Using the Jurkat reporter T cell line for evaluating the functionality of novel chimeric antigen receptors.

Background: T cells that are genetically modified with chimeric antigen receptor (CAR) hold promise for immunotherapy of cancer. Currently, there are intense efforts to improve the safety and efficacy of CAR T cell therapies against liquid and solid tumors. Earlier we designed a novel CAR backbone (FiCAR) where the spacer is derived from immunoglobulin (Ig) -like domains of the signal-regulatory protein alpha (SIRPα). However, the analysis of novel CAR using primary T cells is slow and laborious. Methods: To explore the versatility of the CAR backbone, we designed a set of variant FiCARs with different spacer lengths and targeting antigens. To expedite the analysis of the novel CARs, we transduced the FiCAR genes using lentiviruses into Jurkat reporter T cells carrying fluorescent reporter genes. The expression of fluorescent markers in response to FiCAR engagement with targets was analyzed by flow cytometry, and cytotoxicity was evaluated using killing assays. Furthermore, the killing mechanisms that are employed by FiCAR-equipped Jurkat T cells were investigated by flow cytometry, and the intracellular pathways involved in signaling by FiCAR were analyzed by phosphoproteomic analysis using mass spectrometry. Results: Seven different CARs were designed and transduced into Jurkat reporter cells. We show that the SIRPα derived FiCARs can be detected by flow cytometry using the SE12B6A4 antibody recognizing SIRPα. Furthermore, FiCAR engagement leads to robust activation of NFκß and NFAT signaling, as demonstrated by the expression of the fluorescent reporter genes. Interestingly, the Jurkat reporter system also revealed tonic signaling by a HER-2 targeting FiCAR. FiCAR-equipped Jurkat T cells were cytotoxic in cocultures with target cells and target cell engagement lead to an upregulation of CD107a on the Jurkat reporter T cell surface. Phosphoproteomic analyses confirmed signal transduction via the intracellular CD28/CD3ζ sequences upon the interaction of the FiCAR1 with its antigen. In addition, downstream signaling of CD3ζ/ZAP70- SLP-76-PLCγ, PI3K-AKT-NFκB pathways and activation of NFAT and AP-1 were observed. Conclusion: We conclude that the FiCAR backbone can be shortened and lengthened at will by engineering it with one to three SIRPα derived Ig-like domains, and the FiCARs are functional when equipped with different single chain variable fragment target binding domains. The Jurkat reporter system expedites the analysis of novel CARs as to their expression, signaling function, evaluation of tonic signaling issues and cytotoxic activity.

A novel IgG-based FLT3xCD3 bispecific antibody for the treatment of AML and B-ALL.

BACKGROUND: In lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for acute myeloid leukemia (AML), the most common form of acute leukemia in adults. Common targets in AML such as CD33, CD123, and CLEC12A are highly expressed on both AML blasts and on normal myeloid cells and hematopoietic stem cells (HSCs), thereby raising toxicity concerns. In B-cell acute lymphoblastic leukemia (B-ALL), bsAbs and CAR-T therapy targeting CD19 and CD22 have demonstrated clinical success, but resistance via antigen loss is common, motivating the development of agents focused on alternative targets. An attractive emerging target is FLT3, a proto-oncogene expressed in both AML and B-ALL, with low and limited expression on myeloid dendritic cells and HSCs. METHODS: We developed and characterized CLN-049, a T cell-activating bsAb targeting CD3 and FLT3, constructed as an IgG heavy chain/scFv fusion. CLN-049 binds the membrane proximal extracellular domain of the FLT3 protein tyrosine kinase, which facilitates the targeting of leukemic blasts regardless of FLT3 mutational status. CLN-049 was evaluated for preclinical safety and efficacy in vitro and in vivo. RESULTS: CLN-049 induced target-restricted activation of CD4+ and CD8+ T cells. AML cell lines expressing a broad range of surface levels of FLT3 were efficiently lysed on treatment with subnanomolar concentrations of CLN-049, whereas FLT3-expressing hematopoietic progenitor cells and dendritic cells were not sensitive to CLN-049 killing. Treatment with CLN-049 also induced lysis of AML and B-ALL patient blasts by autologous T cells at the low effector-to-target ratios typically observed in patients with overt disease. Lysis of leukemic cells was not affected by supraphysiological levels of soluble FLT3 or FLT3 ligand. In mouse xenograft models, CLN-049 was highly active against human leukemic cell lines and patient-derived AML and B-ALL blasts. CONCLUSIONS: CLN-049 has a favorable efficacy and safety profile in preclinical models, warranting evaluation of its antileukemic activity in the clinic.

