Porcine Sialoadhesin (CD169/Siglec-1) Is an Endocytic Receptor that Allows Targeted Delivery of Toxins and Antigens to Macrophages

Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab')2 fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages

Identification of essential genes in human lymphopoieis

Hematopoiesis is the highly orchestrated process of blood cell formation from a small pool of multipotent hematopoietic stem cells in the bone marrow. The defining properties of hematopoietic stem cells, their self-renewal capacity and multilineage differentiation potential, form the basis for the successful restoration of the hematopoietic system by bone marrow transplantation or hematopoietic stem cell transplantation after chemotherapy or radiation therapy used to treat cancer. However, due to the slow kinetics of the restoration of the T-cell compartment, patients are temporally vulnerable to infection. Therefore, strategies to enhance T-cell development from HSCs could be of great therapeutic value. Signalling through the Notch-1 transmembrane receptor has been identified as a critical determinant for the lineage choice between B- and T-cell development. Overexpression of the active form of Notch-1 (ICN) in human CD34+ hematopoietic stem cells blocks B-cell development and drives them into T-lymphoid differentiation. In this work we show that physiological stimulation of the Notch pathway by coculturing human CD34+ progenitor cells on a stromal cell layer ectopically expressing the Notch ligand Delta-like-1 also induces T-cell differentiation of human cells. Inversely, by inhibiting physiological Notch signalling during in vitro T-cell differentiation in fetal thymus organ culture using γ- secretase inhibitors we show that Notch signalling is essential for human T-cell development. Because constitutive Notch-1 expression ultimately leads to the development of T-cell leukemias, manipulation of stem cells with ICN cannot be applied clinically. Therefore we investigated whether the Notch-1 target gene HES-1 is able to substitute for Notch-1 signalling in inducing T-cell differentiation of human CD34+ hematopoietic stem cells. Our results demonstrate that overexpression of HES-1 alone is not sufficient to impose T-cell differentiation on human hematopoietic stem cells. The identification of a small lymphoid-committed cell fraction in human umbilical cord blood may also lead to therapeutic applications. We show that CD34+CD38-CD7+ cells have strong T-cell differentiation potential. To identify genes that regulate the lymphoid commitment step we compared the gene expression between CD34+CD38-CD7+ lymphoidcommitted progenitors and CD34+CD38-CD7- multipotent stem cells using Affymetrix oligonucleotide microarrays. Overexpression and silencing studies of selected differentially expressed genes will have to be performed to determine their role in lymphoid development and whether they can be used to instruct lymphoid development

Different thresholds of Notch signaling bias human precursor cells toward B-, NK-, monocytic/dendritic-, or T-cell lineage in thymus microenvironment

Notch receptors are involved in lineage decisions in multiple developmental scenarios, including hematopoiesis. Here, we treated hybrid human-mouse fetal thymus organ culture with the gamma-secretase inhibitor 7 (N-[N-(3,5-difluorophenyl)-Lalanyl]-S-phenyl-glycine t-butyl ester) (DAPT) to establish the role of Notch signaling in human hematopoietic lineage decisions. The effect of inhibition of Notch signaling was studied starting from cord blood CD34(+) or thymic CD34(+)CD1(-), CD34(+)CD1(+), or CD4ISP progenitors. Treatment of cord blood CD34(+) cells with low DAPT concentrations results in aberrant CD4ISP and CD4/CD8 double-positive (DP) thymocytes, which are negative for intracellular T-cell receptor beta (TCR beta). On culture with intermediate and high DAPT concentrations, thymic CD34(+)CD1(-) cells still generate aberrant intracellular TCR beta(-) DIP cells that have undergone DJ but not VDJ recombination. Inhibition of Notch signaling shifts differentiation into non-T cells in a thymic microenvironment, depending on the starting progenitor cells: thymic CD34(+)CD1(+) cells do not generate non-T cells, thymic CD34(+)CD1(-) cells generate INK cells and monocytic/dendritic cells, and cord blood CD34(+)Lin(-) cells generate B, INK, and monocytic/dendritic cells in the presence of DAPT Our data indicate that Notch signaling is crucial to direct human progenitor cells into the T-cell lineage, whereas it has a negative impact on B, NK, and monocytic/dendritic cell generation in a dose-dependent fashion

Overexpression of HES-1 is not sufficient to impose T-cell differentiation on human hematopoietic stem cells.

By retroviral overexpression of the Notch-1 intracellular domain (ICN) in human CD34+ hematopoietic stem cells (HSCs), we have shown previously that Notch-1 signaling promotes the T-cell fate and inhibits the monocyte and B-cell fate in several in vitro and in vivo differentiation assays. Here, we investigated whether the effects of constitutively active Notch-1 can be mimicked by overexpression of its downstream target gene HES1. Upon HES-1 retroviral transduction, human CD34+ stem cells had a different outcome in the differentiation assays as compared to ICN-transduced cells. Although HES-1 induced a partial block in B-cell development, it did not inhibit monocyte development and did not promote T/NK-cell-lineage differentiation. On the contrary, a higher percentage of HES-1-transduced stem cells remained CD34+. These experiments indicate that HES-1 alone is not able to substitute for Notch-1 signaling to induce T-cell differentiation of human CD34+ hematopoietic stem cells.Journal Articleinfo:eu-repo/semantics/publishe

T-, B- and NK-lymphoid, but not myeloid cells arise from human CD34+CD38-CD7+ common lymphoid progenitors expressing lymphoid-specific genes

Hematopoietic stem cells in the bone marrow ( BM) give rise to all blood cells. According to the classic model of hematopoiesis, the differentiation paths leading to the myeloid and lymphoid lineages segregate early. A candidate 'common lymphoid progenitor' (CLP) has been isolated from CD34(+)CD38(-) human cord blood cells based on CD7 expression. Here, we confirm the B- and NK-differentiation potential of CD34(+)CD38(-)CD7(-) cells and show in addition that this population has strong capacity to differentiate into T cells. As CD34(+)CD38(-)CD7(+) cells are virtually devoid of myeloid differentiation potential, these cells represent true CLPs. To unravel the molecular mechanisms underlying lymphoid commitment, we performed genome-wide gene expression profiling on sorted CD34(+)CD38(-)CD7(+) and CD34(+)CD38(-)CD7(+) cells. Interestingly, lymphoid-affiliated genes were mainly upregulated in the CD7(+) population, while myeloid-specific genes were downregulated. This supports the hypothesis that lineage commitment is accompanied by the shutdown of inappropriate gene expression and the upregulation of lineage-specific genes. In addition, we identified several highly expressed genes that have not been described in hematopoiesis before