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Michael C Jensen

Publications and source records attributed to Michael C Jensen.

16 recordsLinked to original sources
PubMed2006

Loss of tumor suppressor PTEN function increases B7-H1 expression and immunoresistance in glioma.

Cancer immunoresistance and immune escape may play important roles in tumor progression and pose obstacles for immunotherapy. Expression of the immunosuppressive protein B7 homolog 1 (B7-H1), also known as programmed death ligand-1 (PD-L1), is increased in many pathological conditions, including cancer. Here we show that expression of the gene encoding B7-H1 increases post transcriptionally in human glioma after loss of phosphatase and tensin homolog (PTEN) and activation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway. Tumor specimens from individuals with glioblastoma multiforme (GBM) had levels of B7-H1 protein that correlated with PTEN loss, and tumor-specific T cells lysed human glioma targets expressing wild-type PTEN more effectively than those expressing mutant PTEN. These data identify a previously unrecognized mechanism linking loss of the tumor suppressor PTEN with immunoresistance, mediated in part by B7-H1.

Analysis of Variance↗
PubMed2006

CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells.

Chimeric antigen receptors (CAR) combine an antigen-binding domain with a CD3-zeta signaling motif to redirect T-cell specificity to clinically important targets. First-generation CAR, such as the CD19-specific CAR (designated CD19R), may fail to fully engage genetically modified T cells because activation is initiated by antigen-dependent signaling through chimeric CD3-zeta, independent of costimulation through accessory molecules. We show that enforced expression of the full-length costimulatory molecule CD28 in CD8(+)CD19R(+)CD28(-) T cells can restore fully competent antigen-dependent T-cell activation upon binding CD19(+) targets expressing CD80/CD86. Thus, to provide costimulation to T cells through a CD19-specific CAR, independent of binding to CD80/CD86, we developed a second-generation CAR (designated CD19RCD28), which includes a modified chimeric CD28 signaling domain fused to chimeric CD3-zeta. CD19R(+) and CD19RCD28(+) CD8(+) T cells specifically lyse CD19(+) tumor cells. However, the CD19RCD28(+) CD8(+) T cells proliferate in absence of exogenous recombinant human interleukin-2, produce interleukin-2, propagate, and up-regulate antiapoptotic Bcl-X(L) after stimulation by CD19(+) tumor cells. For the first time, we show in vivo that adoptively transferred CD19RCD28(+) T cells show an improved persistence and antitumor effect compared with CD19R(+) T cells. These data imply that modifications to the CAR can result in improved therapeutic potential of CD19-specific T cells expressing this second-generation CAR.

Animals↗
PubMed2005

Differentiation of naive cord-blood T cells into CD19-specific cytolytic effectors for posttransplantation adoptive immunotherapy.

Disease relapse is a barrier to achieving therapeutic success after unrelated umbilical cord-blood transplantation (UCBT) for B-lineage acute lymphoblastic leukemia (B-ALL). While adoptive transfer of donor-derived tumor-specific T cells is a conceptually attractive approach to eliminating residual disease after allogeneic hematopoietic stem cell transplantation, adoptive immunotherapy after UCBT is constrained by the difficulty of generating antigen-specific T cells from functionally naive umbilical cord-blood (UCB)-derived T cells. Therefore, to generate T cells that recognize B-ALL, we have developed a chimeric immunoreceptor to redirect the specificity of T cells for CD19, a B-lineage antigen, and expressed this transgene in UCB-derived T cells. An ex vivo process, which is compliant with current good manufacturing practice for T-cell trials, has been developed to genetically modify and numerically expand UCB-derived T cells into CD19-specific effector cells. These are capable of CD19-restricted cytokine production and cytolysis in vitro, as well as mediating regression of CD19+ tumor and being selectively eliminated in vivo. Moreover, time-lapse microscopy of the genetically modified T-cell clones revealed an ability to lyse CD19+ tumor cells specifically and repetitively. These data provide the rationale for infusing UCB-derived CD19-specific T cells after UCBT to reduce the incidence of CD19+ B-ALL relapse.

Animals↗
PubMed2005

Biophotonic cytotoxicity assay for high-throughput screening of cytolytic killing.

