Sarah E. Funk

Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice.

SPARC (Secreted Protein that is Acidic and Rich in Cysteine), a Ca++-binding glycoprotein also known as osteonectin, is produced in significant amounts by injured or proliferating cells in vitro. To elucidate the possible function of SPARC in growth and remodeling, we examined its distribution in embryonic and adult murine tissues. Immunohistochemistry on adult mouse tissues revealed a preferential association of SPARC protein with epithelia exhibiting high rates of turnover (gut, skin, and glandular tissue). Fetal tissues containing high levels of SPARC included heart, thymus, lung, and gut. In the 14-18-day developing fetus, SPARC expression was particularly enhanced in areas undergoing chondrogenesis, osteogenesis, and somitogenesis, whereas 10-day embryos exhibited selective staining for this protein in Reicherts membrane, maternal sinuses, and trophoblastic giant cells. SPARC displayed a Ca++-dependent affinity for hydrophobic surfaces and was not incorporated into the extracellular matrix produced by cells in vitro. We propose that in some tissues SPARC associates with cell surfaces to facilitate proliferation during embryonic morphogenesis and normal cell turnover in the adult.

Regulation of human monocyte matrix metalloproteinases by SPARC

SPARC (secreted protein, acidic and rich in cysteine), also called osteonectin or BM‐40, is a collagen‐binding glycoprotein secreted by a variety of cells and is associated with functional responses involving tissue remodeling, cell movement and proliferation. Because SPARC and monocytes/macrophages are prevalent at sites of inflammation and remodeling in which there is connective tissue turnover, we examined the effect of SPARC on monocyte matrix metalloproteinase (MMP) production. Treatment of human peripheral blood monocytes with SPARC stimulated the production of gelatinase B (MMP‐9) and interstitial collagenase (MMP‐1). Experiments with synthetic peptides indicated that peptide 3.2, belonging to the alpha helical domain III of SPARC, is the major peptide mediating the MMP production by monocytes. SPARC and peptide 3.2 were also shown to induce prostaglandin synthase (PGHS)‐2 as determined by Western and Northern blot analyses. The increase in PGHS‐2 stimulated by SPARC or peptide 3.2 correlated with substantially elevated levels of prostaglandin E2 (PGE2) and other arachidonic acid metabolites as measured by radioimmunoassay and high performance liquid chromatography (HPLC), respectively. Moreover, the synthesis of MMP was dependent on the generation of PGE2 by PGHS‐2, since indomethacin inhibited the production of these enzymes and their synthesis was restored by addition of exogenous PGE2 or dibutyryl cAMP (Bt2cAMP). These results demonstrate that SPARC might play a significant role in the modulation of connective tissue turnover due to its stimulation of PGHS‐2 and the subsequent release of PGE2, a pathway that leads to the production of MMP by monocytes. J. Cell. Physiol. 173:327–334, 1997. Published 1997 Wiley‐Liss, Inc. This article was prepared by a group of United States government employees and non‐United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

SPARC: a Ca2+-binding extracellular protein associated with endothelial cell injury and proliferation

SPARC (Secreted Protein that is Acidic and Rich in Cysteine) is a Ca2+-binding, stress-related protein released in vitro by both malignant and normal cells derived from all primordial germ layers. It is specifically elevated in endothelial cells as a result of culture shock (characterized by high levels of proliferation and migration) and exhibits density-dependent secretion. Exposure of bovine aortic endothelial cells to endotoxin results in a 70-100% increase in secreted protein, with a three-fold increase in SPARC. Immunofluorescence histochemistry on mouse tissues revealed (a) a preferential association of SPARC with highly proliferative cells (e.g., gut epithelia, mammary gland, and epidermis), (b) a cell surface or stromal location in thymus, lung, and salivary gland, (c) staining of epididymidal epithelium and testicular cells, and (d) an association with somites of 14 d mouse embryos. We envision SPARC as an extracellular modulator of Ca2+ and other cation-sensitive proteins/proteinases, that facilitates cellular proliferation in response to injury and to developmental signals.

HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells

Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this missing-self response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8+ T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.

Cell-Intrinsic Abrogation of TGF-β Signaling Delays but Does Not Prevent Dysfunction of Self/Tumor-Specific CD8 T Cells in a Murine Model of Autochthonous Prostate Cancer

Adoptive T cell therapy (ACT) for the treatment of established cancers is actively being pursued in clinical trials. However, poor in vivo persistence and maintenance of antitumor activity of transferred T cells remain major problems. TGF-β is a potent immunosuppressive cytokine that is often expressed at high levels within the tumor microenvironment, potentially limiting T cell-mediated antitumor activity. In this study, we used a model of autochthonous murine prostate cancer to evaluate the effect of cell-intrinsic abrogation of TGF-β signaling in self/tumor-specific CD8 T cells used in ACT to target the tumor in situ. We found that persistence and antitumor activity of adoptively transferred effector T cells deficient in TGF-β signaling were significantly improved in the cancerous prostate. However, over time, despite persistence in peripheral lymphoid organs, the numbers of transferred cells in the prostate decreased and the residual prostate-infiltrating T cells were no longer functional. These findings reveal that TGF-β negatively regulates the accumulation and effector function of transferred self/tumor-specific CD8 T cells and highlight that, when targeting a tumor Ag that is also expressed as a self-protein, additional substantive obstacles are operative within the tumor microenvironment, potentially hampering the success of ACT for solid tumors.

Nuclease-free Adeno-Associated Virus-Mediated Il2rg Gene Editing in X-SCID Mice

X-linked severe combined immunodeficiency (X-SCID) has been successfully treated by hematopoietic stem cell (HSC) transduction with retroviral vectors expressing the interleukin-2 receptor subunit gamma gene (IL2RG), but several patients developed malignancies due to vector integration near cellular oncogenes. This adverse side effect could in principle be avoided by accurate IL2RG gene editing with a vector that does not contain a functional promoter or IL2RG gene. Here, we show that adeno-associated virus (AAV) gene editing vectors can insert a partial Il2rg cDNA at the endogenous Il2rg locus in X-SCID murine bone marrow cells and that these exxa0vivo-edited cells repopulate transplant recipients and produce CD4+ and CD8+ Txa0cells. Circulating, edited lymphocytes increased over time and appeared in secondary transplant recipients, demonstrating successful editing in long-term repopulating cells. Random vector integration events were nearly undetectable, and malignant transformation of the transplanted cells was not observed. Similar editing frequencies were observed in human hematopoietic cells. Our results demonstrate that therapeutically relevant HSC gene editing can be achieved by AAV vectors in the absence of site-specific nucleases and suggest that this may be a safe and effective therapy for hematopoietic diseases where inxa0vivo selection can increase edited cell numbers.