Normal Homing Research Articles

Analysis of time sequence and load of pulling rope mechanism of type D-knotter

To solve the problem that the rope pulling plate may be delayed in the actual work of the knotter, the influence of the change of the timing of the rope pulling mechanism on the knotter forming performance was studied and analyzed. The dynamic model of the flexible bundling of the knotting machine is established using the Bushing connection of RecurDyn. The timing of the bundling machine homing is controlled by modifying the driving function of the bundling machine, and the positive pressure of the hook pliers is obtained by measuring the contact force between the bundle and the hook pliers. The results of the contact force curve show that the maximum positive pressure of the hook pliers for the normal homing of the rope plate is 290.43 N. The maximum value of the sudden change of tension can reach 837.17 N, which proves that the change in the timing of the rope pulling mechanism has a significant influence on the positive pressure of the hook pliers.

IFNg Increases HSC Homing and Induces Relocalization within the Bone Marrow Niche

Infections affect hematopoietic stem cell (HSC) biology, with significant implications in ageing, cancer development, and bone marrow failure syndromes. We previously showed that some infections induce HSCs to proliferate through a cell autonomous response to interferon gamma (IFNg). Given that interactions of HSCs with their bone marrow niche are a critical factor in maintaining quiescence, we considered how IFNg affects the interaction between HSCs and the niche. We hypothesized that infection would displace HSCs from the niche and reduce homing in an IFNg-dependent manner, thereby contributing to activation.We used a Mycobacterium avium model of infection to characterize the effects of infection on HSC homing. In this model HSCs are activated to divide and differentiate via an IFNg-dependent mechanism with depletion of HSCs after several months of infection. We compared the homing of hematopoietic stem and progenitor cells (HSPCs) from M. avium infected versus non-infected animals by IV administration of CSFE-labeled cKit+ cells into lethally irradiated recipient mice. Homing was determined by the frequency with which labeled cells could be found in the bone marrow 17 hours later, relative to a DDAO-stained internal control. Surprisingly, HSPCs from M. avium -infected animals displayed improved homing compared to control HSPCs. A similar phenotype was seen for cKit+ cells isolated from mice treated with recombinant IFNg, whereas cKit+ cells from Ifngr1-/- mice showed decreased homing compared to wildtype. Using transplantation assays, we found decreased engraftment of Ifngr1-deficient whole bone marrow when cells were administered intravenously; but engraftment was normal when cells were infused intrafemorally. Altogether, these findings show that basal IFNg signaling contributes to normal HSPC homing and that IFNg induced during M. avium infection increases homing to the bone marrow.The above studies reflect homing of HSPCs to the bone marrow, not cellular interactions within the niche. Since proximity to Cxcl12-expressing cells in the perivascular niche can affect HSC activation status, we used 3-D intravital imaging to study the effects of IFNg on HSC-niche interactions. We injected CMTMR-stained HSCs into Cxcl12-GFP transgenic mice and measured the distance of the HSCs from quiescence-associated Cxcl12-abundant reticular (CAR) cells. CMTMR-stained HSCs were seen moving upon IFNg treatment, and 24hrs post-IFNg HSCs were located significantly further away from CAR cells compared to untreated or TGF-b treated controls. These results suggest that IFNg can induce movement away from CAR cells, thereby allowing HSC proliferation during inflammatory conditions.To define the mechanism by which IFNg causes HSC relocalization, we performed gene expression profiling of HSCs from control or IFNg-treated mice. Gene set enrichment analysis of differentially regulated genes showed modulation of the expected immune and cytokine response pathways, but also genes related to adhesion and extracellular matrix remodeling. Of note, the glycoprotein Bst2 (Tetherin) was upregulated in HSCs following IFNg treatment. Bst2 has been shown to bind to E-selectin, and localization to E-selectin+ endothelial cells is associated with increased HSC proliferation. Thus we hypothesized that IFNg-induced upregulation of surface Bst2 may facilitate proliferation by promoting HSC binding to E-selectin. We confirmed induction of Bst2 mRNA and protein by qPCR and flow cytometry, and we found that Bst2 was upregulated following M. avium infection via an IFNg-dependent mechanism. In vitro binding assays showed increased HSPC binding to recombinant E-selectin following IFNg treatment. Thus, IFNg-dependent cell surface expression of Bst2 may facilitate the displacement of HSCs from the quiescence-enforcing CAR niche to an active E-selectin-positive niche. Because E-selectin expression along blood vessel walls is also important for adhesion and transendothelial migration of HSCs, increased Bst2 on HSCs may also contribute to more efficient homing. In summary, IFNg improves HSPC homing but displaces HSCs from quiescence-enforcing CAR cells in the niche, potentially by inducing Bst2-mediated movement to the E-selectin rich proliferative niche. Future studies will ascertain whether blocking Bst2 is a feasible method to protect HSCs from the deleterious effects of chronic inflammation. DisclosuresNo relevant conflicts of interest to declare.