Evidence supporting a role for the immune checkpoint protein B7-H3 in NK cell-mediated cytotoxicity against AML.

We observed that the immune checkpoint protein B7-H3 is overexpressed in acute myeloid leukemia (AML) patients with poor treatment outcomes. Inhibition of B7-H3 expression or blocking of its activity using a novel monoclonal antibody (T-1A5) in AML cells significantly enhanced natural killer (NK) cell-mediated cytotoxicity in AML cells in vitro and in vivo. Moreover, a human-mouse chimera of this antibody (ChT-1A5) induced antibody-dependent cell-mediated cytotoxicity (ADCC) in B7-H3+ primary AML cells, but not in normal hematopoietic cells, suggesting the specify of this antibody for AML cells. Epitope mapping studies identified that both T-1A5 and ChT-1A5 antibodies bind to the FG-loop region of B7-H3, which is known to regulate the immunosuppressive function of B7-H3. Furthermore, treatment with ChT-1A5 in combination with human NK cells significantly prolonged survival in AML patient-derived xenograft (PDX) models. Our results suggest that the ChT-1A5 antibody can inhibit the immunosuppressive function of B7-H3 protein as well as induce ADCC in B7-H3+ AML.

The stem cell revolution: on the role of CD164 as a human stem cell marker.

Accurately defining hierarchical relationships between human stem cells and their progeny, and using this knowledge for new cellular therapies, will undoubtedly lead to further successful treatments for life threatening and chronic diseases, which represent substantial burdens on patient quality of life and to healthcare systems globally. Clinical translation relies in part on appropriate biomarker, in vitro manipulation and transplantation strategies. CD164 has recently been cited as an important biomarker for enriching both human haematopoietic and skeletal stem cells, yet a thorough description of extant human CD164 monoclonal antibody (Mab) characteristics, which are critical for identifying and purifying these stem cells, was not discussed in these articles. Here, we highlight earlier but crucial research describing these relevant characteristics, including the differing human CD164 Mab avidities and their binding sites on the human CD164 sialomucin, which importantly may affect subsequent stem cell function and fate.

Removal of CD276+ cells from haploidentical memory T-cell grafts significantly lowers the risk of GVHD.

Detrimental graft-versus-host disease (GVHD) still remains a major cause of death in hematopoietic stem cell transplantation (HSCT). The recently explored depletion of naive cells from mobilized grafts (CD45RA depletion) has shown considerable promise, yet is unable to eliminate the incidence of GVHD. Analysis of CD45RA-depleted haploidentical mixed lymphocytes culture (haplo-MLC) revealed insufficient suppression of alloresponses in the CD4+ compartment and identified CD276 as a marker for alloreactive memory Th1 T cells. Conclusively, depleting CD276+ cells from CD45RA-depleted haplo-MLC significantly attenuated alloreactivity to recipient cells while increasing antiviral reactivity and maintaining anti-third party reactivity in vitro. To evaluate these findings in vivo, bulk, CD45RA-depleted, or CD45RA/CD276-depleted CD4+ T cells from HLA-DR4negative healthy humans were transplanted into NSG-Ab°DR4 mice, a sensitive human allo-GVHD model. Compellingly, CD45RA/CD276-depleted grafts from HLA-DR4negative donors or in vivo depletion of CD276+ cells after transplant of HLA-DR4negative memory CD4 T cells significantly delay the onset of GVHD symptoms and significantly alleviate its severity in NSG-Ab°DR4 mice. The clinical courses correlated with diminished Th1-cytokine secretion and downregulated CXCR6 expression of engrafted peripheral T cells. Collectively, mismatched HLA-mediated GVHD can be controlled by depleting recipient-specific CD276+ alloreacting T cells from the graft, highlighting its application in haplo-HSCT.

