CD34 expression on murine marrow-derived mesenchymal stromal cells: impact on neovascularization.

OBJECTIVE: CD34 is a sialomucin often expressed by cells with hemangiopoietic potential and widely serves as a surrogate marker of stem cell potential. Mesenchymal stromal cells (MSCs) also express CD34, although the functional significance of its expression remains undefined. In this study, we determined whether CD34(pos) MSCs are functionally distinct from CD34(null) MSCs. MATERIALS AND METHODS: MSCs derived from C57Bl/6 mice were transduced to express the green fluorescent protein (GFP) from which pure CD34(pos) MSC and CD34(null) MSC clones were selected. In vitro, clones were examined by microarray analysis, while in vivo subcutaneous implantation of matrix-embedded MSCs was used to assess cell survival, differentiation, and neovascularization. RESULTS: The flow cytometric phenotype of CD34(pos) and CD34(null) MSCs were similar, as was gene expression of vascular endothelial growth factors (VEGFs) A and B. However, CD34(pos) MSCs upregulated a number of supplementary angiogenesis-associated genes and showed a greater expression of gene associated with vascular differentiation. At 15 days postimplantation, cell survival between CD34(pos) and CD34(null) MSCs was similar, however, CD34(pos) MSCs evoked a significantly greater host-derived response (4.2 +/- 0.7 vs 1.9 +/- 0.5 x 10(6) cells; p < 0.05). GFP-expressing CD34(pos) MSC implants acquired significantly more CD31 expression compared to CD34(null) MSC cells (10.7% +/- 8.4% vs 3.1% +/- 0.6%; p < 0.05), as well as a significantly greater host-derived endothelial cell influx (CD31(+)/CD45(-)). CONCLUSION: CD34 expression by MSCs correlates with enhanced vasculogenic and angiogenic potential in vivo.

Plasminogen Kringle 5 blocks tumor progression by antiangiogenic and proinflammatory pathways.

Proteolytic processing of human plasminogen generates potent antiangiogenic peptides such as angiostatin. The plasminogen kringle 5 (K5) domain, which is distinct from angiostatin, possesses potent antiangiogenic properties on its own, which can be exploited in cancer therapy. It has been recently observed that antiangiogenic agents promote leukocyte-vessel wall interaction as part of their antitumor effect. Although we have previously shown that K5 suppresses cancer growth in tumor xenograft models, its modulation of inflammation in experimental mice with intact immune systems is unknown. To determine whether K5 possesses immune proinflammatory properties, we investigated the effects of K5 in an immune competent model of breast cancer and observed that tumor rejection is substantially reduced in nonobese diabetic/severe combined immunodeficient and BALB/c nude when compared with wild-type BALB/c mice, suggesting an important role for T-lymphoid cells in the antitumor effect of K5. Tumor explant analysis shows that K5 enhances tumor recruitment of CD3(+) lymphoid cells, in particular, the NKT phenotype. We also observed a significant decrease in tumor-associated microvessel length and density consistent with antiangiogenic activity. Histologic analysis of K5 tumors also revealed a robust neutrophilic infiltrate, which may be explained by the neutrophil chemotactic activity of K5 as well as its ability to promote CD64 up-regulation within the CD11b(+) adhesive neutrophil population. In sum, our findings confirm that the K5 protein acts as a potent angiostatic agent and possesses a novel proinflammatory role via its ability to recruit tumor-associated neutrophils and NKT lymphocytes, leading to a potent antitumor response.

A GMCSF and IL-15 fusokine leads to paradoxical immunosuppression in vivo via asymmetrical JAK/STAT signaling through the IL-15 receptor complex.

