CCL19-sorted mature dendritic cells have enhanced lymph node migratory capacity and function

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CCR7+ dendritic cells sorted by binding of CCL19 show enhanced Ag-presenting capacity and antitumor potency

Dendritic cell therapy has been a promising addition to the current armory of therapeutic options in cancer for more than 20 years but has not yet achieved breakthrough success. To successfully initiate immunity, dendritic cells have to enter the lymph nodes. However, experience to date of therapeutic dendritic cell administration indicates that this is frequently an extremely inefficient process. The major regulator of dendritic cell migration to the lymph nodes is the chemokine receptor CCR7 and in vitro generated dendritic cells typically display heterogeneous expression of this receptor. Here we demonstrate that positive selection for the dendritic cell subpopulation expressing CCR7, using a chemically-synthesized ligand:CCL19, enriches for cells with enhanced lymph node migration and Ag presentation competence as well as a chemokine expression profile indicative of improved interactions with T cells. This enhanced lymph node homing capacity of enriched CCR7+ cells is seen in comparison to a population of unsorted dendritic cells containing an equivalent number of CCR7+ dendritic cells. Importantly, this indicates that separating the CCR7+ dendritic cells from the CCR7− cells, rather than simple CCL19 exposure, is required to affect the enhanced lymph node migration of the CCR7+ cells. In models of both subcutaneous and metastatic melanoma, we demonstrate that the dendritic cells sorted for CCR7 expression trigger enhanced CD8 T-cell driven antitumor immune responses which correlate with reduced tumor burden and increased survival. Finally, we demonstrate that this approach is directly translatable to human dendritic cell therapy using the same reagents coupled with clinical-grade flow-cytometric sorting

The GATA1s isoform is normally down-regulated during terminal haematopoietic differentiation and over-expression leads to failure to repress MYB, CCND2 and SKI during erythroid differentiation of K562 cells

Background: Although GATA1 is one of the most extensively studied haematopoietic transcription factors little is currently known about the physiological functions of its naturally occurring isoforms GATA1s and GATA1FL in humans—particularly whether the isoforms have distinct roles in different lineages and whether they have non-redundant roles in haematopoietic differentiation. As well as being of general interest to understanding of haematopoiesis, GATA1 isoform biology is important for children with Down syndrome associated acute megakaryoblastic leukaemia (DS-AMKL) where GATA1FL mutations are an essential driver for disease pathogenesis. <p/>Methods: Human primary cells and cell lines were analyzed using GATA1 isoform specific PCR. K562 cells expressing GATA1s or GATA1FL transgenes were used to model the effects of the two isoforms on in vitro haematopoietic differentiation. <p/>Results: We found no evidence for lineage specific use of GATA1 isoforms; however GATA1s transcripts, but not GATA1FL transcripts, are down-regulated during in vitro induction of terminal megakaryocytic and erythroid differentiation in the cell line K562. In addition, transgenic K562-GATA1s and K562-GATA1FL cells have distinct gene expression profiles both in steady state and during terminal erythroid differentiation, with GATA1s expression characterised by lack of repression of MYB, CCND2 and SKI. <p/>Conclusions: These findings support the theory that the GATA1s isoform plays a role in the maintenance of proliferative multipotent megakaryocyte-erythroid precursor cells and must be down-regulated prior to terminal differentiation. In addition our data suggest that SKI may be a potential therapeutic target for the treatment of children with DS-AMKL

Spread of psoriasiform inflammation to remote tissues is restricted by the atypical chemokine receptor ACKR2

Elucidating the poorly defined mechanisms by which inflammatory lesions are spatially restricted in vivo, is of critical importance in understanding skin disease. Chemokines are the principal regulators of leukocyte migration and are essential in the initiation and maintenance of inflammation. The membrane-bound psoriasis associated atypical chemokine receptor ACKR2 binds, internalises and degrades most pro-inflammatory CC-chemokines. Here we investigate the role of ACKR2 in limiting the spread of cutaneous psoriasiform inflammation to sites that are remote from the primary lesion.  Circulating factors capable of regulating ACKR2 function at remote sites were identified and examined using a combination of clinical samples, relevant primary human cell cultures, in vitro migration assays and the imiquimod-induced model of psoriasiform skin inflammation. Localised inflammation and IFN together upregulate ACKR2 in remote tissues, protecting them from the spread of inflammation. ACKR2 controls inflammatory T-cell chemotaxis and positioning within the skin, preventing an epidermal influx that is associated with lesion development. Our results have important implications for our understanding of how spatial restriction is imposed on the spread of inflammatory lesions, and highlight systemic ACKR2 induction as a therapeutic strategy in the treatment and prevention of psoriasis and potentially a broad range of other immune-mediated diseases

Advanced glycation end product precusors in diabetes : a crucial link between oxidative stress and inflammation?