Unexpected Novel Findings That Caspase-1-KO Mice Are Poor Mobilizers and Engraft Poorly with Wild Type Bone Marrow Cells - Indicating a Presence of an Autocrine Feedback Loop Involving Interleukin 1b and Interleukin 18 Signaling That Potentiates Nlrp3 Inflammasome Activity, Both in HSPCs and in the BM Microenvironment for Optimal Stem Cell Trafficking

Background . Our recent results indicate that Nlrp3 inflammasome expressed in hematopoietic stem/progenitor cells (HSPCs) and in the bone marrow (BM) microenvironment are required for optimal mobilization of murine cells (Leukemia 2020 Jun;34(6):1512-1523, Stem Cell Rev Rep. 2019 Jun;15(3):391-403). Moreover, Nlrp3 inflammasome expressed in these cellular contexts are also required for normal homing and engraftment after hematopoietic transplantation (Stem Cell Rev and Rep (2020) in press. https://doi.org/10.1007/s12015-020-10005-w). Consistent with these results, Nlrp3-KO mice are poor mobilizers, cells from Nlrp3-KO mice engraft poorly, and, as transplant recipients, Nlrp3-KO mice show a delay in hematopoietic recovery after transplantation of normal BM cells. Activation of Nlrp3 inflammasomes leads to the release in caspase-1-dependent manner from HSPCs and cells in the BM microenvironment of two potent pro-inflammatory cytokines, interleukin 1b (IL-1b) and interleukin 18 (IL-18). Hypothesis. Based on the aforementioned results, we envisioned that the activated Nlrp3 inflammasome caspase-1 autocrine feedback loop involving IL-1bor IL-18 signaling potentiates HSPCs trafficking by amplifying Nlrp3 inflammasome activation in HSPCs and the BM microenvironment.Materials and Methods. We employed normal control and caspase-1-KO mice for mobilization and homing/engraftment experiments. Mice were mobilized with G-CSF or AMD3100 in the absence or presence of administered IL-1b or IL-18, and we measured i) the total number of white blood cells (WBCs) and ii) the number of clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. Next, caspase-1-KO cells were transplanted into lethally irradiated wild type animals, while caspase-1-KO mice were transplanted with wild type BMMNCs. We then evaluated homing and engraftment by measuring the number of PKH27-labeled cells, the number of clonogenic progenitors 24 hours after transplantation, and the numbers of day-12 CFU-S colonies and day-12 CFU-GM progenitors in the BM of recipient mice. In control experiments, to provide proof of our hypothesis, we also mobilized Nlrp3-KO mice with AMD3100 or G-CSF in the presence of administered IL-1b and IL-18. Results. We found that IL-1b or IL-18 alone mobilizes HSPCs and, significantly, that caspase-1-KO mice were poor mobilizers. Radiation chimera experiments revealed that this pro-mobilizing defect is dependent on the lack of caspase-1 in hematopoietic cells. To our surprise, administration of IL-1b and IL-18 did not improve G-CSF- or AMD3100-induced mobilization in Nlrp3-KO animals. We also found that both of these pro-inflammatory cytokines activate Nlrp3 inflammasomes in an Myd88-dependent manner. Moreover, HSPCs from caspase-1-KO mice show defective migration in response to a BM-released SDF-1 gradient and other supportive homing chemoattractants (sphingosine-1 phosphate; S1P and extracellular adenosine triphosphate; eATP). Finally, caspase-1-KO mice recipients were also engrafted poorly with normal HSPCs. To explain this phenomenon, we observed that the Nlrp3 inflammasome-caspase-1-IL-1b axis is important in upregulating SDF-1 and other key HSPC chemoattractants in the BM microenvironment conditioned for transplantation. Conclusions. We demonstrate for the first time that an Nlrp3 inflammasome-caspase-1-IL-1b/IL18 autocrine feedback mechanism operating in HSPCs and the BM microenvironment is involved in the normal trafficking of HSPCs during mobilization and homing/engraftment. This research demonstrates the unexpected role of caspase-1, which is situated downstream of the Nlrp3 inflammasome and directs the release of the potent pro-inflammatory cytokines IL-1b and IL18 that by feedback mechanism, in turn, activate Nlrp3 inflammasomes in HSPCs and the BM microenvironment, which is required for optimal trafficking of HSPCs. These results are important for understanding the novel casapase-1-mediated autocrine mechanisms involved not only in pharmacological mobilization but also in the inflammatory response of the organism to external challenges, such as infection and tissue/organ damage. Disclosures No relevant conflicts of interest to declare.

Distinct integrin activation pathways for effector and regulatory T cell trafficking and function.

Integrin activation mediates lymphocyte trafficking and immune functions. Conventional T cell (Tconv cell) integrin activation requires Rap1-interacting adaptor molecule (RIAM). Here, we report that Apbb1ip-/- (RIAM-null) mice are protected from spontaneous colitis due to IL-10 deficiency, a model of inflammatory bowel disease (IBD). Protection is ascribable to reduced accumulation and homing of Tconv cells in gut-associated lymphoid tissue (GALT). Surprisingly, there are abundant RIAM-null regulatory T cells (T reg cells) in the GALT. RIAM-null T reg cells exhibit normal homing to GALT and lymph nodes due to preserved activation of integrins αLβ2, α4β1, and α4β7. Similar to Tconv cells, T reg cell integrin activation and immune function require Rap1; however, lamellipodin (Raph1), a RIAM paralogue, compensates for RIAM deficiency. Thus, in contrast to Tconv cells, RIAM is dispensable for T reg cell integrin activation and suppressive function. In consequence, inhibition of RIAM can inhibit spontaneous Tconv cell-mediated autoimmune colitis while preserving T reg cell trafficking and function.

