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

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