Advanced glycated end-products (AGEs) are a heterogenous group of compounds formed through the Maillard reaction. During AGE formation, reactive α-dicarbonyls are formed, such as glyoxal (GO) and methylglyoxal (MG). These α-dicarbonyls are present at elevated concentrations in diabetes, and are frequently implicated in the initiation and progression of diabetic complications. Previous research has linked α-dicarbonyls with formation of reactive oxygen species (ROS) and inflammation. However, much of the prior work has been conducted using concentrations of α-dicarbonyls that are substantially higher than can be generated in vivo, and the biochemistry has been investigated under conditions (e.g. pH) outside the physiological range. The aim of the work presented in this thesis was to test the hypothesis that GO and MG are pro-oxidant and pro-inflammatory at (patho)physiological concentrations in both monocytes and pancreatic β-cells. In this work several new and important observations have been made regarding the action of α-dicarbonyls on oxidative stress and inflammation. 1) The amount of oxidative species production by α-dicarbonyls in glycation reactions with amino acids and proteins may be so low as to be negligible in vivo, despite previous evidence to the contrary. 2) α-dicarbonyls did not appear to generate oxidative stress within inflammatory cells nor pancreatic β-cells by depleting the levels of GSH. 3) At least in the β-cell model, the mechanism of action of the α-dicarbonyls did not involve dysregulation of the antioxidant SOD enzymes. 4) Neither α-dicarbonyl significantly affected insulin production by β-cells, except at cytotoxic concentrations. 5) Treatment of inflammatory cells with α-dicarbonyls induced release of the proinflammatory cytokine IL-8. 6) In both immune cells and pancreatic β-cells, α-dicarbonyls were involved in O2.- generation by activation and/or upregulation of NADPH oxidase. 7) Despite the structural similarities of α-dicarbonyls, they have distinct mechanisms of action with respect to oxidative stress.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The <it>GATA1s</it> isoform is normally down-regulated during terminal haematopoietic differentiation and over-expression leads to failure to repress <it>MYB, CCND2</it> and <it>SKI</it> during erythroid differentiation of K562 cells

Abstract Background Although GATA1 is one of the most extensively studied haematopoietic transcription factors little is currently known about the physiological functions of its naturally occurring isoforms GATA1s and GATA1FL in humans—particularly whether the isoforms have distinct roles in different lineages and whether they have non-redundant roles in haematopoietic differentiation. As well as being of general interest to understanding of haematopoiesis, GATA1 isoform biology is important for children with Down syndrome associated acute megakaryoblastic leukaemia (DS-AMKL) where GATA1FL mutations are an essential driver for disease pathogenesis. Methods Human primary cells and cell lines were analyzed using GATA1 isoform specific PCR. K562 cells expressing GATA1s or GATA1FL transgenes were used to model the effects of the two isoforms on in vitro haematopoietic differentiation. Results We found no evidence for lineage specific use of GATA1 isoforms; however GATA1s transcripts, but not GATA1FL transcripts, are down-regulated during in vitro induction of terminal megakaryocytic and erythroid differentiation in the cell line K562. In addition, transgenic K562-GATA1s and K562-GATA1FL cells have distinct gene expression profiles both in steady state and during terminal erythroid differentiation, with GATA1s expression characterised by lack of repression of MYB, CCND2 and SKI. Conclusions These findings support the theory that the GATA1s isoform plays a role in the maintenance of proliferative multipotent megakaryocyte-erythroid precursor cells and must be down-regulated prior to terminal differentiation. In addition our data suggest that SKI may be a potential therapeutic target for the treatment of children with DS-AMKL.</p

