Effects of APC De-targeting and GAr modification on the duration of luciferase expression from plasmid DNA delivered to skeletal muscle.

Immune responses to expressed foreign transgenes continue to hamper progress of gene therapy development. Translated foreign proteins with intracellular location are generally less accessible to the immune system, nevertheless they can be presented to the immune system through both MHC Class I and Class II pathways. When the foreign protein luciferase was expressed following intramuscular delivery of plasmid DNA in outbred mice, expression rapidly declined over 4 weeks. Through modifications to the expression plasmid and the luciferase transgene we examined the effect of detargeting expression away from antigen-presenting cells (APCs), targeting expression to skeletal muscle and fusion with glycine-alanine repeats (GAr) that block MHC-Class I presentation on the duration of luciferase expression. De-targeting expression from APCs with miR142-3p target sequences incorporated into the luciferase 3'UTR reduced the humoral immune response to both native and luciferase modified with a short GAr sequence but did not prolong the duration of expression. When a skeletal muscle specific promoter was combined with the miR target sequences the humoral immune response was dampened and luciferase expression persisted at higher levels for longer. Interestingly, fusion of luciferase with a longer GAr sequence promoted the decline in luciferase expression and increased the humoral immune response to luciferase. These studies demonstrate that expression elements and transgene modifications can alter the duration of transgene expression but other factors will need to overcome before foreign transgenes expressed in skeletal muscle are immunologically silent.

Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-ß.

OBJECTIVES: Targeting cytokines to sites of disease has clear advantages because it increases their therapeutic index. We designed fusion proteins of the latent-associated peptide (LAP) derived from TGF-ß with various cytokines via a matrix metalloproteinase (MMP) cleavage site. This design confers latency, increased half-life and targeting to sites of inflammation. The aim of this study is to determine whether this approach can be applied to cytokines of different molecular structures and sizes. METHODS: Mature cytokines cloned downstream of LAP and a MMP cleavage site were expressed in 293T cells and assessed for latency and biological activity by Western blotting and bioassay. RESULTS: We demonstrate here that fusion proteins of TGF-ß, erythropoietin, IL-1ra, IL-10, IL-4, BMP-7, IGF1 and IL-17 were rendered latent by fusion to LAP, requiring cleavage to become active in respective bioassays. As further proof of principle, we also show that delivery of engineered TGF-ß can inhibit experimental autoimmune encephalomyelitis and that this approach can be used to efficiently deliver cytokines to the brain and spinal cord in mice with this disease. CONCLUSIONS: The latent cytokine approach can be successfully applied to a range of molecules, including cytokines of different molecular structure and mass, growth factors and a cytokine antagonist.

Activated pancreatic stellate cells sequester CD8+ T cells to reduce their infiltration of the juxtatumoral compartment of pancreatic ductal adenocarcinoma.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic microenvironment that contains many different immune cells. Activated pancreatic stellate cells (PSCs) contribute to the desmoplasia. We investigated whether distinct stromal compartments are differentially infiltrated by different types of immune cells. METHODS: We used tissue microarray analysis to compare immune cell infiltration of different pancreaticobiliary diseased tissues (PDAC, ampullary carcinoma, cholangiocarcinoma, mucinous cystic neoplasm, chronic inflammation, and chronic pancreatitis) and juxtatumoral stromal (<100 µm from tumor) and panstromal compartments. We investigated the association between immune infiltrate and patient survival times. We also analyzed T-cell migration and tumor infiltration in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice and the effects of all-trans retinoic acid (ATRA) on these processes. RESULTS: Juxtatumoral compartments in PDAC samples from 2 independent groups of patients contained increased numbers of myeloperoxidase(+) and CD68(+) cells compared with panstromal compartments. However, juxtatumoral compartments of PDACs contained fewer CD8(+), FoxP3(+), CD56(+), or CD20(+) cells than panstromal compartments, a distinction absent in ampullary carcinomas and cholangiocarcinomas. Patients with PDACs that had high densities of CD8(+) T cells in the juxtatumoral compartment had longer survival times than patients with lower densities. In KPC mice, administration of ATRA, which renders PSCs quiescent, increased numbers of CD8(+) T cells in juxtatumoral compartments. We found that activated PSCs express cytokines, chemokines, and adhesion molecules that regulate T-cell migration. In vitro migration assays showed that CD8(+) T cells, from patients with PDAC, had increased chemotaxis toward activated PSCs, which secrete CXCL12, compared with quiescent PSCs or tumor cells. These effects could be reversed by knockdown of CXCL12 or treatment of PSCs with ATRA. CONCLUSIONS: Based on studies of human PDAC samples and KPC mice, activated PSCs appear to reduce migration of CD8(+) T cells to juxtatumoral stromal compartments, preventing their access to cancer cells. Deregulated signaling by activated PSCs could prevent an effective antitumor immune response.