The osteogenic commitment of CD271+CD56+ bone marrow stromal cells (BMSCs) in osteoarthritic femoral head bone.

Osteoarthritis (OA), the most common joint disorder, is characterised by progressive structural changes in both the cartilage and the underlying subchondral bone. In late disease stages, subchondral bone sclerosis has been linked to heightened osteogenic commitment of bone marrow stromal cells (BMSCs). This study utilised cell sorting and immunohistochemistry to identify a phenotypically-distinct, osteogenically-committed BMSC subset in human OA trabecular bone. Femoral head trabecular bone tissue digests were sorted into CD45-CD271+CD56+CD146-, CD45-CD271+CD56-CD146+ and CD45-CD271+CD56-CD146-(termed double-negative, DN) subsets, and CD45+CD271-hematopoietic-lineage cells served as control. Compared to the CD146+ subset, the CD56+ subset possessed a lower-level expression of adipocyte-associated genes and significantly over 100-fold higher-level expression of many osteoblast-related genes including osteopontin and osteocalcin, whilst the DN subset presented a transcriptionally 'intermediate' BMSC population. All subsets were tri-potential following culture-expansion and were present in control non-OA trabecular bone. However, while in non-OA bone CD56+ cells only localised on the bone surface, in OA bone they were additionally present in the areas of new bone formation rich in osteoblasts and newly-embedded osteocytes. In summary, this study reveals a distinct osteogenically-committed CD271+CD56+ BMSC subset and implicates it in subchondral bone sclerosis in hip OA. CD271+CD56+ subset may represent a future therapeutic target for OA and other bone-associated pathologies.

Induction of NK Cell Reactivity against B-Cell Acute Lymphoblastic Leukemia by an Fc-Optimized FLT3 Antibody.

Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism by which antitumor antibodies mediate therapeutic efficacy. At present, we evaluate an Fc-optimized (amino acid substitutions S239D/I332E) FLT3 antibody termed 4G8-SDIEM (FLYSYN) in patients with acute myeloid leukemia (NCT02789254). Here we studied the possibility to induce NK cell ADCC against B-cell acute lymphoblastic leukemia (B-ALL) by Fc-optimized FLT3 antibody treatment. Flow cytometric analysis confirmed that FLT3 is widely expressed on B-ALL cell lines and leukemic cells of B-ALL patients. FLT3 expression did not correlate with that of CD20, which is targeted by Rituximab, a therapeutic monoclonal antibody (mAb) employed in B-ALL treatment regimens. Our FLT3 mAb with enhanced affinity to the Fc receptor CD16a termed 4G8-SDIE potently induced NK cell reactivity against FLT3-transfectants, the B-ALL cell line SEM and primary leukemic cells of adult B-ALL patients in a target-antigen dependent manner as revealed by analyses of NK cell activation and degranulation. This was mirrored by potent 4G8-SDIE mediated NK cell ADCC in experiments with FLT3-transfectants, the cell line SEM and primary cells as target cells. Taken together, the findings presented in this study provide evidence that 4G8-SDIE may be a promising agent for the treatment of B-ALL, particularly in CD20-negative cases.

An Fc-Optimized CD133 Antibody for Induction of Natural Killer Cell Reactivity against Colorectal Cancer.