We have developed a highly sensitive biophotonic luciferase assay as an alternative to (51)Cr-release for assessment of cell-mediated cytotoxicity. The luciferin/ATP-dependent luminescent signal of target cells stably or transiently transfected with a firefly luciferase reporter gene (fLuc:Zeo) linearly correlates with viable target cell number. Upon incubation of fLuc:Zeo(+) target cells with CD8(+) CTLs, a rapid decrease in bioluminescence was detected that correlated with antigen-specific target cell lysis. The levels of specific lysis measured by (51)Cr-release assays correlated with the attenuation in biophotonic target cell signal, thus validating this approach as a sensitive and accurate method for the measurement of cytolysis. We show that this luminescent-based cytolytic assay (LCA) is amenable for high-throughput screening of effector cell cytolytic activity, allows for the rate of cytolysis to be measured in a single micro-plate, and permits the multiplexing of cytolytic killing with other lymphocyte functional assays such as cytokine release. Importantly, this method accurately measures the cytolytic killing of target cells that are either stably or transiently transfected with a fLuc reporter gene, and thus is ideal for monitoring cytolysis of both primary autologous and immortalized target cell lines. The versatility of the non-radioactive, high-throughput, biophotonic cytolytic assay should make this method an attractive alternative to chromium-release for quantifying effector cell cytolytic activity.

CD8-Positive T-Lymphocytes↗
PubMed2005

A quantitative high-throughput chemotaxis assay using bioluminescent reporter cells.

Here we report on a novel biophotonic assay system for the detection and quantitation of chemotaxis, the directed movement of cells in response to chemokine concentration gradients. Our assay employs a firefly luciferase (ffLuc)-generated biophotonic signal to quantify cellular migration in 96-well microplate chemotaxis instruments. When compared to direct cell enumeration, the biophotonic reporter method is superior in accuracy, reproducibility, and sensitivity. As a proof-of-concept, we demonstrate the utility of this assay for quantifying the chemotactic response of ex vivo expanded ffLuc(+) primary human T-cells to recombinant human chemokines MCP-1, RANTES, and IP-10. The 96-well microplate format and in situ biophotonic detection of cells are amenable to high-throughput screening of peptides and small molecule libraries to identify agonists and antagonists of cellular chemotaxis, to analyze biological fluids for chemotactic activity, and to study chemotaxis in a variety of cell types.

Animals↗
PubMed2005

Amplification of RNAi--targeting HLA mRNAs.

Posttranscriptional suppression of gene expression can be achieved by introduction of sequence-specific small interfering (si) RNA duplexes and by de novo intracellular synthesis of short sequence-specific double-stranded RNAs. However, achieving desired levels of knockdown is a barrier to successful analytic and therapeutic application. We demonstrate that increasing expression of introduced short hairpin RNA (shRNA) can markedly enhance RNA interference (RNAi) and that this approach can be used to achieve maximal target down-regulation, when the choice of optimal siRNA-binding sites is restricted or when multiple genes are simultaneously targeted and the amount of siRNA is limiting. A dose-dependent RNAi effect was accomplished by placing copies of shRNA under control of the Pol III U6 small nuclear RNA promoter in tandem in a DNA vector. Using this system, we achieved simultaneous down-regulation of expression of classical human leukocyte antigen (HLA) class I genes in cultured and primary human T cells, which might be applied to help circumvent T-cell-mediated rejection of immunogenic and/or HLA-disparate allografts.

Base Sequence↗
PubMed2004

Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells.

The interleukin (IL) 13 receptor alpha2 (IL13Ralpha2) is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. Here, we describe a novel approach for targeting glioblastoma multiforme (GBM) with IL13Ralpha2-specific cytolytic T cells (CTLs) by their genetic modification to express a membrane-tethered IL13 cytokine chimeric T-cell antigen receptor, or zetakine. Our prototype zetakine incorporates an IL13 E13Y mutein for selective binding to IL13Ralpha2. Human IL13-zetakine(+)CD8(+) CTL transfectants display IL13Ralpha2-specific antitumor effector function including tumor cell cytolysis, T(C)1 cytokine production, and zetakine-regulated autocrine proliferation. The E13Y amino acid substitution of the IL13 mutein of the zetakine endows CTL transfectants with the capacity to discriminate between IL13Ralpha2(+) GBM targets from targets expressing IL13Ralpha1. In vivo, the adoptive transfer of IL13-zetakine(+)CD8(+) CTL clones results in the regression of established human glioblastoma orthotopic xenografts. Pilot clinical trials have been initiated to evaluate the feasibility and safety of local-regional delivery of autologous IL13-zetakine redirected CTL clones in patients with recurrent GBM. Our IL13-zetakine is a prototype of a new class of chimeric immunoreceptors that signal through an engineered immune synapse composed of membrane-tethered cytokine muteins bound to cell-surface cytokine receptors on tumors.

