ALBERT

Description: P-selectin was shown to bind committed human hematopoietic progenitors (colony-forming unit-granulocyte-macrophage [CFU-GM] and burst-forming unit-erythroid [BFU-E]) as identified by their expression of the CD34 antigen and by in vitro clonogenic assays. In addition, P-selectin bound all precursors (pre-CFU) of committed myeloid progenitors assayed by their ability to sustain hematopoiesis in both conventional stroma-containing and stroma-free, cytokine-dependent systems. Binding of CD34+ cells to P-selectin was temperature-independent and shear-resistant, occurred only in the presence of divalent cations, was protease sensitive, and was completely blocked by anti-P-selectin antibody. Neuraminidase treatment of CD34+ cells completely abrogated their binding to P-selectin, implying a prominent role for sialic acid in the structure and function of the P-selectin ligand on hematopoietic progenitors. Monoclonal antibodies (MoAbs) CSLEX-1 and HECA-452, which identify carbohydrate epitopes involving sialic acid, bound to 33% and 35% of CD34+ cells, respectively, and included the majority of CFU-GM and pre-CFU. Three-color flow cytometric analysis showed a precise codistribution of CSLEX-1 and HECA-452 antigens on CD34+ cells, implying recognition of the same glycoprotein antigen by the two MoAbs. Treatment of CD34+ cells with neuraminidase completely abolished binding of both MoAbs. In addition, HECA-452 partially blocked the adhesion of CD34+ cells to P-selectin. P-selectin glycoprotein ligand (PSGL-1), recently molecularly cloned from the promyelocytic leukemia cell line HL60, was expressed by CD34+ cells as determined by reverse transcription polymerase chain reaction. Combined with the functional and biochemical characteristics, these data suggest that PSGL-1 may comprise an important P-selectin ligand expressed by primitive hematopoietic cells, but do not preclude the existence of additional P-selectin ligands on these cells.

Description: The selectins are lectin-like cell surface glycoproteins that have been implicated in playing a crucial role in the initiation of leukocyte adhesion to endothelial cells (ECs) during inflammation. Binding of selectins under conditions of flow mediates leukocyte rolling, which in vivo is almost exclusively observed in venular microvessels. We have shown in previous experiments that intraperitoneal treatment of rabbits with interleukin-1 beta (IL-1) increases leukocyte rolling in exteriorized mesenteries. In the present study, we used immunohistochemistry of mesenteries and found that IL-1 induced a marked E-selectin immunoreactivity, preferentially in venules. We therefore hypothesized that the increased rolling in response to IL-1 may be related to the induction of E-selectin on venular ECs. Intravital microscopy was used to investigate interactions between leukocytes and ECs after intraperitoneal application of IL-1. The rabbit E-selectin monoclonal antibody (MoAb) 9H9 significantly reduced rolling of leukocytes by approximately 40%. Vehicle alone, class- matched control MoAb or the nonblocking anti-E-selectin MoAb 14G2 had no effect on rolling. These results indicate that leukocytes roll on inflamed venular ECs partly through interactions with E-selectin. Furthermore, we propose that the restricted E-selectin immunoreactivity by venular ECs contributes to the remarkable difference seen between arterioles and venules in exhibiting leukocyte rolling in vivo.

Description: Circulating lymphocytes leave the blood by binding to specialized high endothelial cells lining postcapillary venules in lymphoid organs or sites of chronic inflammations, migrating through the vessel wall into the surrounding tissue. The capacity of lymphocytes to recognize and bind to high endothelial venules (HEVs) is thus central to the overall process of lymphocyte traffic and recirculation. We show that viable human lymphocytes bind selectively to HEVs in frozen sections of normal human lymph nodes, thus defining a simple in vitro model for the study of human lymphocyte homing properties. Optimal conditions for the quantitative analysis of lymphocyte-HEV interaction are described. Furthermore, by using this assay, we demonstrate that the ability of human lymphocyte populations to bind to HEVs parallels their presumed migratory status in vivo. Thus, thymocytes and bone marrow cells, which are sessile in vivo, bind poorly to HEVs in comparison with mature circulating lymphocytes in peripheral blood or in peripheral lymphoid tissues. These results indicate that HEV-binding ability is a regulated property of mature lymphocytes and, as demonstrated previously in animal models, probably plays a fundamental role in controlling lymphocyte traffic in humans. The in vitro model of lymphocyte-HEV interaction thus provides a unique means to assay the migratory properties of normal and neoplastic human lymphocyte subsets, to analyze the role of lymphocyte traffic mechanisms in normal and pathologic inflammatory reactions, and to define some of the molecular mechanisms responsible for the control of lymphocyte migration and positioning in humans.