Abstract B42: Impact of EZH2 inhibition on the immune microenvironment of squamous lung cancers

Abstract Squamous cell carcinoma (SCC) is a subtype of non-small cell lung cancer (NSCLC) that often resists treatment, resulting in poor prognosis for the patient. Immunotherapies, including those targeting the PD1/PD-L1 immune checkpoint, have become popular treatment options, but they are effective for only 20% of squamous lung cancer patients. The likely cause of differential responses is tumor heterogeneity. This heterogeneity can present in the tumor cells, for example, if more or fewer tumor cells express PD-L1, and also in the microenvironment, for example, if the tumor is infiltrated by more immune-suppressive neutrophils or more immune-activating CD8+ T cells. In order to study SCC in immune-intact mice, our laboratory utilizes a mouse model whereby tumors are induced through biallelic deletion of Lkb1 (aka Stk11) and Pten in the mouse lung. These SCCs express PD-L1 at high levels on the surface of the tumor cells; however, treatment with anti-PD1 antibody caused only a delay in tumor growth, not a reduction. We believe the ineffectiveness of immunotherapy in this model is due to the presence of large populations of tumor-associated neutrophils (TANs). TANs have been shown to produce Arginase1, which has repressive effects on the T-cell populations by inhibiting proper signaling and activation. An inverse relationship between TAN and CD8+ T cell abundance has also been reported for squamous lung cancers. Finding a second therapy that can prime squamous lung cancers to respond to immunotherapy would be beneficial for a large number of patients. EZH2 is a histone methyl transferase that promotes differentiation of cells via lineage-specific gene repression. We hypothesize that using an anti-PD-1 immunotherapeutic in combination with an EZH2 inhibitor will increase the PD-L1 expression on the tumor cells, decrease the normal homing or T-cell suppression of neutrophil populations, and increase the efficacy of the immunotherapy. In order to test our hypothesis, we have treated lung cancer cells with EZH2 inhibitor both in vitro and in vivo. In vitro, treatment of lung cancer cell lines or patient-derived organoids of squamous lung cancers caused increased expression of PD-L1. In mice harboring Lkb1/Pten tumors, treatment with EZH2 inhibitor increased the proportion of tumor cells expressing PD-L1 but did not prevent the tumor from growing. In contrast, the mouse treated with EZH2 inhibitor and anti-PD-1 immunotherapy had tumor regression that was durable up to 8 weeks post-treatment initiation. We isolated TANs from untreated tumors and observed that they express very high levels of Arginase 1. Lung neutrophils isolated from EZH2 conditional knockout mice had significantly lower levels of Arginase 1 than WT lung neutrophils or Lkb1/Pten TANs. Together these data suggest that EZH2 inhibition will be an effective strategy to both increase the PD-L1+ tumor cell population, and to decrease TAN-mediate T cell suppression by decreasing TAN-derived Arginase 1 expression. Citation Format: Tanner J. DuCote, Xiulong Song, Christine F. Brainson. Impact of EZH2 inhibition on the immune microenvironment of squamous lung cancers [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B42.

The CXCL12-CXCR4/CXCR7 axis as a mechanism of immune resistance in gastrointestinal malignancies.

Single agent checkpoint inhibitor therapy has not been effective for most gastrointestinal solid tumors, but combination therapy with drugs targeting additional immunosuppressive pathways is being attempted. One such pathway, the CXCL12-CXCR4/CXCR7 chemokine axis, has attracted attention due to its effects on tumor cell survival and metastasis as well as immune cell migration. CXCL12 is a small protein that functions in normal hematopoietic stem cell homing in addition to repair of damaged tissue. Binding of CXCL12 to CXCR4 leads to activation of G protein signaling kinases such as P13K/mTOR and MEK/ERK while binding to CXCR7 leads to β-arrestin mediated signaling. While some gastric and colorectal carcinoma cells have been shown to make CXCL12, the primary source in pancreatic cancer and peritoneal metastases is cancer-associated fibroblasts. Binding of CXCL12 to CXCR4 and CXCR7 on tumor cells leads to anti-apoptotic signaling through Bcl-2 and survivin upregulation, as well as promotion of the epithelial-to-mesechymal transition through the Rho-ROCK pathway and alterations in cell adhesion molecules. High levels of CXCL12 seen in the bone marrow, liver, and spleen could partially explain why these are popular sites of metastases for many tumors. CXCL12 is a chemoattractant for lymphocytes at lower levels, but becomes chemorepellant at higher levels; it is unclear exactly what gradient exists in the tumor microenvironment and how this influences tumor-infiltrating lymphocytes. AMD3100 (Plerixafor or Mozobil) is a small molecule CXCR4 antagonist and is the most frequently used drug targeting the CXCL12-CXCR4/CXCR7 axis in clinical trials for gastrointestinal solid tumors currently. Other small molecules and monoclonal antibodies against CXCR4 are being trialed. Further understanding of the CXCL12- CXCR4/CXCR7 chemokine axis in the tumor microenvironment will allow more effective targeting of this pathway in combination immunotherapy.