Animals↗
PubMed2004

Enhanced antilymphoma efficacy of CD19-redirected influenza MP1-specific CTLs by cotransfer of T cells modified to present influenza MP1.

To enhance the in vivo antitumor activity of adoptively transferred, CD19-specific chimeric antigen receptor (CAR)-redirected cytotoxic T lymphocytes (CTLs), we studied the effect of restimulating CAR(+) CTLs through their endogenous virus-specific T-cell antigen receptor (TcR) by the cotransfer of engineered T-cell antigen-presenting cells (T-APCs). Using influenza A matrix protein 1 (MP1) as a model antigen, we show that ex vivo-expanded CD4(+) and CD8(+) T-APCs expressing a hygromycin phosphotransferase-MP1 fusion protein (HyMP1) process and present MP1 to autologous human leukocyte antigen (HLA)-restricted, MP1-specific CD4(+) and CD8(+) CTL precursors. The MP1-specific CTLs are amenable to subsequent genetic modification to express a CD19-specific CAR, designated CD19R, and acquire HLA-unrestricted reactivity toward CD19(+) leukemia and lymphoma tumor targets while maintaining HLA-restricted MP1 specificity. The restimulation of MP1xCD19 dual-specific CTLs in vivo by the adoptive transfer of irradiated HyMP1(+) T-APCs resulted in the enhanced antilymphoma potency of bispecific effector cells, as measured by elimination of the biophotonic signal of established firefly luciferase-expressing Burkitt lymphoma xenografts in nonobese diabetic/severe combined immunodeficiency (NOD/scid) animals compared with control groups restimulated by Hy(+)MP1(neg) T-APCs. Engineered T-APCs are a novel and versatile antigen-delivery system for generating antigen-specific T cells in vitro and enhancing the in vivo effector functioning of CAR-redirected antitumor effector cells.

Adoptive Transfer↗
PubMed2004

Genetic engineering of cytolytic T lymphocytes for adoptive T-cell therapy of neuroblastoma.

BACKGROUND: Disease relapse is the leading cause of mortality for children diagnosed with disseminated neuroblastoma. The adoptive transfer of tumor-specific T cells is an attractive approach to target minimal residual disease following conventional therapies. We describe here the genetic engineering of human cytotoxic T lymphocytes (CTL) to express a chimeric immunoreceptor for re-directed HLA-independent recognition of neuroblastoma. METHODS: The CE7R chimeric immunoreceptor was constructed by PCR splice overlap extension and is composed of a single-chain antibody extracellular domain (scFv) derived from the L1-CAM-specific murine CE7 hybridoma fused to human IgG1 hinge-Fc, the transmembrane portion of human CD4, and the cytoplasmic tail of huCD3-zeta chain (scFvFc:zeta). Primary human T cells were genetically modified by naked DNA electrotransfer of plasmid expression vector CE7R-pMG then analyzed by Western blotting, flow cytometry for CE7R expression and cell surface trafficking, 4-h chromium release assay for re-directed neuroblastoma lysis, and ELISA for tumor-specific activation of cytokine production. RESULTS: CE7R is expressed as an intact chimeric protein that trafficks to the cell surface as a type I transmembrane protein. Primary human CE7R-expressing CD8(+) CTL clones specifically recognize human neuroblastoma tumor cells and are activated for tumor cell lysis and T(c)1 cytokine production. CONCLUSIONS: These data demonstrate the utility of CE7R for re-directing the effector function of CTL to neuroblastoma and have provided the rationale to initiate a FDA-authorized (BB-IND#9149) pilot clinical trial to establish the feasibility and safety of adoptive transfer of autologous CE7R(+)CD8(+) CTL clones to children with recurrent/refractory neuroblastoma.