The introduction of monoclonal antibodies (mAbs) has largely improved treatment options for cancer patients. The ability of antitumor mAbs to elicit antibody-dependent cellular cytotoxicity (ADCC) contributes to a large extent to their therapeutic efficacy. Many efforts accordingly aim to improve this important function by engineering mAbs with Fc parts that display enhanced affinity to the Fc receptor CD16 expressed, e.g., on natural killer (NK) cells. Here we characterized the CD133 mAb 293C3-SDIE that contains an engineered Fc part modified by the amino acid exchanges S239D/I332E-that reportedly increase the affinity to CD16-with regard to its ability to induce NK reactivity against colorectal cancer (CRC). 293C3-SDIE was found to be a stable protein with favorable binding characteristics achieving saturating binding to CRC cells at concentrations of approximately 1 µg/mL. While not directly affecting CRC cell growth and viability, 293C3-SDIE potently induced NK cell activation, degranulation, secretion of Interferon-γ, as well as ADCC resulting in potent lysis of CRC cell lines. Based on the preclinical characterization presented in this study and the available data indicating that CD133 is broadly expressed in CRC and represents a negative prognostic marker, we conclude that 293C3-SDIE constitutes a promising therapeutic agent for the treatment of CRC and thus warrants clinical evaluation.

Bone Marrow-Harvesting Technique Influences Functional Heterogeneity of Mesenchymal Stem/Stromal Cells and Cartilage Regeneration.

BACKGROUND: Connective tissue progenitors (CTPs) from native bone marrow (BM) or their culture-expanded progeny, often referred to as mesenchymal stem/stromal cells, represents a promising strategy for treatment of cartilage injuries. But the cartilage regeneration capacity of these cells remains unpredictable because of cell heterogeneity. HYPOTHESIS: The harvest technique of BM may highly influence stem cell heterogeneity and, thus, cartilage formation because these cells have distinct spatial localization within BM from the same bone. STUDY DESIGN: Controlled laboratory study. METHODS: CTPs obtained from the femur of patients undergoing total hip replacement by 2 harvest techniques-BM aspiration and BM collection-after bone rasping were immunophenotyped by flow cytometry and evaluated for chondrogenic ability. The spatial localization of different CTP subsets in BM was verified by immunohistochemistry. RESULTS: Cells from the BM after rasping were significantly more chondrogenic than the donor-matched aspirate, whereas no notable difference in their osteogenic or adipogenic potential was observed. The authors then assessed whether distinct immunophenotypically defined CTP subsets were responsible for the different chondrogenic capacity. Cells directly isolated from BM after rasping contained a higher percentage (mean, 7.2-fold) of CD45-CD271+CD56+ CTPs as compared with BM aspirates. The presence of this subset in the harvested BM strongly correlated with chondrogenic ability, showing that CD271+CD56+ cells are enriched in chondroprogenitors. Furthermore, evaluation of these CTP subsets in BM revealed that CD271+CD56+ cells were localized in the bone-lining regions whereas CD271+CD56- cells were found in the perivascular regions. Since the iliac crest remains a frequent site of BM harvest for musculoskeletal regeneration, the authors also compared the spatial distribution of these subsets in trabeculae of femoral head and iliac crest and found CD271+CD56+ bone-lining cells in both tissues. CONCLUSION: Chondrogenically distinct CTP subsets have distinct spatial localization in BM; hence, the harvest technique of BM determines the efficiency of cartilage formation. CLINICAL RELEVANCE: The harvest technique of BM may be of major importance in determining the clinical success of BM mesenchymal stem/stromal cells in cartilage repair.

Molecular Signatures of Primary Human Spermatogonial Progenitors and Its Neighboring Peritubular Stromal Compartment.

In-depth understanding of human spermatogenesis requires studying specific molecular signatures and interactions of spermatogonia with other testicular cell populations, for which isolation of pure populations of different cell types is crucial. Here, we describe a technique to simultaneously enrich pure, multiple testicular cell populations, including spermatogonia, endothelial (TECs), and perivascular mesenchymal stem/stromal cells (TMSCs), from testicular tissue by flow cytometry using a combination of defined markers. Immunohistochemical studies, multicolor staining, and cell sorting followed by multiplex quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that spermatogonia were highly enriched in the CD49f+CD49a-HLA-ABC-SSEA-4+ fraction of primary testicular cells. In contrast to spermatogonia, TMSCs and TECs were highly enriched in the CD49f+CD49a+HLA-ABC+CD144- and CD49f+CD49a+HLA-ABC+CD144+subsets, respectively. The delineation was confirmed by the expression of specific stromal and endothelial key markers as well as by the differentiation and angiogenic capacity of the sorted populations. In this article, for the first time, we performed transcriptome profiling of highly enriched, freshly isolated human spermatogonia and compared their expression profile with that of TMSCs. Our RNA sequencing data favor the hypothesis that TMSCs are candidate niche components for spermatogonia. The composite genotype and phenotype of defined testicular cell populations combined with a robust isolation procedure from small biopsies contributes to a better understanding of cellular interactions and for the establishment of efficient culture techniques to maintain spermatogonial progenitors.

