vessel walls, connective tissue, smooth muscle, kidney mesangial cells, and some non-cellular matrix tissue, had material cross-reactive with the 210,000 m.w. protein on activated T lymphocytes.

A monoclonal antibody that recognizes B cells and B cell precursors in mice.

The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

Formation of B Lymphocytes in Fetal and Adult Life

Publisher Summary were regarded Lymphoid and hematopoietic stem cells are highly specialized and antigens recently identified by several laboratories may be useful in resolving categories and stages of differentiation. This chapter discusses some procedures that may not only allow the differentiative potential of B-cell precursors to be assessed but provide some insight into the inductive signals and regulatory mechanisms involved. The extraordinary progress of experimental hematologists in resolving the precursors of erythroid and myeloid cells has created a false sense of understanding about the nature and fate of stem cells relevant to the humoral immune system. The time when B cells or their precursors first appear during ontogeny or during regeneration of irradiated animals, they are diluted among nonlymphoid hematopoietic cells. The use of cytoplasmic immunoglobulin as a marker for pre-B cells and various cell surface antigens are discussed. Cells dissected from spleen colonies have been used to restore humoral immunity to irradiated secondary recipients, and the time required was because of transition of stem cells. Extensive examinations of spleen colonies have not revealed any that had predominantly lymphoid morphology. However, the possibility of a minor entry of stem cells into B and T pathways would be difficult to rule out, particularly if the newly formed cells rapidly migrated elsewhere.

Polycythemia vera. Increased expression of normal committed granulocytic stem cells in vitro after exposure of marrow to tritiated thymidine.

In previous studies of two patients with polycythemia vera (PV) and heterozygous at the X-linked locus for glucose-6-phosphate dehydrogenase (G-6-PD), only type A isoenzyme was found in non-lymphoid hematopoietic cells. However, some granulocytic and erythrocytic colonies grown in vitro had type B G-6-PD and therefore arose from presumably normal progenitors. In this study we exposed marrow cells from these same two patients to high-specific activity tritiated thymidine (3HTdR) before culture to kill cells actively synthesizing DNA. Individual granulocytic colonies were plucked and tested for G-6-PD after 14 d of culture. The frequency of type B colonies rose after exposure to 3HTdR from 8/101 to 11/36 in patient 1 and from 0/32 to 6/31 in patient 2 (P less than 0.003). No increase in the frequency of normal erythroid bursts after 3HTdR exposure was seen, implying that in PV, early granulopoiesis, and erythropoiesis are regulated differently. The results demonstrated that only type A granulocytic colonies, arising from the abnormal clone, were removed by the 3HTdR. In addition, for patient 2, statistical analysis indicated there was an absolute increase in normal granulocytic colonies detected in culture. Thus, PV clonal colony-forming units in culture (CFU-C) cycle more rapidly than do normal CFU-C and may suppress proliferation of normal CFU-C in vitro.