CD3 Complex↗
PubMed2004

Cellular immunotherapy for follicular lymphoma using genetically modified CD20-specific CD8+ cytotoxic T lymphocytes.

Humoral immunotherapy using the monoclonal anti-CD20 antibody rituximab induces remissions in approximately 60% of patients with relapsed follicular lymphoma; however, most patients eventually relapse despite continued expression of CD20 on lymphoma cells. We have hypothesized that cellular immunotherapy targeting CD20(+) cells might provide a more effective mechanism for eliminating lymphoma cells than anti-CD20 antibodies and are therefore investigating the utility of cytotoxic T lymphocytes (CTL) genetically modified to target the CD20 antigen. Peripheral blood mononuclear cells were activated with anti-CD3 antibody (OKT3) and recombinant human interleukin-2 and electroporated with a plasmid containing a CD20-specific scFvFc:zeta chimeric T cell receptor gene and a neomycin phosphotransferase gene (neo(R)). Transfected cells were selected using the antibiotic G418 and cloned by limiting dilution. Using this approach, we have generated CD8(+) CTL clones with CD20-specific cytotoxicity, which specifically lysed CD20(+) target cells, including actual tumor cells from patients with follicular lymphoma, small lymphocytic lymphoma, splenic marginal zone lymphoma, diffuse large B cell lymphoma, and chronic lymphocytic leukemia. The CTL clones have been expanded to numbers sufficient for therapy ( approximately 10(9) cells). Our data indicate the feasibility of generating and expanding CD20-specific CTL and, for the first time, demonstrate that such CTL exhibit specific cytotoxicity against actual tumor cells isolated from patients with a variety of B lymphoid malignancies. In view of these promising findings, a Phase I clinical trial for relapsed follicular lymphoma is being initiated.

Antigens, CD20↗
PubMed2004

Enhanced transgene expression in quiescent and activated human CD8+ T cells.

The level of expression of retroviral vector-encoded proteins in T cells, decreasing during periods of quiescence, could be an obstacle to their clinical utility. To identify promoter systems that could increase the strength and persistence of transgene expression in primary human CD8(+) T cells, we designed a panel of Moloney retroviral vectors to express a destabilized enhanced green fluorescent protein (d4EGFP) reporter protein (t(1/2) = 4 hr). We found that the promoters phosphoglycerate kinase (Pgk), beta-actin, and long terminal repeat (LTR) produced the highest levels of d4EGFP expression in proliferating T cells, but that expression dramatically declined in quiescent cells with all promoters. To improve gene expression, we examined the effect of the beta-interferon (IFN) scaffold attachment region (SAR). This SAR augmented expression from mammalian promoters in cycling T cells, but had a small effect on maintenance of expression in resting T cells. However, when the SAR was combined with the LTR promoter, it significantly enhanced expression in resting and cycling cells. These data support use of the IFN-beta SAR with the LTR in Moloney retroviral vectors to help sustain gene expression in resting primary human CD8(+) T cells and to enhance gene expression in activated T cells.

CD8-Positive T-Lymphocytes↗
PubMed2003

Restoration of CD28 expression in CD28- CD8+ memory effector T cells reconstitutes antigen-induced IL-2 production.

The control of many persistent viral infections by Ag-specific cytolytic CD8+ T cells requires a concurrent virus-specific CD4+ Th cell response. This reflects in part a requirement of activated effector CD8+ T cells for paracrine IL-2 production as a growth and survival factor. In human CMV and HIV infection, the majority of differentiated virus-specific CD8+ T cells notably lose the ability to produce IL-2 but also lose expression of CD28, a costimulatory molecule. Analysis of the fraction of memory CD8+ T cells that continue to express CD28 revealed these cells retain the ability to produce IL-2. Therefore, we examined if IL-2 production by CD28- CD8+ T cells could be restored by introduction of a constitutively expressed CD28 gene. Expression of CD28 in CD28- CD8+ CMV- and HIV-specific CD8+ T cells reconstituted the ability to produce IL-2, which could sustain an autocrine proliferative response after Ag recognition. These results suggest that the loss of CD28 expression during differentiation of memory/effector CD8+ T cells represents a decisive step in establishing regulation of responding CD8+ T cells, increasing the dependence on CD4+ Th for proliferation after target recognition, and has implications for the treatment of viral disease with adoptively transferred CD8+ T cells.