IL-7-dependent maintenance of ILC3s is required for normal entry of lymphocytes into lymph nodes.

IL-7 is essential for the development and homeostasis of T and B lymphocytes and is critical for neonatal lymph node organogenesis because Il7-/- mice lack normal lymph nodes. Whether IL-7 is a continued requirement for normal lymph node structure and function is unknown. To address this, we ablated IL-7 function in normal adult hosts. Either inducible Il7 gene deletion or IL-7R blockade in adults resulted in a rapid loss of lymph node cellularity and a corresponding defect in lymphocyte entry into lymph nodes. Although stromal and dendritic cell components of lymph nodes were present in normal numbers and representation, innate lymphoid cell (ILC) subpopulations were substantially decreased after IL-7 ablation. Testing lymphocyte homing in bone marrow chimeras reconstituted with Rorc-/- bone marrow confirmed that ILC3s in lymph nodes are required for normal lymphocyte homing. Collectively, our data suggest that maintenance of intact lymph nodes relies on IL-7-dependent maintenance of ILC3 cells.

Novel Evidence That Hematopoietic-Specific PLC-β2 Is Required for Normal Homing and Engraftment of Hematopoietic Stem Cells

Background. We recently identified phospholipase C-β2 (PLC-β2) as the first lipolytic enzyme known to be involved in the mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB) (Leukemia 2016, 4:919-28). The phospholipase C (PLC) family of enzymes consists of 13 members in six subfamilies, including the PLC-δ (1, 3, 4), -β (1-4), -γ (1, 2), -ε, -ζ, and -η isoforms. PLC enzymes are associated with cell surface receptors that convert phosphatidyloinositol-4,5-biphosphate into two important second messengers, diacylglycerol (DAG) and inositol-1,4.5-triphosphate (IP3). Among these isoforms, PLC-β2 is somewhat unique in being a hematopoietic-specific enzyme. Moreover, PLC-β2, when released extracellularly from granulocytes and HSPCs upon stimulation, cleaves glycolipid glycosylphosphatidylinositol anchor (GPI-A) in cell membranes and thus disrupts the structure of membrane lipid rafts, which are important in the retention of HSPCs in BM niches. It is well known that the major BM-retention receptors for HSPCs, CXCR4 and VLA-4, are membrane lipid raft-associated receptors. What is important for the current study, while performing mobilization studies, we previously found that BM cells from these animals show somewhat reduced chemotaxis in response to several chemoattractants involved in BM homing.Aim of the Study. Based on this latter observation, we became interested in the potential role of PLC-β2 in regulating the migration of HSPCs, as this enzyme could be potentially involved in BM homing and engraftment of HSPCs after transplantation. We also explored the potential relationship between PLC-β2 and heme oxygenase 1 (HO-1), which is, as we recently demonstrated, a negative regulator of HSPC trafficking (Cell Transplant. 2016 epub).Materials and Methods. We performed i) homing, ii) short-term engraftment, and iii) long-term competitive engraftment studies in WT mice transplanted with BM cells derived from PLC-β2-/- mice and control (WT) animals. Chemotaxis and adhesion of PLC-β2-/- HSPCs was evaluated in Transwell dishes in response to SDF-1, S1P, C1P, and ATP. The expression of HO-1 was evaluated by real-time PCR and western blotting. Finally, both HSPCs and Gr-1+ cells were evaluated for release of Ca2+ upon stimulation by homing factors.Results. We report here for first time that, besides its role in the release of HSPCs from BM niches, PLC-β2 also regulates the migration of HSPCs in response to chemotactic gradients of BM homing factors, including stromal derived factor-1 (SDF-1), sphingosine-1 phosphate (S1P), ceramide-1 phosphate C1P, and adenosine triphosphate (ATP). Specifically, HSPCs from PLC-β2-KO mice show impaired homing and engraftment in vivo after transplantation into lethally irradiated mice. This decrease in migration of HSPCs can be explained by impaired calcium release and calcium signaling in cells from PLC-β2-KO mice and a high baseline level of HO-1, an enzyme that negatively regulates cell migration, in PLC-β2-KO mouse cells. Downregulation of HO-1 activity in HSPCs improved homing of PLC-β2-/- BM cells.Conclusions. PLC-β2 plays an important role in homing and engraftment of HSPCs. The decrease in migration of HSPCs in PLC-β2-KO mice can be explained by impaired calcium release and a high baseline level of HO-1, an enzyme that negatively regulates cell migration and our data provides evidence that downregulation of HO-1 activity in HSPCs by small molecular inhibitors could become a new strategy to improve homing and engraftment after transplantation. DisclosuresNo relevant conflicts of interest to declare.