We hypothesized that a granulocyte macrophage colony-stimulating factor (GMCSF) and interleukin 15 (IL-15) fusokine (GIFT15) would possess greater immune-stimulatory properties than their combined use. Unexpectedly, tumor cells engineered to secrete GIFT15 protein led to suppression of natural killer (NK) and NKT-cell recruitment in vivo, suggesting an unanticipated immune-suppressive effect. We found GIFT15 to have pleiotropic effects on an array of immune-competent cells. Among these, macrophages treated with GIFT15 secrete de novo the tissue inhibitor of metalloproteinase-2 (TIMP-2); activated matrix metalloproteinase-2 (MMP-2); transforming growth factor-beta (TGF-beta); as well as vascular endothelial growth factor (VEGF). We show that the GIFT15 fusokine has increased affinity for the alpha chain component of the IL-15R, leading to aberrant signaling through the beta chain manifested by the hyperphosphorylation of STAT3 both in macrophages and splenocytes. Suppression of common gamma chain-mediated STAT5 phosphorylation and blockade of the IL-15-dependent IFN-gamma response in mouse splenocytes were also observed. We tested GIFT15 as an immunosuppressor and demonstrated that it allowed engraftment of allogeneic B16F0 and human xenograft U87GM glioma cells in immunocompetent mice. Thus, GIFT15 defines a new class of fusokine that mediates proangiogenic and immunosuppressive effects via aberrant signaling by the IL-15R in lymphomyeloid cells.

A self-inactivating retrovector incorporating the IL-2 promoter for activation-induced transgene expression in genetically engineered T-cells.

BACKGROUND: T-cell activation leads to signaling pathways that ultimately result in induction of gene transcription from the interleukin-2 (IL-2) promoter. We hypothesized that the IL-2 promoter or its synthetic derivatives can lead to T-cell specific, activation-induced transgene expression. Our objective was to develop a retroviral vector for stable and activation-induced transgene expression in T-lymphocytes. RESULTS: First, we compared the transcriptional potency of the full-length IL-2 promoter with that of a synthetic promoter composed of 3 repeats of the Nuclear Factor of Activated T-Cells (NFAT) element following activation of transfected Jurkat T-cells expressing the large SV40 T antigen (Jurkat TAg). Although the NFAT3 promoter resulted in a stronger induction of luciferase reporter expression post stimulation, the basal levels of the IL-2 promoter-driven reporter expression were much lower indicating that the IL-2 promoter can serve as a more stringent activation-dependent promoter in T-cells. Based on this data, we generated a self-inactivating retroviral vector with the full-length human IL-2 promoter, namely SINIL-2pr that incorporated the enhanced green fluorescent protein (EGFP) fused to herpes simplex virus thymidine kinase as a reporter/suicide "bifunctional" gene. Subsequently, Vesicular Stomatitis Virus-G Protein pseudotyped retroparticles were generated for SINIL-2pr and used to transduce the Jurkat T-cell line and the ZAP-70-deficient P116 cell line. Flow cytometry analysis showed that EGFP expression was markedly enhanced post co-stimulation of the gene-modified cells with 1 muM ionomycin and 10 ng/ml phorbol 12-myristate 13-acetate (PMA). This activation-induced expression was abrogated when the cells were pretreated with 300 nM cyclosporin A. CONCLUSION: These results demonstrate that the SINIL-2pr retrovector leads to activation-inducible transgene expression in Jurkat T-cell lines. We propose that this design can be potentially exploited in several cellular immunotherapy applications.

Cooperation of matrix metalloproteinases with the RhoA/Rho kinase and mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase signaling pathways is required for the sphingosine-1-phosphate-induced mobilization of marrow-derived stromal cells.