T cells from CLL patients exhibit features of T-cell exhaustion but retain capacity for cytokine production.

T-cell exhaustion, originally described in chronic viral infections, was recently reported in solid and hematologic cancers. It is not defined whether exhaustion contributes to T-cell dysfunction observed in chronic lymphocytic leukemia (CLL). We investigated the phenotype and function of T cells from CLL patients and age-matched controls. CD8+ and CD4+ T cells from CLL patients had increased expression of exhaustion markers CD244, CD160, and PD1, with expansion of a PD1+BLIMP1HI subset. These molecules were most highly expressed in the expanded population of effector T cells in CLL. CLL CD8+ T cells showed functional defects in proliferation and cytotoxicity, with the cytolytic defect caused by impaired granzyme packaging into vesicles and nonpolarized degranulation. In contrast to virally induced exhaustion, CLL T cells showed increased production of interferon-γ and TNFα and increased expression of TBET, and normal IL2 production. These defects were not restricted to expanded populations of cytomegalovirus (CMV)­specific cells, although CMV seropositivity modulated the distribution of lymphocyte subsets, the functional defects were present irrespective of CMV serostatus. Therefore, although CLL CD8+ T cells exhibit features of T-cell exhaustion, they retain the ability to produce cytokines. These findings also exclude CMV as the sole cause of T-cell defects in CLL.

Multiple inhibitory ligands induce impaired T-cell immunologic synapse function in chronic lymphocytic leukemia that can be blocked with lenalidomide: establishing a reversible immune evasion mechanism in human cancer.

Cancer immune evasion is an emerging hallmark of disease progression. We have demonstrated previously that impaired actin polymerization at the T-cell immunologic synapse is a global immune dysfunction in chronic lymphocytic leukemia (CLL). Direct contact with tumor cells induces defective actin polarization at the synapse in previously healthy T cells, but the molecules mediating this dysfunction were not known. In the present study, we show via functional screening assays that CD200, CD270, CD274, and CD276 are coopted by CLL cells to induce impaired actin synapse formation in both allogeneic and autologous T cells. We also show that inhibitory ligand-induced impairment of T-cell actin dynamics is a common immunosuppressive strategy used by both hematologic (including lymphoma) and solid carcinoma cells. This immunosuppressive signaling targets T-cell Rho-GTPase activation. Of clinical relevance, the immunomodulatory drug lenalidomide prevented the induction of these defects by down-regulating tumor cell-inhibitory molecule expression. These results using human CLL as a model cancer establish a novel evasion mechanism whereby malignant cells exploit multiple inhibitory ligand signaling to down-regulate small GTPases and lytic synapse function in global T-cell populations. These findings should contribute to the design of immunotherapeutic strategies to reverse T-cell tolerance in cancer.

A novel hybrid promoter responsive to pathophysiological and pharmacological regulation.