Description: CD31 (PECAM-1) is an immunoglobulin gene superfamily cell adhesion molecule found on vascular endothelium, platelets, and leukocytes. Lymphocyte expression of CD31 is most closely associated with the CD45RA+CD8+ naive T phenotype. CD31 has recently been shown to play a role in leukocyte egress to inflammatory sites. The mechanism of CD31 adhesion remains under investigation. Several investigators have reported evidence for a heterotypic ligand. We have previously shown that CD31 is phosphorylated with cell activation, which suggests a possible role for CD31 in cell activation events. We therefore studied the effects of CD31 antibodies on in vitro assays of lymphocyte activation. One CD31 antibody, LYP21, inhibited the mixed lymphocyte reaction (MLR) in a specific and dose-dependent fashion. An LYP21 epitope was localized to the sixth Ig domain of CD31. This peptide and a scrambled control peptide were synthesized and used to study effects of this epitope on lymphocyte activation. The CD31 peptide strongly inhibited the MLR. Because CD31 is expressed on both stimulator and responder populations, stimulator peripheral blood leukocytes and responder lymphocyte populations were separately incubated with CD31 peptide or control peptide and then washed before mixing. The CD31 peptide inhibited the MLR equally when either stimulator or responder cells were preincubated with the CD31 peptide. We further sorted responder cells into CD31-high and CD31-low populations and separately incubated these subsets with peptides. The CD31 peptide strongly inhibited MLRs, regardless of level of responder-cell CD31 expression. Examination of MLR reactions involving the CD31 peptide showed dispersed small aggregates of cells, rather than the single large aggregate observed in control MLRs. The CD31 peptide did not affect activation of lymphocytes by phorbol myristate acetate (PMA) and ionomycin. These results suggest that a surface CD31-ligand interaction may have a functional role in alloimmune lymphocyte activation and identify a functionally important domain of CD31.

Description: P-selectin expressed on the surface of endothelium mediates leukocyte adhesion in vitro and rolling in vivo. Several inducers of cell-surface P-selectin expression on endothelial cells (EC) have previously been identified, all of which yield transient cell-surface expression of P- selectin lasting minutes to a few hours. We now show that a T- lymphocyte product, interleukin-3 (IL-3), stimulates the long-term endothelial cells (HUVEC). IL-3 induced cell-surface P-selectin expression in two phases. An initial peak at 10 minutes was followed by a prolonged upregulation beginning 16 hours after IL-3 addition and lasting at least 4 days. The level of P-selectin expression induced by IL-3 added for 48 hours was similar to that induced by treatment of HUVEC for 10 minutes with thrombin, and the effect of adding IL-3 for 48 hours followed by thrombin for 10 minutes was additive. Induction of cell-surface P-selectin expression by IL-3 was blocked by pretreatment of EC with a blocking monoclonal antibody against the IL-3 receptor alpha-chain. Lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) and a mutant form of IL-3 with decreased potency did not induce cell-surface P-selectin expression after 48 hours' incubation with HUVEC, suggesting that the effect was specific to IL-3. The increase in cell-surface P-selectin expression occurring after 16 hours of incubation with IL-3 was accompanied by a similar prolonged increase in the steady-state mRNA level that was not observed at 10 minutes after IL-3 addition. As T-lymphocyte infiltration is a hallmark of chronic inflammation, our observations suggest that the secretion of IL-3 by T lymphocytes may serve to maintain the inflammatory state during chronic inflammation.