The ease of isolation and ex vivo culture of marrow-derived stromal cells (MSCs) from adult bone marrow renders them a very promising source of adult stem cells for gene transfer and cell therapy. However, little is known about the signaling pathways that control their in vivo mobilization and subsequent biodistribution. Platelet-derived sphingosine-1-phosphate (S1P), a bioactive lipid that acts via G-protein-coupled-receptors, exerts strong chemoattraction upon MSCs through yet-uncharacterized signaling pathways. We show that the S1P-induced migration and morphological changes of MSCs in vitro require the activities of extracellular signal-regulated kinase (ERK), Rho kinase (ROCK), and matrix metalloproteinase (MMP) signaling molecules. Specifically, S1P-induced remodeling of the MSC cytoskeleton led to the rapid (<1 minute) formation of actin stress fibers via activation of the RhoA/ROCK pathway and required the catalytic activity of MMPs. S1P-induced activation of the mitogen-activated protein kinase kinase-1 (MEK1)/ERK pathway also contributed to the induction of the actin stress fibers and to the redistribution of paxillin at the focal adhesions through tyrosine phosphorylation of focal adhesion kinase in an MMP-dependent manner. Moreover, MMP- and ROCK-dependent molecular events are implicated in the regulation of the S1P-induced activation of ERK. Our results demonstrate that MSC mobilization in response to S1P requires cooperation between MMP-mediated signaling events and the RhoA/ROCK and MEK1/ERK intracellular pathways. Therefore, the characterization of the cellular factors and the intracellular signaling pathways underlying MSC mobilization is crucial to achieve high efficacy in therapeutic use.

Plasminogen kringle 5-engineered glioma cells block migration of tumor-associated macrophages and suppress tumor vascularization and progression.

Angiostatin, a well-characterized angiostatic agent, is a proteolytic cleavage product of human plasminogen encompassing the first four kringle structures. The fifth kringle domain (K5) of human plasminogen is distinct from angiostatin and has been shown, on its own, to act as a potent endothelial cell inhibitor. We propose that tumor-targeted K5 cDNA expression may act as an effective therapeutic intervention as part of a cancer gene therapy strategy. In this study, we provide evidence that eukaryotically expressed His-tagged human K5 cDNA (hK5His) is exported extracellularly and maintains predicted disulfide bridging conformation in solution. Functionally, hK5His protein produced by retrovirally engineered human U87MG glioma cells suppresses in vitro migration of both human umbilical vein endothelial cells and human macrophages. Subcutaneous implantation of Matrigel-embedded hK5His-producing glioma cells in nonobese diabetic/severe combined immunodeficient mice reveals that hK5His induces a marked reduction in blood vessel formation and significantly suppresses the recruitment of tumor-infiltrating CD45+ Mac3+ Gr1- macrophages. Therapeutically, we show in a nude mouse orthotopic brain cancer model that tumor-targeted K5 expression is capable of effectively suppressing glioma growth and promotes significant long-term survival (>120 days) of test animals. These data suggest that plasminogen K5 acts as a novel two-pronged anticancer agent, mediating its inhibitory effect via its action on host-derived endothelial cells and tumor-associated macrophages, resulting in a potent, clinically relevant antitumor effect.

Allogeneic marrow stromal cells are immune rejected by MHC class I- and class II-mismatched recipient mice.

It has been suggested that marrow stromal cells (MSCs) may be immunoprivileged and can engraft in allogeneic recipients with intact immune systems. We determined if the implantation of murine MSCs engineered to release erythropoietin (Epo) would be feasible in major histocompatibility complex (MHC)-mismatched allogeneic mice without immunosuppression, and we monitored hematocrit (Hct) as a reporter of MSC graft survival. MSCs from C57Bl/6 mice were engineered to release murine Epo (Epo+ MSCs) and implanted subcutaneously in either syngeneic C57Bl/6 mice or MHC-mismatched Balb/c mice. In syngeneic recipients, the Hct rapidly rose from baseline level and remained higher than .88 (88%) for more than 200 days. However, in MHC-mismatched recipient Balb/c mice, the Hct rose transiently and rapidly declined to baseline values. Repeat implantations in these same mice were associated with an acquired refractoriness in the Hct response consistent with alloimmunization to donor Epo+ MSCs. Allogeneic MSC implants had an increased proportion of host-derived lymphoid CD8+, natural killer T (NKT), and NK infiltrating cells compared with syngeneic controls, and splenocytes isolated from Balb/c mice that had received implants also displayed a significant interferon-gamma (IFNgamma) response to C57Bl/6 MSCs in vitro. These results strongly suggest that MSCs are not intrinsically immunoprivileged and cannot serve as a "universal donor" in immunocompetent MHC-mismatched recipients.