Lymphocyte recruitment via high endothelial venules in lymphoid stroma of Warthin’s tumour

Warthin's tumour is composed of bilayered oncocytic epithelium and organised lymphoid stroma, which resembles mucosa-associated lymphoid tissue (MALT); however, the histogenesis of the lymphoid stroma is not fully understood. We hypothesised that lymphocytes consisting of the stroma are recruited via high endothelial venules (HEVs) by the mechanism operating in normal lymphocyte homing in secondary lymphoid organs. The aim of this study was to determine immunohistochemically the molecules expressed on these HEVs. Tissue sections of Warthin's tumour (n = 10) were immunostained for vascular addressin-related antigens including peripheral lymph node addressin (PNAd) and mucosal addressin cell adhesion molecule 1 (MAdCAM-1). An L-selectin·IgM chimera in situ binding assay was also carried out. Triple immunostaining for PNAd, CD3, and CD20/CD79α was performed to determine which lymphocyte subsets are closely associated with these HEVs. HEVs in the lymphoid stroma of Warthin's tumour express PNAd, which is detected by MECA-79 as well as recently developed monoclonal antibodies S1 and S2. These HEVs were bound by L-selectin·IgM chimeras in a calcium-dependent manner, and numbers of lymphocytes, particularly T cells, attached to these HEVs. The lymphoid stroma of Warthin's tumour is most likely developed by lymphocytes recruited via HEVs.

Smurf1 ubiquitin ligase targets Kruppel-like factor KLF2 for ubiquitination and degradation in human lung cancer H1299 cells

Krüppel-like factor 2 (KLF2) has been demonstrated to be essential for normal lung development, erythroid differentiation, T-cell differentiation, migration and homing. However, the mechanisms underlying the regulation of KLF2, in particular its responsible E3 ligase is still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets KLF2 for poly-ubiquitination and proteasomal degradation specifically in lung cancer H1299 cells. The catalytic ligase activity of Smurf1 is required for it to regulate KLF2. Consequently, Smurf1 represses the transcriptional factor activity of KLF2 and regulates the expression its downstream genes such as CD62L and Wee1. This study provided the first evidence that Smurf1 functions as an E3 ligase to promote the ubiquitination and proteasomal degradation of KLF2.

Hypermethylation Is A Key Feature of the Transition of Multiple Myeloma to Plasma Cell Leukemia

AbstractAbstract 535PCL is associated with a failure of the plasma cells to retain their normal homing patterns to the bone marrow, is difficult to treat and developing targeted treatments based on its pathogenesis would be a step forward. PCL lies at the end of the multistep pathway from normal to malignant plasma cells and little is known about the genetic mechanisms mediating the final stages of this pathway. The methylation status of genes in myeloma can change as the malignancy progresses and as such identifying genes deregulated by methylation that mediate the progression of MM to PCL may offer epigenetically relevant therapeutic targets. We have previously analyzed 181 samples including B cell, normal plasma cells, MGUS and MM samples for methylation differences using the Illumina Infinium humanmethylation27 array, which interrogates 27,578 highly informative CpG sites per sample at the single-nucleotide resolution using bisulfite converted DNA. Here we have added 32 PCL samples to our dataset in order to further investigate the methylation changes that occur on the transition from MM to PCL. Data are presented as an average beta-score where 1.0 is fully methylated and 0 is fully unmethylated. Data were analyzed in GenomeStudio using the methylation module (Illumina). Further analyses were performed using R and the LIMMA package. Differential methylation between samples was identified using an empirical Bayes moderated t-test and the resulting p-values were adjusted using the Benjamini and Hochberg method. P-values below 0.05 were considered significant. We have shown genome-wide hypomethylation at the transition from MGUS to MM with gene-specific hypermethylation of tumor suppressor genes. The methylation patterns within myeloma samples correlate with translocations or hyperdiploidy, where the latter are split into 2 distinct clusters with a significant difference in overall survival, p=0.03. However, the t(4;14) subgroup had the most distinct methylation profile with hypermethylation of genes specific to the t(4;14) subgroup including CD79A, FAM49A and SOCS2 which correlate with loss of expression in this subgroup. Herein, we analyzed presentation MM (n=161) and PCL (n=32) samples for methylation changes which may contribute to expression and phenotypic changes. When comparing all MM and PCL samples, irrespective of cytogenetic subgroup, we find hypermethylation of the genome in PCL samples. Upon clustering we find that PCL samples segregate within the translocation groups with which they belong i.e. t(11;14) PCL samples cluster with t(11;14) myeloma samples. However, when MM and PCL samples from the same cytogenetic group are compared there is exclusive hypermethylation of genes occurring as the disease progresses. Although t(4;14) myeloma samples have considerable more methylation than t(11;14) samples, the increase in methylation from MM to PCL is consistent in both groups, resulting in t(4;14) PCL samples with increased methylation over t(11;14) PCL samples. The genes which are hypermethylated in the transition of MM to PCL can be classified as either tumor suppressor genes, genes involved in cell-cell signaling, or as cell adhesion molecules. The further analysis of these genes will allow us to identify genes which are down-regulated through methylation and mediate the progression of MM to PCL allowing the clone to become independent of the bone marrow microenvironment. In addition, these analyses will enable us to identify targets which may be sensitive to modulation by epigenetic therapies in vivo. In conclusion, we have demonstrated that methylation changes play a significant part in differentiating the various cytogenetic subgroups of MM and in mediating the transition to PCL. Hypermethylation affects genes and pathways important in retaining plasma cells in the bone marrow as well as in their growth factor independent growth in the absence of stromal cell support. Disclosures:No relevant conflicts of interest to declare.