The aim of this study was to construct a promoter containing DNA motifs for an endogenous transcription factor associated with inflammation along with motifs for pharmacological regulation factors. We demonstrate in transfected cells that expression of a gene of interest is induced by hypoxic conditions or through pharmacological induction, and also show pharmacological repression. In vivo studies utilised electroporation of plasmid to mouse paws, a delivery method shown to be effective by bioluminescence imaging. For gene therapy, the promoter was used to drive expression of IL-1Ra in a paw inflammation model with therapeutic effect observed which was further enhanced when the promoter was additionally induced with a pharmacological activator. One of the most important observations from this study was that promoter induction by hypoxia or inflammation could be prevented by the pharmacological repressor in the absence of doxycycline. These studies demonstrate that hybrid promoters enable pharmacological adjustment to the pathophysiological level of gene expression and, importantly, that they allow termination of gene expression even in the presence of pathophysiological stimuli.

Follicular lymphoma cells induce T-cell immunologic synapse dysfunction that can be repaired with lenalidomide: implications for the tumor microenvironment and immunotherapy.

An important hallmark of cancer progression is the ability of tumor cells to evade immune recognition. Understanding the relationship between neoplastic cells and the immune microenvironment should facilitate the design of improved immunotherapies. Here we identify impaired T-cell immunologic synapse formation as an active immunosuppressive mechanism in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). We found a significant reduction in formation of the F-actin immune synapse in tumor-infiltrating T cells (P < .01) from lymphoma patients compared with age-matched healthy donor cells. Peripheral blood T cells exhibited this defect only in patients with leukemic-phase disease. Moreover, we demonstrate that this T-cell defect is induced after short-term tumor cell contact. After 24-hour coculture with FL cells, previously healthy T cells showed suppressed recruitment of critical signaling proteins to the synapse. We further demonstrate repair of this defect after treatment of both FL cells and T cells with the immunomodulatory drug lenalidomide. Tissue microarray analysis identified reduced expression of the T-cell synapse signature proteins, including the cytolytic effector molecule Rab27A associated with poor prognosis, in addition to reduced T-cell numbers and activity with disease transformation. Our results highlight the importance of identifying biomarkers and immunotherapeutic treatments for repairing T-cell responses in lymphoma.

Peripheral blood T cells in acute myeloid leukemia (AML) patients at diagnosis have abnormal phenotype and genotype and form defective immune synapses with AML blasts.

Understanding how the immune system in patients with cancer interacts with malignant cells is critical for the development of successful immunotherapeutic strategies. We studied peripheral blood from newly diagnosed patients with acute myeloid leukemia (AML) to assess the impact of this disease on the patients' T cells. The absolute number of peripheral blood T cells is increased in AML compared with healthy controls. An increase in the absolute number of CD3+56+ cells was also noted. Gene expression profiling on T cells from AML patients compared with healthy donors demonstrated global differences in transcription suggesting aberrant T-cell activation patterns. These gene expression changes differ from those observed in chronic lymphocytic leukemia (CLL), indicating the heterogeneous means by which different tumors evade the host immune response. However, in common with CLL, differentially regulated genes involved in actin cytoskeletal formation were identified, and therefore the ability of T cells from AML patients to form immunologic synapses was assessed. Although AML T cells could form conjugates with autologous blasts, their ability to form immune synapses and recruit phosphotyrosine signaling molecules to the synapse was significantly impaired. These findings identify T-cell dysfunction in AML that may contribute to the failure of a host immune response against leukemic blasts.

The inflammatory cytokine tumor necrosis factor-alpha generates an autocrine tumor-promoting network in epithelial ovarian cancer cells.