Expression of the Wnt Receptor Frizzled-4 in the Human Enteric Nervous System of Infants.

The Wnt signalling pathway plays a crucial role in the development of the nervous system. This signalling cascade is initiated upon binding of the secreted Wnt ligand to a member of the family of frizzled receptors. In the present study, we analysed the presence of frizzled-4 in the enteric nervous system of human infants. Frizzled-4 could be identified by immunohistochemistry in a subpopulation of enteric neuronal and glial cells in the small and large intestine. Detection of frizzled-4 in the tunica muscularis by RT-PCR confirmed this receptor's expression on the mRNA level. Interestingly, we observed distinct cell populations that co-expressed frizzled-4 with the intermediate filament protein nestin and the neurotrophin receptor p75(NTR), which have been reported to be expressed in neural progenitor cells. Flow cytometry analysis revealed that 60% of p75(NTR) positive cells of the tunica muscularis were positive for frizzled-4. Additionally, in pathological samples of Hirschsprung's disease, the expression of this Wnt receptor correlated with the number of myenteric ganglion cells and decreased from normoganglionic to aganglionic areas of large intestine. The expression pattern of frizzled-4 indicates that this Wnt receptor could be involved in postnatal development and/or function of the enteric nervous system.

Profiling of primary peripheral blood- and monocyte-derived dendritic cells using monoclonal antibodies from the HLDA10 Workshop in Wollongong, Australia.

Dendritic cells (DCs) arise from hematopoietic stem cells and develop into a discrete cellular lineage distinct from other leucocytes. Mainly three phenotypically and functionally distinct DC subsets are described in the human peripheral blood (PB): plasmacytoid DCs (pDCs), which express the key marker CD303 (BDCA-2), and two myeloid DC subsets (CD1c(+) DC (mDC1) and CD141(+) DC (mDC2)), which express the key markers CD1c (BDCA-1) and CD141 (BDCA-3), respectively. In addition to these primary cell subsets, DCs can also be generated in vitro from either CD34(+) stem/progenitor cells in the presence of Flt3 (Fms-related tyrosine kinase 3) ligand or from CD14(+) monocytes (monocyte-derived DCs (mo-DCs)) in the presence of granulocyte-macrophage colony-stimulating factor+interleukin-4 (GM-CSF+IL-4). Here we compare the reactivity patterns of HLDA10 antibodies (monoclonal antibody (mAb)) with pDCs, CD1c(+) DCs and CD141(+) DCs, as well as with CD14(+)-derived mo-DCs cultured for 7 days in the presence of 100 ng/ml GM-CSF plus 20 ng/ml IL-4. A detailed profiling of these DC subsets based on immunophenotyping and multicolour flow cytometry analysis is presented. Using the panel of HLDA10 Workshop mAb, we could verify known targets selectively expressed on discrete DC subsets including CD370 as a selective marker for CD141(+) DCs and CD366 as a marker for both myeloid subsets. In addition, vimentin and other markers are heterogeneously expressed on all three subsets, suggesting the existence of so far not identified DC subsets.

Innate immune system favors emergency monopoiesis at the expense of DC-differentiation to control systemic bacterial infection in mice.

DCs are professional APCs playing a crucial role in the initiation of T-cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC-depletion in humans and mice. The mechanisms of pathogen-induced DC-depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC-development, one reason for DC-depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC-depletion and the impact of different bacteria on DC-development. We show that the number of bone marrow (BM) hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram-positive and Gram-negative bacteria. This is associated with a TLR4- and IFN-γ-signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye-infection. Adoptive transfer experiments revealed that infection-induced monopoiesis occurs at the expense of DC-development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC-development leads to DC-depletion, possibly driving transient immunosuppression in bacterial sepsis.