CXCR4 and cancer

The chemokine receptor CXCR4 belongs to the large superfamily of G protein-coupled receptors and has been identified to play a crucial role in a number of biological processes, including the trafficking and homeostasis of immune cells such as T lymphocytes. CXCR4 has also been found to be a prognostic marker in various types of cancer, including leukemia and breast cancer, and recent evidence has highlighted the role of CXCR4 in prostate cancer. Furthermore, CXCR4 expression is upregulated in cancer metastasis, leading to enhanced signaling. These observations suggest that CXCR4 is important for the progression of cancer. The CXCR4-CXCL12 (stromal cell-derived factor 1 (SDF-1)) axis has additionally been identified to have a role in normal stem cell homing. Interestingly, cancer stem cells also express CXCR4, indicating that the CXCR4-SDF-1 axis may direct the trafficking and metastasis of these cells to organs that express high levels of SDF-1, such as the lymph nodes, lungs, liver, and bone. This review focuses on the current knowledge of CXCR4 regulation and how deregulation of this protein may contribute to the progression of cancer.

Regulation of Rho GTPases by the Hematopoietic-Specific Guanine Nucleotide Exchange Factor Vav1 Is Critical for Hematopoietic Stem Cell Retention in the Endosteal Niche and Engraftment.

Abstract 80 Background:Rho GTPases are molecular switches that regulate actin cytoskeleton dynamics, cell proliferation and survival. In hematopoietic stem cells and progenitors (HSC/P), several Rho GTPases (including Rac1, Rac2 and Cdc42) function as critical regulators of engraftment through the integration of diverse extracellular signals, such as those transmitted by growth factor, chemokine and adhesion receptors. In addition, Rac-deficient mice show significantly increased numbers of mobilized HSC/P. GTPase activation downstream of these and other receptors is mediated by a large family of guanine nucleotide exchange factors (GEF). Functional interactions between receptors, GEF and Rho GTPases are potentially complex and the crucial biochemical pathways regulating HSC activity have not been defined. Among the Rho/Rac GEFs, Vav1 shows hematopoietic-specific expression and has been previously implicated in immune cell processes, such as immunoreceptor signaling in lymphocytes and neutrophil migration. To further explore the mechanism of Rho GTPase regulation of HSC engraftment, we investigated the role of Vav1 GEF in Rho GTPase activation after ligation of multiple HSC receptors and the effect of genetic deletion of Vav1 on HSC homing, retention and engraftment in the hematopoietic microenvironment. Methods: GTPase activation (Rac, Cdc42, RhoA) was analyzed by in vitro pulldown assays. The HSC/P compartment of Vav1−/− mice was studied by flow cytometry, colony forming cell (CFC) assays, progenitor (CFC) homing, competitive and non-competitive repopulation assays. HSC localization in the endosteal niche was determined by intravital microscopy 1 h and 48 h after transplant. Results: At the biochemical level, Vav1−/− hematopoietic progenitors showed a dysfunctional Rho GTPase activation pattern, with increased baseline levels of GTP-bound Rac, Cdc42 and RhoA; however, in the absence of Vav1, these GTPases were unresponsive to stimulation by stem cell factor and SDF1α, critical proteins in HSC engraftment. In spite of this biochemical abnormality, Vav1−/− mice at baseline had nearly normal numbers of immunophenotypically defined HSC, myeloid and lymphoid progenitors in the bone marrow (BM), and normal hematopoietic progenitor content as defined by CFC, although reduced rather than increased circulating HSC/P. Vav1−/− HSC/P transplanted into irradiated recipients exhibited normal BM CFC homing efficiency (∼5%) and normal early endosteal localization of HSC in vivo (1 h after injection) as determined by intravital microscopy. Surprisingly-but in concordance with the normal BM homing of HSC/P in vivo- the loss of Vav1 did not affect hematopoietic progenitor chemotaxis or short-term adhesion to fibronectin in vitro. However, there was a significant decrease in the retention of HSC in the endosteal space at 48 h after transplant (Vav1−/− HSC numbers were reduced to 46%, relative to WT HSC) and this defect was associated with a profound loss of short- and long-term engraftment. In competitive repopulation assays, Vav1−/− cells virtually did not contribute to the graft (Table 1), whereas in a non-competitive setting, they either failed to rescue the recipient (60% survival vs 100% at 1 month, Vav1−/− vs WT) or showed significantly delayed hematopoietic reconstitution (Table 2). Conclusions: The hematopoietic-specific GEF Vav1 is essential for the appropriate microenvironment-induced Rho GTPase activation in HSC/P after transplant and is required for the retention of HSC/P in the BM endosteal niche and subsequent engraftment.Table 1Percentage of donor chimerism in lethally irradiated recipients transplanted with either WT or Vav1−/− CD45.2 BM cells (3 × 106 cells/recipient) and an equal number of WT CD45.1 competitor BM cells. Values represent mean ± SD. (*) p < 0.05, (**) p < 0.005.time after transplant (months)WTVav1−/−Peripheral blood153.4 ± 6.01.1 ± 2.4 (**)263.2 ± 5.10.9 ± 1.2 (**)360.1 ± 14.01.4 ± 1.7 (**)457.4 ± 18.50.1 ± 0.1 (**)Bone marrow466.3 ± 33.00.2 ± 0.4 (*)Spleen455.0 ± 13.30.4 ± 0.5 (**)Table 2Peripheral blood leukocyte count (× 103/μl) of lethally irradiated recipients transplanted with either WT or Vav1−/− BM cells (3 × 106 cells/recipient). Values represent mean ± SD. (**) p < 0.005.time after transplant (months)WTVav1−/−Peripheral blood0.54.1 ± 1.41.1 ± 0.6 (**)114.5 ± 3.85.6 ± 4.7 (**)214.2 ± 6.410.7 ± 6.4 Disclosures:No relevant conflicts of interest to declare.