Constitutive expression of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is characteristic of malignant ovarian surface epithelium. We investigated the hypothesis that this autocrine action of TNF-alpha generates and sustains a network of other mediators that promote peritoneal cancer growth and spread. When compared with two ovarian cancer cell lines that did not make TNF-alpha, constitutive production of TNF-alpha was associated with greater release of the chemokines CCL2 and CXCL12, the cytokines interleukin-6 (IL-6) and macrophage migration-inhibitory factor (MIF), and the angiogenic factor vascular endothelial growth factor (VEGF). TNF-alpha production was associated also with increased peritoneal dissemination when the ovarian cancer cells were xenografted. We next used RNA interference to generate stable knockdown of TNF-alpha in ovarian cancer cells. Production of CCL2, CXCL12, VEGF, IL-6, and MIF was decreased significantly in these cells compared with wild-type or mock-transfected cells, but in vitro growth rates were unaltered. Tumor growth and dissemination in vivo were significantly reduced when stable knockdown of TNF-alpha was achieved. Tumors derived from TNF-alpha knockdown cells were noninvasive and well circumscribed and showed high levels of apoptosis, even in the smallest deposits. This was reflected in reduced vascularization of TNF-alpha knockdown tumors. Furthermore, culture supernatants from such cells failed to stimulate endothelial cell growth in vitro. We conclude that autocrine production of TNF-alpha by ovarian cancer cells stimulates a constitutive network of other cytokines, angiogenic factors, and chemokines that may act in an autocrine/paracrine manner to promote colonization of the peritoneum and neovascularization of developing tumor deposits.

Gene therapy with an improved doxycycline-regulated plasmid encoding a tumour necrosis factor-alpha inhibitor in experimental arthritis.

Inhibition of tumour necrosis factor (TNF)-alpha with biological molecules has proven an effective treatment for rheumatoid arthritis, achieving a 20% improvement in American College of Rheumatology score in up to 65% of patients. The main drawback to these and many other biological treatments has been their expense, which has precluded their widespread application. Biological molecules could alternatively be delivered by gene therapy as the encoding DNA. We have developed novel plasmid vectors termed pGTLMIK and pGTTMIK, from which luciferase and a dimeric TNF receptor II (dTNFR) are respectively expressed in a doxycycline (Dox)-regulated manner. Regulated expression of luciferase from the self-contained plasmid pGTLMIK was examined in vitro in a variety of cell lines and in vivo following intramuscular delivery with electroporation in DBA/1 mice. Dox-regulated expression of luciferase from pGTLMIK of approximately 1,000-fold was demonstrated in vitro, and efficient regulation was observed in vivo. The vector pGTTMIK encoding dTNFR was delivered by the same route with and without administration of Dox to mice with collagen-induced arthritis. When pGTTMIK was delivered after the onset of arthritis, progression of the disease in terms of both paw thickness and clinical score was inhibited when Dox was also administered. Vectors with similar regulation characteristics may be suitable for clinical application.

Generation of neoantigenic epitopes after posttranslational modification of type II collagen by factors present within the inflamed joint.

OBJECTIVE: Collagen-induced arthritis is a commonly accepted model of rheumatoid arthritis (RA). However, it has been difficult to substantiate the involvement of autoimmunity to type II collagen (CII) in the pathogenesis of RA. The aim of this investigation was to determine if CII, modified by reactive oxidant species present within the inflamed joint, could generate neoantigenic epitopes. METHODS: Oxidants that play a role in acute and chronic inflammation and are present in the rheumatoid joint (hydroxyl radical, hypochlorous acid, and peroxynitrite) were used for modification of native CII. In addition, CII was glycated with ribose, since nonenzymatic oxidative reactions by glycation are evident in RA. Modifications were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 3-dimensional fluorescence followed by enzyme-linked immunosorbent assay (ELISA) and Western blotting, using, as probes, sera from patients with RA and from patients with other inflammatory and noninflammatory joint diseases. RESULTS: Only 1 RA serum sample showed strong binding to native CII. In contrast, binding to modified CII was increased in 14 of 31 RA sera, of which 7 were strong binders and 7 were moderate binders. Among the non-RA serum samples, only 1 yielded a strong reaction to modified CII and 5 of 41 were moderate binders. Samples that showed the strongest binding to modified CII in ELISA also showed strong binding to various fragmented or aggregated forms of CII in Western blots, as well as strong binding to fragmented CII present in RA synovial fluid. CONCLUSION: When modified by conditions found within the inflamed joint, CII acts as an autoantigen in RA.