Transgenic expression of the Ly49A natural killer cell receptor confers class I major histocompatibility complex (MHC)-specific inhibition and prevents bone marrow allograft rejection.

Natural killer (NK) cells and some T cells are endowed with receptors specific for class I major histocompatibility complex (MHC) molecules that can inhibit cellular effector functions. The function of the Ly49 receptor family has been studied in vitro, but no gene transfer experiments have directly established the role of these receptors in NK cell functions. We show here that transgenic expression of the H-2Dd-specific Ly49A receptor in all NK cells and T cells conferred class I-specific inhibition of NK cell-mediated target cell lysis as well as of T cell proliferation. Furthermore, transgene expression prevented NK cell-mediated rejection of allogeneic H-2d bone marrow grafts by irradiated mice. These results demonstrate the function and specificity of Ly49 receptors in vivo, and establish that their subset-specific expression is necessary for the discrimination of MHC-different cells by NK cells in unmanipulated mice

A novel element upstream of the Vgamma2 gene in the murine T cell receptor gamma locus cooperates with the 3 enhancer to act as a locus control region.

Transgenic expression constructs were employed to identify a cis-acting transcription element in the T cell receptor (TCR)-gamma locus, called HsA, between the Vgamma5 and Vgamma2 genes. In constructs lacking the previously defined enhancer (3E(Cgamma1)), HsA supports transcription in mature but not immature T cells in a largely position-independent fashion. 3E(Cgamma1), without HsA, supports transcription in immature and mature T cells but is subject to severe position effects. Together, the two elements support expression in immature and mature T cells in a copy number-dependent, position-independent fashion. Furthermore, HsA was necessary for consistent rearrangement of transgenic recombination substrates. These data suggest that HsA provides chromatin-opening activity and, together with 3E(Cgamma1), constitutes a T cell-specific locus control region for the TCR-gamma locus

Developmentally Programmed Rearrangement of T Cell Receptor Vγ Genes Is Controlled by Sequences Immediately Upstream of the Vγ Genes

AbstractDistinct subsets of γδ T cells expressing different Vγ and Vδ chains arise in ordered waves during thymic development. In the murine Jγ1–Cγ1 cluster, the Vγ3 gene segment is utilized earliest in fetal thymic development, in progenitors of dendritic epidermal T cells (DECs). The Vγ2 gene segment predominates in the late fetal stages and beyond, in cells destined for the secondary lymphoid organs. Using transgenic TCRγ recombination substrates, we demonstrate that this restricted Vγ gene usage is determined by developmentally targeted gene rearrangement. We show that sequences immediately upstream of the Vγ2 and Vγ3 genes direct the rearrangement pattern in adult thymocytes. Thus, the choice of Vγ gene for recombination is coordinated with distinct differentiation programs in γδ subsets

A Novel Element Upstream of the Vγ2 Gene in the Murine T Cell Receptor γ Locus Cooperates with the 3′ Enhancer to Act as a Locus Control Region

Transgenic expression constructs were employed to identify a cis-acting transcription element in the T cell receptor (TCR)-γ locus, called HsA, between the Vγ5 and Vγ2 genes. In constructs lacking the previously defined enhancer (3′ECγ1), HsA supports transcription in mature but not immature T cells in a largely position-independent fashion. 3′ECγ1, without HsA, supports transcription in immature and mature T cells but is subject to severe position effects. Together, the two elements support expression in immature and mature T cells in a copy number–dependent, position-independent fashion. Furthermore, HsA was necessary for consistent rearrangement of transgenic recombination substrates. These data suggest that HsA provides chromatin-opening activity and, together with 3′ECγ1, constitutes a T cell–specific locus control region for the TCR-γ locus

Polysaccharide films built by simultaneous or alternate spray: a rapid way to engineer biomaterial surfaces.

We investigated polysaccharide films obtained by simultaneous and alternate spraying of a chitosan (CHI) solution as polycation and hyaluronic acid (HA), alginate (ALG), and chondroitin sulfate (CS) solutions as polyanions. For simultaneous spraying, the film thickness increases linearly with the cumulative spraying time and passes through a maximum for polyanion/CHI molar charge ratios lying between 0.6 and 1.2. The size of polyanion/CHI complexes formed in solution was compared with the simultaneously sprayed film growth rate as a function of the polyanion/CHI molar charge ratio. A good correlation was found. This suggests the importance of polyanion/polycation complexation in the simultaneous spraying process. Depending on the system, the film topography is either liquid-like or granular. Film biocompatibility was evaluated using human gingival fibroblasts. A small or no difference is observed in cell viability and adhesion between the two deposition processes. The CHI/HA system appears to be the best for cell adhesion inducing the clustering of CD44, a cell surface HA receptor, at the membrane of cells. Simultaneous or alternate spraying of CHI/HA appears thus to be a convenient and fast procedure for biomaterial surface modifications.journal articleresearch support, non-u.s. gov't2012 Jun 052012 05 23importe

Comparative Study of Hematopoietic Differentiation between Human Embryonic Stem Cell Lines

Directed differentiation of human embryonic stem cells (hESCs) into any desired cell type has been hailed as a therapeutic promise to cure many human diseases. However, substantial roadblocks still exist for in vitro differentiation of hESCs into distinct cell types, including T lymphocytes. Here we examined the hematopoietic differentiation potential of six different hESC lines. We compare their ability to develop into CD34+ or CD34+CD45+ hematopoietic precursor populations under several differentiation conditions. Comparison of lymphoid potential of hESC derived- and fetal tissue derived-hematopoietic precursors was also made. We found diverse hematopoietic potential between hESC lines depending on the culture or passage conditions. In contrast to fetal-derived hematopoietic precursors, none of the CD34+ precursors differentiated from hESCs were able to develop further into T cells. These data underscore the difficulties in the current strategy of hESC forward differentiation and highlight distinct differences between CD34+ hematopoietic precursors generated in vitro versus in vivo

Transgenic expression of the Ly49A natural killer cell receptor confers class I major histocompatibility complex (MHC)-specific inhibition and prevents bone marrow allograft rejection.

Natural killer (NK) cells and some T cells are endowed with receptors specific for class I major histocompatibility complex (MHC) molecules that can inhibit cellular effector functions. The function of the Ly49 receptor family has been studied in vitro, but no gene transfer experiments have directly established the role of these receptors in NK cell functions. We show here that transgenic expression of the H-2Dd-specific Ly49A receptor in all NK cells and T cells conferred class I-specific inhibition of NK cell-mediated target cell lysis as well as of T cell proliferation. Furthermore, transgene expression prevented NK cell-mediated rejection of allogeneic H-2d bone marrow grafts by irradiated mice. These results demonstrate the function and specificity of Ly49 receptors in vivo, and establish that their subset-specific expression is necessary for the discrimination of MHC-different cells by NK cells in unmanipulated mice