Expression of stage-specific embryonic antigen-4 (SSEA-4) defines spontaneous loss of epithelial phenotype in human solid tumor cells.

Stage-specific embryonic antigen-4 (SSEA-4) is a glycosphingolipid, which is overexpressed in some cancers and has been linked to disease progression. However, little is known about the functions of SSEA-4 and the characteristics of SSEA-4 expressing tumor cells. Our studies identified SSEA-4 expression on a subpopulation of cells in many solid tumor cell lines but not in leukemic cell lines. Fluorescence-activated cell sorting-sorted SSEA-4(+) prostate cancer cells formed fibroblast-like colonies with limited cell-cell contacts, whereas SSEA-4(-) cells formed cobblestone-like epithelial colonies. Only colonies derived from SSEA-4(+) cells were enriched for pluripotent embryonic stem cell markers. Moreover, major epithelial cell-associated markers Claudin-7, E-cadherin, ESRP1 and GRHL2 were down-regulated in the SSEA-4(+) fraction of DU145 and HCT-116 cells. Similar to cell lines, SSEA-4(+) primary prostate tumor cells also showed down-regulation of epithelial cell-associated markers. In addition, they showed up-regulation of epithelial-to-mesenchymal transition as well as mesenchymal markers. Furthermore, SSEA-4(+) cells escape from adhesive colonies spontaneously and form invadopodia-like migratory structures, in which SSEA-4, cortactin as well as active pPI3K, pAkt and pSrc are enriched and colocalized. Finally, SSEA-4(+) cells displayed strong tumorigenic ability and stable knockdown of SSEA-4 synthesis resulted in decreased cellular adhesion to different extracellular matrices. In conclusion, we introduce SSEA-4 as a novel marker to identify heterogeneous, invasive subpopulations of tumor cells. Moreover, increased cell-surface SSEA-4 expression is associated with the loss of cell-cell interactions and the gain of a migratory phenotype, suggesting an important role of SSEA-4 in cancer invasion by influencing cellular adhesion to the extracellular matrix.

Characterization of a bispecific FLT3 X CD3 antibody in an improved, recombinant format for the treatment of leukemia.

FLT3 is a receptor-tyrosine-kinase that is expressed on leukemic cells of the myeloid and lymphoid lineage rather specifically. We here report on the construction and selection of bispecific FLT3 X CD3 antibodies in a new recombinant format, termed Fabsc, that resembles the normal antibody structure more closely than the well-established bispecific single chain (bssc)-format. Our preferred antibody, which emerged from an initial selection procedure utilizing different FLT3- and CD3-antibodies, contains the FLT3-antibody 4G8 and the CD3-antibody UCHT1. The 4G8 X UCHT1 Fabsc-antibody was found to be superior to a bssc-antibody with identical specificities with respect to (i) affinity to the target antigen FLT3, (ii) production yield by transfected cells, and (iii) the diminished formation of aggregates. T-cell activation in the presence and absence of cultured leukemic cells and killing of these cells was comparable for both molecules. In addition, the 4G8 X UCHT1 Fabsc-antibody was found to induce T-cell activation and efficient killing of leukemic blasts in primary peripheral blood mononuclear cell (PBMC) cultures of acute myeloid leukemia (AML) patients. In these experiments, the bispecific molecule was clearly superior to an Fc-optimized monospecific FLT3-antibody described previously, indicating that within PBMC of AML patients the recruitment of T cells is more effective than that of natural killer cells.

Both mature KIR+ and immature KIR- NK cells control pediatric acute B-cell precursor leukemia in NOD.Cg-Prkdcscid IL2rgtmWjl/Sz mice.