The chemokine receptor CXCR4 demonstrates several features of a cancer stem cell marker in OSCC

Introduction: The concept of cancer stem cells (CSCs) has accumulated plenty of recent evidence derived from work on solid epithelial tumours such as breast, ovary, prostate and lung. The chemokine receptor CXCR4 and its ligand SDF-1 have been identified as key to normal stem cell tissue homing and their role in CSC populations is suggested. The aim of this study was to investigate the possibility of the SDF1-CXCR4 axis defining a population of CSCs in oral squamous cell carcinoma (OSCC).

Menin regulates the function of hematopoietic stem cells and lymphoid progenitors

AbstractMen1 is a tumor suppressor gene mutated in endocrine neoplasms. Besides its endocrine role, the Men1 gene product menin interacts with the mixed lineage leukemia (MLL) protein, a histone H3 lysine 4 methyltransferase. Although menin and MLL fusion proteins cooperate to activate Homeobox (Hox) gene expression during transformation, little is known about the normal hematopoietic functions of menin. Here, we studied hematopoiesis after Men1 ablation. Menin loss modestly impaired blood neutrophil, lymphocyte, and platelet counts. Without hematopoietic stress, multilineage and myelo-erythroid bone marrow progenitor numbers were preserved, while B lymphoid progenitors were decreased. In contrast, competitive transplantation revealed a marked functional defect of long-term hematopoietic stem cells (HSC) in the absence of menin, despite normal initial homing of progenitors to the bone marrow. HoxA9 gene expression was only modestly decreased in menin-deficient HSCs. These observations reveal a novel and essential role for menin in HSC homeostasis that was most apparent during situations of hematopoietic recovery, suggesting that menin regulates molecular pathways that are essential during the adaptive HSC response to stress.

Menin regulates the function of hematopoietic stem cells and lymphoid progenitors

Men1 is a tumor suppressor gene mutated in endocrine neoplasms. Besides its endocrine role, the Men1 gene product menin interacts with the mixed lineage leukemia (MLL) protein, a histone H3 lysine 4 methyltransferase. Although menin and MLL fusion proteins cooperate to activate Homeobox (Hox) gene expression during transformation, little is known about the normal hematopoietic functions of menin. Here, we studied hematopoiesis after Men1 ablation. Menin loss modestly impaired blood neutrophil, lymphocyte, and platelet counts. Without hematopoietic stress, multilineage and myelo-erythroid bone marrow progenitor numbers were preserved, while B lymphoid progenitors were decreased. In contrast, competitive transplantation revealed a marked functional defect of long-term hematopoietic stem cells (HSC) in the absence of menin, despite normal initial homing of progenitors to the bone marrow. HoxA9 gene expression was only modestly decreased in menin-deficient HSCs. These observations reveal a novel and essential role for menin in HSC homeostasis that was most apparent during situations of hematopoietic recovery, suggesting that menin regulates molecular pathways that are essential during the adaptive HSC response to stress.

Knockdown of CCR7 or Its Ligands Causes a Loss of Central Nervous System Involvement in Notch1 Induced T-ALL

Intensive chemotherapy helps achieve remission in the vast majority of acute lymphoblastic leukemia (ALL) patients. A subset of these cases, T-cell ALL (T-ALL), fare significantly worse, presenting with more severe disease, lower remission rates and a greater likelihood of relapse, particularly within the central nervous system (CNS). All modern treatment protocols involve intensive prophylactic CNS chemotherapy and cranial irradiation. The dramatic increase in overall survival is thought to be worth the significant long term side effects associated with CNS treatment such as secondary tumors, neurocognitive deficits and neuroendocrine disorders. Despite its significance, very little is known about the basic mechanisms of leukemic lymphocyte infiltration into the CNS. Over 50% of T-ALL patients have activating mutations in the Notch1 protein. According to our gene expression profiling results, Notch1 activation leads to the induction of CCR7, a chemokine receptor known to play a role in normal lymphocyte homing in extramedullary tissues and CNS. To study CNS infiltration, we generated two murine models expressing activated Notch1 (N1-IC), a bone marrow transplant (WT/WTN1) and a conditional knockin. Both developed an aggressive T-ALL with immature T-cells infiltrating into secondary lymphoid tissues, but more importantly, they had CNS involvement. Numerous malignant appearing CD3 positive cells were found in the leptomeningeal space of the brain in both mouse models. When we transplanted CCR7 knock out bone marrow cells expressing N1-IC, the recipient mice (WT/CCR7KON1) developed an aggressive T-ALL as before. However, a close examination of the brain failed to show any leukemic infiltration in the host mice. In agreement, wild-type N1-IC positive cells transplanted into plt mice (plt/ WTN1) lacking the CCR7 ligands, CCL19 and CCL21, induced T-cell leukemia, but again, these cells failed to infiltrate into the CNS. In addition, WT/CCR7KON1 and plt/WTN1 mice survived longer than WT/WTN1 mice (61 and 71 vs 47 days, p<0.0001). Using real time PCR and FACS, we quantified the levels of CCR7 in 7 human T-ALL cell lines and found that all but one expressed CCR7 at varying levels. The highest expressing, CEM, and non-expressing, DND41, cell lines were transplanted into alymphoid Rag2−/−gc−/− hosts and rapid leukemic infiltration of both lines was monitored by bioluminescent imaging. We found dramatic infiltration of CEM cells, but not DND41 cells into the CNS of the recipients. Moreover, the overexpression of CCR7 in the non-expressing DND41 cells caused them to acquire the ability to infiltrate into the CNS of the hosts. These results strongly implicate CCR7 as a major mediator of CNS involvement in T-cell leukemia and would suggest that targeting the Notch1 or CCR7 pathways could provide an effective CNS therapy with reduced long term morbidity.