Description: Chemerin is a potent chemoattractant for cells that express the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. Chemerin circulates in the blood in a relatively inactive form (prochemerin). Its chemotactic activity is unleashed following proteolytic cleavage of carboxyl-terminal amino acids by serine proteases including plasmin, factor XIIa and VIIa. Recruitment of plasmacytoid dendritic cells and macrophages by chemerin may play a role in local tissue immune and inflammatory responses. The shortest bioactive chemerin peptide NH2-YFPGQFAFS-COOH (9mer) is present in the carboxyl-terminal domain. In this work, we show that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activatable fibrinolysis inhibitor (TAFIa)) (30 nM) remove the C-terminal lysine (K) from YFPGQFAFSK (10mer) (200nM) and enhance the migration of CMKLR1-transfected cells by ∼16-fold. To investigate if sequential proteolysis by plasmin and carboxypeptidases can modulate the activity of chemerin peptides, we generated the carboxyl-terminal 15mer of prochemerin, NH2-YFPGQFAFSKALPRS-COOH. Plasmin cleavage (1 μM) generated a 10mer, which was further processed to 9mer by CPN or CPB (30 nM) cleavage. These sequential cleavages were paralleled by corresponding increases in chemotactic activity. At concentrations as high as 1 μM the 15 mer did not induce chemotaxis; after plasmin cleavage and conversion to 10mer, however, significant chemotactic activity was demonstrated. Treatment with CPN or CPB further enhanced this chemotactic activity. We observed a similar enhancement in bioactivity following sequential plasmin/CPN-or-CPB cleavage of full-length prochemerin. Endogenous plasma CPN supports the activation (∼2.5 fold increase in bioactivity) of plasmin-cleaved prochemerin (0.2 nM). This activation was blocked by incubating plasma with MGTA (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid), a specific CPN inhibitor. In addition, we show that platelets are a rich source of chemerin (20–30 ng of chemerin/5x108 platelets). Chemerin released from activated platelets triggers CMKLR1-transfectant chemotaxis, which was blocked by anti-chemerin antibodies. Thus, the circulating humoral factors reported here (platelets, serine proteases, and carboxypeptidases) may contribute to the regulation of chemerin bioactivity in vivo and therefore play a critical role in CMKLR1-mediated immune responses.