Human-compatible collagen matrix for prolonged and reversible systemic delivery of erythropoietin in mice from gene-modified marrow stromal cells.

Bone marrow stromal cells (MSCs) can be exploited therapeutically in transgenic cell therapy approaches. Our aim was to determine if gene-modified MSCs sequestered within a clinically approved, bovine type I collagen-based viscous bulking material could serve as a retrievable implant for systemic delivery of erythropoietin (Epo). To test this hypothesis, we embedded Epo-secreting MSCs in viscous collagen (Contigen) and determined the pharmacological effect following implantation in normal mice. Primary MSCs from C57Bl/6 mice were retrovirally engineered to express murine Epo (mEpo) and 10(7) cells of a clonal population secreting 3 U of mEpo/10(6) cells/24 h were implanted subcutaneously in normal C57Bl/6 mice with and without viscous collagen. Without matrix support, Hct rose to >70% for <25 days and returned to baseline by 60 days. However, in mice implanted with viscous collagen-embedded MSCs, the Hct rose to >70% up to 203 days postimplantation (P < 0.0001). In parallel, plasma Epo concentration was significantly increased (P < 0.05) for >145 days. Moreover, surgical removal of the viscous collagen organoid 24 days after implantation led to reduction of Hct to baseline levels within 14 days. In conclusion, this investigation demonstrates that mEpo(+) MSCs embedded in a human-compatible viscous collagen matrix offers a potent, durable, and reversible approach for delivery of plasma-soluble therapeutic proteins.

Marrow stromal cells for interleukin-2 delivery in cancer immunotherapy.

Marrow stromal cells (MSCs) can be easily gene-modified and clonally expanded making them ideal candidates for transgenic cell therapy. However, recent reports suggest that MSCs possess immunosuppressive effects, which may limit their clinical applications. We investigated whether interleukin (IL)-2 gene-modified MSCs can be used to mount an effective immune response against the poorly immunogenic B16 melanoma model. We first show that primary MSCs mixed with B16 cells and injected subcutaneously in syngeneic recipients do not affect tumor growth. On the other hand, IL-2-producing MSCs mixed with B16 cells significantly delayed tumor growth in an IL-2 dose-dependent manner. Furthermore, we observed that matrix-embedded IL-2-producing MSCs injected in the vicinity of preestablished B16 tumors led to absence of tumor growth in 90% of treated mice (p < 0.001). We demonstrated that tumor-bearing mice treated with IL-2-producing MSCs developed CD8-mediated tumor-specific immunity and significantly delayed tumor growth of a B16 cell challenge (p < 0.05). In addition, treatment of cd8-/-, cd4-/- and beige mice revealed that CD8+ and natural killer (NK) cells, but not CD4+ cells, were required to achieve antitumor effect. In conclusion, MSCs can be exploited to deliver IL-2 and generate effective immune responses against melanoma in mice with normal immune systems.

Human CD4 expression at the late single-positive stage of thymic development supports T cell maturation and peripheral export in CD4-deficient mice.

Positive selection of developing thymocytes is initiated at the double-positive (DP) CD4(+)CD8(+) stage of their maturation. Accordingly, expression of a human CD4 (hCD4) transgene beginning at the DP stage has been shown to restore normal T cell development and function in CD4-deficient mice. However, it is unclear whether later onset CD4 expression would still allow such a restoration. To investigate this issue, we used transgenic mice in which a hCD4 transgene is not expressed on DP, but only on single-positive cells. By crossing these animals with CD4-deficient mice, we show that late hCD4 expression supports the maturation of T cell precursors and the peripheral export of mature TCRalphabeta(+) CD8(-) T cells. These results were confirmed in two different MHC class II-restricted TCR transgenic mice. T cells arising by this process were functional in the periphery because they responded to agonist peptide in vivo. Interestingly, thymocytes of these mice appeared refractory to peptide-induced negative selection. Together, these results indicate that the effect of CD4 on positive selection of class II-restricted T cells extends surprisingly late into the maturation process by a previously unrecognized pathway of differentiation, which might contribute to the generation of autoreactive T cells.