Leukocyte trafficking in a mouse model for leukocyte adhesion deficiency II/congenital disorder of glycosylation IIc

AbstractLeukocyte adhesion deficiency II (LAD II), also known as congenital disorder of glycosylation IIc (CDG-IIc), is a human disease in which a defective GDP-fucose transporter (SLC35C1) causes developmental defects and an immunodeficiency that is based on the lack of fucosylated selectin ligands. Since the study of in vivo leukocyte trafficking in patients with LAD II is experimentally limited, we analyzed this process in mice deficient for Slc35c1. We found that E-, L-, and P-selectin–dependent leukocyte rolling in cremaster muscle venules was virtually absent. This was accompanied by a strong but not complete decrease in firm leukocyte adhesion. Moreover, neutrophil migration to the inflamed peritoneum was strongly reduced by 89%. Previous reports showed surprisingly normal lymphocyte functions in LAD II, which indicated sufficient lymphocyte trafficking to secondary lymphoid organs. We now found that while lymphocyte homing to lymph nodes was reduced to 1% to 2% in Slc35c1−/− mice, trafficking to the spleen was completely normal. In accordance with this, we found a defect in the humoral response to a T cell–dependent antigen in lymph nodes but not in the spleen. Taken together, Slc35c1−/− mice show strongly defective leukocyte trafficking but normal lymphocyte homing to the spleen, which may explain normal lymphocyte functions in LAD II.

The “mode” of lymphocyte extravasation through HEV of Peyer's patches and its role in normal homing and inflammation

The mode of lymphocyte transendothelial migration in the postcapillary high endothelial venules (HEVs) of Peyer's patches during normal homing and acute inflammation in the guinea pig was studied. It is common opinion that the lymphocyte transendothelial passage from the blood stream into the extravasal lymphoid tissue calls for a multistep process of endothelial and lymphocyte molecules favoring tethering, rolling, activation, arrest and its firm adhesion to the endothelial luminal surface. Ultrastructural serial pictures and the three-dimensional reconstruction of HEVs with lymphocytes during different moments of their transmigration through the endothelial wall enabled us to demonstrate in vivo the morphological modality of their extravasation in lymphoid tissue. The latter is accomplished by means of an intraendothelial canalicular formation (6.8–7.2 μm long and 2.1–2.2 μm in diameter), whose creation depends on the particular behavior of adjacent endothelial cells, without compromising the interendothelial contacts. This new canalicular pathway of lymphocyte extravasation, particularly selective for the B cell, does not permit confirmation of the dogmas of the transcellular and paracellular (open interendothelial junctions) modes that have prevailed in recent decades. The lack of knowledge regarding the molecular bases that would induce constitution of this intraendothelial canalicular formation is a critical point for stimulating future interdisciplinary research aimed at developing strategies for modulating normal lymphocyte homing and in inflammation.

Chemokine CXCL12 enhances proliferation in pre‐B‐ALL via STAT5 activation

CXCL12 (SDF-1alpha) is a chemokine, which plays an important role in normal B-cell lymphopoesis, migration and homing to the bone marrow (BM) and previous studies have suggested a role for CXCL12 and its receptor CXCR4 in the pathogenesis of ALL. CXCL12 levels in serum were evaluated from ALL-children and controls. The biological effect of recombinant CXCL12 on primary leukaemic cells was investigated. Signalling via the CXCL12/CXCR4 axis was further characterized in an in vitro model using the pre-B leukaemic cell line Nalm-6. The serum level of CXCL12 in children at diagnosis of pre-B-ALL is significantly higher than in healthy children (4.8 (0-32) ng/ml vs. 0 (0-3.2) ng/ml, P < 0.001). After completed chemotherapy, CXCL12 decreases to levels comparable to those found in the control group. In addition, we found that recombinant CXCL12 enhances pre-B leukaemic cell proliferation in vitro. The CXCL12/CXCR4 axis is able to initiate functional signalling and we show that STAT5 is activated in CD19+ leukaemic cells from BM of ALL patients and in the leukaemic cell line Nalm-6. Our findings suggest that CXCL12 may have a role in leukaemic cell proliferation and survival during childhood ALL.