Chemokines as novel and versatile reagents for flow cytometry and cell sorting

Cell therapy regimens are frequently compromised by low-efficiency cell homing to therapeutic niches. Improvements in this regard would enhance effectiveness of clinically applicable cell therapy. The major regulators of tissue-specific cellular migration are chemokines, and therefore selection of therapeutic cellular populations for appropriate chemokine receptor expression would enhance tissue-homing competence. A number of practical considerations preclude the use of Abs in this context, and alternative approaches are required. In this study, we demonstrate that appropriately labeled chemokines are at least as effective in detecting their cognate receptors as commercially available Abs. We also demonstrate the utility of biotinylated chemokines as cell-sorting reagents. Specifically, we demonstrate, in the context of CCR7 (essential for lymph node homing of leukocytes), the ability of biotinylated CCL19 with magnetic bead sorting to enrich for CCR7-expressing cells. The sorted cells demonstrate improved CCR7 responsiveness and lymph node–homing capability, and the sorting is effective for both T cells and dendritic cells. Importantly, the ability of chemokines to detect CCR7, and sort for CCR7 positivity, crosses species being effective on murine and human cells. This novel approach to cell sorting is therefore inexpensive, versatile, and applicable to numerous cell therapy contexts. We propose that this represents a significant technological advance with important therapeutic implications

An analysis of mechanisms of central nervous system infiltration in acute lymphoblastic leukaemia using primary cells xenografted into immunodeficient mice

Despite great advances in the treatment of paediatric acute lymphoblastic leukaemia (ALL), disease in the central nervous system (CNS) continues to pose challenges. Current diagnostic tests are insensitive and risk factors for CNS relapse are poorly understood. This results in all children receiving intensive CNS-directed therapy which may be associated with acute and/or chronic neurotoxicity. A better understanding of the mechanisms of CNS engraftment is a necessary pre-requisite for diagnostic and therapeutic advances.  To investigate if primary cells infiltrate the CNS, and whether this property resides in any particular sub-clonal compartment we undertook a comprehensive analysis of the CNS engrafting potential of primary ALL cells in immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. In addition, we used this model to investigate whether chemokine receptor expression drives CNS infiltration in pre-B ALL, as previously shown for T-ALL. CNS engraftment was seen in 18/23 pre-B ALL samples (78%). A consistent pattern of engraftment was observed with plaques of disease in the leptomeninges, relative sparing of the ventricles and complete absence of parenchymal infiltration. This implies ALL transit across the blood-CSF barrier rather than the blood-brain barrier – an important distinction, as leucocytes utilise distinct physiological trafficking mechanisms to cross these two barriers. To examine the frequency of cells with CNS-engrafting potential, intra-femoral transfer of 10 to 10,000 cells was performed.  CNS engraftment was seen with as few as 10 initial cells. In addition, the ability to engraft the CNS was not restricted to any particular immunophenotypic compartment and was seen in CD19+CD10high, CD19+CD10low, CD19+CD20low, CD19+CD20high, CD19+CD34high, CD19+CD34low fractions. These data suggest that CNS engraftment potential is present at high-frequency in the bulk leukaemic population at diagnosis. To examine candidate trafficking molecules governing CNS entry, we investigated chemokine receptor expression on pre-B ALL blasts using quantitative PCR and flow cytometry. The chemokine receptors CXCR3, CXCR4 and CXCR7 were expressed by pre-B ALL but were not up-regulated in cells retrieved from the CNS compared to the bone marrow (BM). In conclusion, in xenograft models of pre-B ALL, CNS engrafting potential is present at high frequency in patient diagnostic BM specimens and does not appear to be restricted to sub-clonal compartments. These findings have important implications for the design of risk-adapted CNS therapy. Firstly, our studies indicate that CNS entry is a common property of leukaemic blasts and therefore the current dogma of CNS-directed therapy for all patients appears to have a rational scientific basis. Secondly, it is unlikely that chemokine receptor expression profiling will be a useful biomarker for CNS disease in pre-B ALL. Finally, identifying factors that facilitate long-term survival of cells in the CNS (which may also enhance long-term survival in the bone marrow) may be a better therapeutic strategy than attempts to block cell entry