Description: The hematopoietic stem and progenitor cell (HSPC) niche is a supportive microenvironment comprised of distinct cell types, including specialized vascular endothelial cells (ECs) that directly interact with HSPCs and promote stem cell function. Utilizing spatial transcriptomics, in combination with tissue-specific RNA-seq, we identified 29 genes selectively enriched in ECs of the zebrafish fetal hematopoietic niche. Using upstream regulatory sequences for two of these genes, mrc1a and selectin E (sele), we generated GFP reporter lines that allowed us to selectively isolate niche ECs for ATAC-seq. This analysis identified 6,848 regions of chromatin that were accessible in niche ECs but not ECs from other tissues. Several of these regions were associated with the 29 genes. To evaluate whether these regions might be enhancers we coupled them to GFP and injected them into embryos. 12/15 sequences drove GFP expression in niche ECs. Upon closer examination of the mrc1a and sele genes, we identified enhancer sequences as short as 125 bp and 158 bp, respectively, which drove niche EC-specific expression. A genome-wide motif enrichment analysis of the 6,848 uniquely open chromatin regions revealed that Ets, SoxF and Nuclear Hormone Receptor (RXRA/NR2F2, specifically) sites were most enriched. In contrast, 4,522 pan-EC elements were enriched for Ets sites but not SoxF or NHR motifs. Using mutant variants of the 125 bp and 158 bp enhancer sequences, we demonstrated that Ets, SoxF and RXRA/NR2F2 sites were independently required for specific transgene expression. Gel shift experiments demonstrated that NR2F2 could bind the 125 bp and 158 bp zebrafish enhancers and this binding was disrupted upon mutation of the NR2F2 binding sites. Knockdown of the endogenous zebrafish nr2f2 gene resulted in a loss of expression of the 125 bp mrc1a enhancer-GFP construct and a significantly reduced number of HSPCs in the fetal niche. We next injected pools of human transcription factors, including at least one member from each of the three families, under the control of a ubiquitous promoter. Strikingly, we found that a combination of ETV2 or ETS1 with SOX7 and NR2F2 generated ectopic patches of mrc1a+ niche ECs that recruited runx1+ HSPCs outside of the endogenous niche. Using high-resolution live cell imaging we could observe HSPCs initially arriving at the ectopic sites, lodging for several hours and then eventually dividing and migrating away from the site through circulation. HSPCs localized to the ectopic regions were found in both intravascular and extravascular spaces, and were often enwrapped by ECs and in contact with cxcl12a+ stromal cells, similar to what is observed in the endogenous niche. Ectopic regions of niche EC gene expression were similarly observed when alternative regulatory elements were used for transcription factor overexpression, including a pan-EC enhancer (nrp1b), a muscle promoter (mylz2) and a heat shock promoter (hsp70). These results suggest the three-factor combinations are sufficient to reprogram niche EC identify in vivo. Lastly, we evaluated by RNA-seq the expression of our niche EC signature in the zebrafish kidney marrow (the site of adult hematopoiesis) and in ECs from multiple organs of the mouse, including the heart, kidney, liver, lung and bone marrow, at multiple stages of development (E11-13, E14-15, E16-17, P2-P4 and adult). Strikingly, 23/29 genes were highly expressed in ECs of the zebrafish kidney and 21/29 genes were enriched in the ECs of a mammalian hematopoietic organ - the fetal liver and/or adult bone marrow - relative to their expression in ECs from non-hematopoietic organs at the same stage. Notably, for a subset of the genes the expression patterns mirrored the temporal dynamics of HSPC ontogeny in the mouse, showing robust expression in fetal liver ECs and then later in adult bone marrow ECs with a concomitant reduction in liver ECs. An analysis of transcription factor expression within these EC populations revealed that Ets1, the SoxF factor Sox18, and Nr2f2 were the most highly expressed members of the Ets, Sox and NHR families. Collectively our work has uncovered a conserved gene expression signature and transcriptional regulatory program unique to the vascular niche of hematopoietic organs. These findings have important implications for designing a synthetic vascular niche for blood stem cells or for modulating the niche in a therapeutic context. Disclosures Zon: Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership; CAMP4: Equity Ownership.

Description: Abstract LBA-3 Mutations in the transcription factor genes, RUNX1 and CEBPA, can lead to an autosomal dominant familial predisposition to MDS/AML. Using a candidate gene approach, we have detected domain specific heterozygous mutations in the GATA2 gene in 4 MDS/AML families which predispose to MDS/AML. The same novel heterozygous T354M missense mutation was observed in 3 families and a 355delT mutation in 1 family, all with multigenerational transmission of MDS and/or MDS/AML. Importantly, these genetic variants segregate with all affected members in each of the families. The 2 mutated threonine residues are in 5 consecutive highly conserved threonine residues at the DNA-binding, protein-protein interacting second zinc finger (ZF2) of GATA2. Neither these mutations, nor any other variants in the GATA2 coding sequence, were seen in a population screen of 695 normal individuals. Haplotype analysis suggests that the T354M mutation has multiple ancestral origins. While mutations in RUNX1 and CEBPA, can also lead to familial predisposition to MDS/AML, these patients with GATA2 mutations are unique in that there is no obvious pre-MDS or pre-leukaemic phenotype such as thrombocytopenia (RUNX1) and eosinophilia (CEBPA) in predisposed carriers. Most patients in these families have had a rapid disease course “appearing out of the blue” leading to death, with a variety of ages of onset from teenagers to early 40s. Yet remarkably, there are still asymptomatic carriers in their 60s. One of these carriers, and his 2 children, has had bone marrow prophylactically stored over 15 years ago in case of disease onset. No pathogenic GATA2 coding sequence changes were found in 268 sporadic MDS/AML patient samples. Additionally, GATA2 mutations were not found in germline samples from 35 other families predisposed to AML and various other hematological malignancies. Both the T354M and 355delT mutants appear to localize appropriately to the nucleus and maintain at least some DNA binding in electrophoretic mobility shift assays. We used the known murine Gata3 ZF2 structure bound to DNA to model the effects of the observed mutations and demonstrated that the T354 residue does not contact DNA but makes polar contact with the adjacent threonines, and via its amino group, with C349 which coordinates the zinc atom. Replacement of the T354 side-chain with the bulky methionine moiety may affect the zinc contacts and is predicted to alter the overall structure of this ZF2. In contrast, 355delT will shorten the conserved threonine string which is predicted to impact on the orientation and position of L359 which directly contacts DNA. Thus, 355delT is likely to have an effect on DNA binding. Luciferase reporter assays indicate that T354M and 355delT greatly reduce the transactivation ability of GATA2 on multiple response elements, impacting on downstream target genes such as RUNX1 and CD34. Of note, T354M shows a markedly lesser synergistic effect than wildtype (WT) GATA2 with PU.1 on the CSF1R promoter. Competition assays show that these mutations may be acting in a dominant negative fashion in some biological contexts. In stable promyelocytic HL-60 cell lines expressing regulatable GATA2 (WT or T354M), T354M allows proliferation to proceed even under stimulus to differentiate with all-trans retinoic acid. Microarray studies indicated that the down regulation of proapoptotic BCL-xS by T354M, but not WT, may be responsible for this phenotype. GATA2 is considered to be a hematopoietic “stemness” gene, highly expressed in haematopoietic stem cells and is required for megakaryocyte and mast cell production. GATA2 is down regulated during myeloid differentiation and forced overexpression prevents such differentiation. Discovery of GATA2 mutants in MDS/AML predisposed families provides new tools for probing the mechanism of GATA2 induced leukemogenesis, and possibly also for clarifying its role in maintenance of stemness. Our findings highlight the power of investigating familial predispositions to cancer identifying specific mutations with unique biological effects. They have immediate implications for diagnostic genetic testing, and longer term therapeutic implications through identification of drugable biological pathways such as apoptosis. The poor outcome associated with these mutations may suggest that an aggressive strategy is appropriate in the treatment of affected individuals in families found to be carrying GATA2 mutations. Disclosures: No relevant conflicts of interest to declare.