Therapeutic natural killer (NK)-cell-mediated alloreactivity toward acute myeloid leukemia has largely been attributed to mismatches between killer immunoglobulin-like receptors (KIRs) on NK cells and their ligands, HLA class I molecules, on target cells. While adult acute B-cell precursor leukemia (BCP-ALL) appears to be resistant to NK-cell-mediated lysis, recent data indicate that pediatric BCP-ALL might yet be a target of NK cells. In this study, we demonstrate in a donor-patient-specific NOD.Cg-Prkdc(scid) IL2rg(tmWjl)/Sz (NSG) xenotransplantation model that NK cells mediate considerable alloreactivity toward pediatric BCP-ALL in vivo. Notably, both adoptively transferred mature KIR(+) NK cells and immature KIR(-) NK cells arising early posttransplantation in humanized NSG mice exerted substantial antileukemic activity. Low-dose and long-term treatment of humanized NSG mice with the DNA-demethylating agent 5-aza-cytidine distinctly enhanced the antitumor response, interestingly without inducing common inhibitory KIR expression but rather by promoting the differentiation of various NK-cell precursor subsets. Collectively, these data indicate that the future design of innovative therapy protocols should consider further exploitation of NK-cell-mediated immune responses for poor prognosis pediatric BCP-ALL patients.

Activated platelets interfere with recruitment of mesenchymal stem cells to apoptotic cardiac cells via high mobility group box 1/Toll-like receptor 4-mediated down-regulation of hepatocyte growth factor receptor MET.

Recruitment of mesenchymal stem cells (MSC) following cardiac injury, such as myocardial infarction, plays a critical role in tissue repair and may contribute to myocardial recovery. However, the mechanisms that regulate migration of MSC to the site of tissue damage remain elusive. Here, we demonstrate in vitro that activated platelets substantially inhibit recruitment of MSC toward apoptotic cardiac myocytes and fibroblasts. The alarmin high mobility group box 1 (HMGB1) was released by platelets upon activation and mediated inhibition of the cell death-dependent migratory response through Toll-like receptor (TLR)-4 expressed on the MSC. Migration of MSC to apoptotic cardiac myocytes and fibroblasts was driven by hepatocyte growth factor (HGF), and platelet activation was followed by HMGB1/TLR-4-dependent down-regulation of HGF receptor MET on MSC, thereby impairing HGF-driven MSC recruitment. We identify a novel mechanism by which platelets, upon activation, interfere with MSC recruitment to apoptotic cardiac cells, a process that may be of particular relevance for myocardial repair and regeneration.

Surface CD24 distinguishes between low differentiated and transit-amplifying cells in the basal layer of human prostate.

BACKGROUND: Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are common abnormalities in elderly men. It is considered that epithelial stem cells are involved in the etiology and development of both diseases. To distinguish aberrant from normal cells, the knowledge about primary epithelial stem/progenitor cells (ES/P) is essential. The aim of this study was to examine the role of surface markers to distinguish between different subsets of prostate basal epithelium. METHODS: The expression pattern of prostate tissue single cell suspensions was analyzed by flow cytometry using different markers. Sorted cell populations were examined for their clonogenic capacity and the resulted colonies were analyzed with flow cytometry, Western blot, and qPCR for stem cell, basal, and luminal epithelium markers. Additionally, the histological localization of the examined markers was determined using immunofluorescence. RESULTS: Using the combination of CD49f, Trop-2, and surface CD24, basal cell subsets with distinct differentiation capacities were dissected (CD49f(+) Trop-2(+) CD24(-) and CD49f(+) Trop-2(+) CD24(+) ). Although cells from the two subsets gave rise to similar basal colonies, qPCR of primary tissue revealed that higher levels of basal marker expression were detected in the CD49f(+) Trop-2(+) CD24(-) subset. Immunofluorescence analysis showed a prominent expression of CD24 by luminal and basal cells. CONCLUSIONS: Subsets with distinct differentiation capacities within the basal epithelium (CD49f(+) Trop-2(+) CD24(-) and CD49f(+) Trop-2(+) CD24(+) ) can be distinguished in human prostate. CD24 is a marker expressed on the basal transit-amplifying cells (transition cells) and may play a role in the differentiation and migration of ES/P cells to the luminal layer. The knowledge of this mechanism is of relevance for treatment of both diseases.