CD28 Antigens↗
PubMed2002

T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B-lineage leukemia effect.

Relapse of B-lineage acute lymphoblastic leukemia (B-ALL) after allogeneic hematopoietic stem cell transplantation (HSCT) commonly results from the failure of a graft-versus-leukemia (GVL) effect to eradicate minimal residual disease. Augmenting the GVL effect by the adoptive transfer of donor-derived B-ALL-specific T-cell clones is a conceptually attractive strategy to decrease relapse rates without exacerbating graft-versus-host disease (GVHD). Toward this end, we investigated whether a genetic engineering approach could render CD8(+) cytotoxic T lymphocytes (CTLs) specific for tumor cells that express the B-cell lineage cell surface molecule CD19. This was accomplished by the genetic modification of CTLs to express a chimeric immunoreceptor composed of a CD19-specific single-chain immunoglobulin extracellular targeting domain fused to a CD3-zeta intracellular signaling domain. CD19-redirected CTL clones display potent CD19-specific lytic activity and chimeric immunoreceptor-regulated cytokine production and proliferation. Because B-ALL cells can evade T-cell/natural killer- cell recognition by down-regulation of cell surface accessory molecules that participate in the formation of a functional immunologic synapse, we compared the CD19-specific effector function of genetically modified CD8(+) CTLs toward CD19(+) cells with disparate levels of intercellular adhesion molecule 1 (ICAM-1), leukocyte function-associated antigen 1 (LFA-1), and LFA-3. We observed that recognition of B-lineage tumor lines by CD19-specific CTLs was not impaired by low levels of ICAM-1, LFA-1, and LFA-3 cell surface expression, a functional attribute that is likely a consequence of our high-affinity CD19-specific chimeric immunoreceptor. Furthermore, the CD19-specific CTLs could lyse primary B-ALL blasts. These preclinical observations form the basis for implementing clinical trials using donor-derived CD19-specific T-cell clones to treat or prevent relapse of B-ALL after allogeneic HSCT.

Adoptive Transfer↗
PubMedDate not supplied

Development and application of CD19-specific T cells for adoptive immunotherapy of B cell malignancies.

The graft-versus-leukemia (GVL)-effect achieved by donor-derived T cells arising from transplanted allogeneic hematopoietic stem cells or given as donor-leukocyte infusions (DLI) after allogeneic transplant, demonstrates that donor-derived T cells can eradicate B-lineage malignancies. However, graft-versus-host-disease (GVHD) occurring after allogeneic hematopoietic stem-cell transplant (HSCT) or polyclonal DLI can limit the efficacy of these interventions. This toxicity can be avoided by using autologous T cells and/or tumor-specific cytotoxic T lymphocytes (CTLs). To generate antigen-specific T cells that can be derived from the allogeneic donor or the patient, we have genetically manipulated T cells to express a CD19-specific chimeric immunoreceptor. This renders T cells specific for CD19, a cell surface molecule found on B-lineage leukemia and lymphoma. This review will demonstrate the redirected specificity of CD19-specific T cells and implementation of clinical trials using these cellular agents.

Antigens, CD19↗
PubMedDate not supplied

In vivo testing of Renilla luciferase substrate analogs in an orthotopic murine model of human glioblastoma.

In vivo bioluminescent imaging using cells expressing Renilla luciferase is becoming increasingly common. Hindrances to the more widespread use of Renilla luciferase are the high autoluminescence of its natural substrate, coelenterazine, in plasma, the relatively high absorbance by tissue of the light emitted by the enzyme-substrate reaction; rapid clearance of the substrate; and significant cost. These factors, save for the cost, which has its own limiting effect on use, can combine to reduce the sensitivity of in vivo assays utilizing this reporter system, and methods of increasing light output or decreasing autoluminescence could be of great benefit. A number of analogs of coelenterazine are being investigated may accomplish one or both of these goals. In this study that we report on the testing of two new substrate analogs, EnduRen and ViViren, manufactured by Promega Corporation, in an orthotopic murine model of human glioblastoma expressing Renilla luciferase. We have tested these analogs in this cell line both in vitro and in vivo, and find that the substrate viviren results in significantly greater light output than the natural substrate or the other analog EnduRen. This new substrate could be valuable for studies where greater sensitivity is important.

Animals↗