Description: The selectins are lectin-like cell surface glycoproteins that have been implicated in playing a crucial role in the initiation of leukocyte adhesion to endothelial cells (ECs) during inflammation. Binding of selectins under conditions of flow mediates leukocyte rolling, which in vivo is almost exclusively observed in venular microvessels. We have shown in previous experiments that intraperitoneal treatment of rabbits with interleukin-1 beta (IL-1) increases leukocyte rolling in exteriorized mesenteries. In the present study, we used immunohistochemistry of mesenteries and found that IL-1 induced a marked E-selectin immunoreactivity, preferentially in venules. We therefore hypothesized that the increased rolling in response to IL-1 may be related to the induction of E-selectin on venular ECs. Intravital microscopy was used to investigate interactions between leukocytes and ECs after intraperitoneal application of IL-1. The rabbit E-selectin monoclonal antibody (MoAb) 9H9 significantly reduced rolling of leukocytes by approximately 40%. Vehicle alone, class- matched control MoAb or the nonblocking anti-E-selectin MoAb 14G2 had no effect on rolling. These results indicate that leukocytes roll on inflamed venular ECs partly through interactions with E-selectin. Furthermore, we propose that the restricted E-selectin immunoreactivity by venular ECs contributes to the remarkable difference seen between arterioles and venules in exhibiting leukocyte rolling in vivo.

Description: The guanosine triphosphatases (GTPases) of the immunity-associated protein (GIMAP) family of putative GTPases has been implicated in the regulation of T-lymphocyte development and survival. A mouse conditional knockout allele was generated for the immune GTPase gene GIMAP1. Homozygous loss of this allele under the influence of the lymphoid-expressed hCD2-iCre recombinase transgene led to severe (〉 85%) deficiency of mature T lymphocytes and, unexpectedly, of mature B lymphocytes. By contrast there was little effect of GIMAP1 deletion on immature lymphocytes in either B or T lineages, although in vitro studies showed a shortening of the survival time of both immature and mature CD4+ single-positive thymocytes. These findings show a vital requirement for GIMAP1 in mature lymphocyte development/survival and draw attention to the nonredundant roles of members of the GIMAP GTPase family in these processes.