Maria C. Courreges

Tumor-infiltrating dendritic cell precursors recruited by a beta-defensin contribute to vasculogenesis under the influence of Vegf-A.

The involvement of immune mechanisms in tumor angiogenesis is unclear. Here we describe a new mechanism of tumor vasculogenesis mediated by dendritic cell (DC) precursors through the cooperation of β-defensins and vascular endothelial growth factor-A (Vegf-A). Expression of mouse β-defensin-29 recruited DC precursors to tumors and enhanced tumor vascularization and growth in the presence of increased Vegf-A expression. A new leukocyte population expressing DC and endothelial markers was uncovered in mouse and human ovarian carcinomas coexpressing Vegf-A and β-defensins. Tumor-infiltrating DCs migrated to tumor vessels and independently assembled neovasculature in vivo. Bone marrow–derived DCs underwent endothelial-like differentiation ex vivo, migrated to blood vessels and promoted the growth of tumors expressing high levels of Vegf-A. We show that β-defensins and Vegf-A cooperate to promote tumor vasculogenesis by carrying out distinct tasks: β-defensins chemoattract DC precursors through CCR6, whereas Vegf-A primarily induces their endothelial-like specialization and migration to vessels, which is mediated by Vegf receptor-2.

Ovarian carcinoma expresses the NKG2D ligand Letal and promotes the survival and expansion of CD28- antitumor T cells

The role of the NKG2D immunoreceptor and its ligands in antitumor immune response is incompletely understood. Here, we report that effector immune cells infiltrating ovarian carcinoma are mostly CD8+ lymphocytes lacking CD28 but expressing the NKG2D costimulatory receptor. Human ovarian carcinoma expresses the novel NKG2D ligand lymphocyte effector cell toxicity-activating ligand (Letal). Letal was found to be an independent prognosticator of improved survival in advanced ovarian cancer. Higher levels of tumor-derived Letal were associated with stronger lymphocyte infiltration. Letal exerted marked costimulatory effects and induced type-1 polarization in CD8+CD28− tumor-infiltrating lymphocytes ex vivo. Letal engagement increased the expression of the glucose transporter Glut-1, enhanced glucose up-take, and protected CD8+ lymphocytes from cisplatin-induced killing. Letal also down-regulated the expression of Fas in CD8+ cells and rendered them resistant to Fas ligand-induced apoptosis. Our results indicate that Letal promotes tumor immune surveillance by promoting the survival and intratumoral expansion of antitumor cytotoxic lymphocytes. We propose that Letal could be used for the ex vivo expansion of apoptosis-resistant tumor-reactive cytotoxic lymphocytes for adoptive transfer.

Letal, A tumor-associated NKG2D immunoreceptor ligand, induces activation and expansion of effector immune cells.

NKG2D serves as one of the most potent activating receptors for effector lymphocytes in peripheral tissues. Here we report the characterization of Letal, the first human transmembrane NKG2D ligand lacking an immunoglobulin-like a-3 ectodomain. Letal is constitutively expressed by a variety of normal tissues, and is up-regulated in tumor cells of different origins. Unlike other NKG2D ligands, Letal mRNA expression progressively decreased after treatment of tumor cells with retinoic acid. Simultaneous T-cell receptor activation and engagement of Letal stimulated proliferation of CD8+ cells and dramatically increased IL-2 and IFN-g secretion. In addition, Letal induced the killing of cancer cells by CD8+ and NK cells. These results suggest that Letal delivers activating signals to NK cells and promotes tumor immune surveillance by inducing the expansion of anti-tumor cytotoxic lymphocytes.

Whole tumor antigen vaccination using dendritic cells: Comparison of RNA electroporation and pulsing with UV-irradiated tumor cells

Because of the lack of full characterization of tumor associated antigens for solid tumors, whole antigen use is a convenient approach to tumor vaccination. Tumor RNA and apoptotic tumor cells have been used as a source of whole tumor antigen to prepare dendritic cell (DC) based tumor vaccines, but their efficacy has not been directly compared. Here we compare directly RNA electroporation and pulsing of DCs with whole tumor cells killed by ultraviolet (UV) B radiation using a convenient tumor model expressing human papilloma virus (HPV) E6 and E7 oncogenes. Although both approaches led to DCs presenting tumor antigen, electroporation with tumor cell total RNA induced a significantly higher frequency of tumor-reactive IFN-gamma secreting T cells, and E7-specific CD8+ lymphocytes compared to pulsing with UV-irradiated tumor cells. DCs electroporated with tumor cell RNA induced a larger tumor infiltration by T cells and produced a significantly stronger delay in tumor growth compared to DCs pulsed with UV-irradiated tumor cells. We conclude that electroporation with whole tumor cell RNA and pulsing with UV-irradiated tumor cells are both effective in eliciting antitumor immune response, but RNA electroporation results in more potent tumor vaccination under the examined experimental conditions.

Herpes virus oncolytic therapy reverses tumor immune dysfunction and facilitates tumor antigen presentation.

We have previously shown that intratumor administration of HSV-1716 (an ICP34.5 null mutant) resulted in significant reduction of tumor growth and a significant survival advantage in a murine model of ovarian cancer. Herewith we report that oncolytic HSV-1716 generates vaccination effects in the same model. Upon HSV-1716 infection, mouse ovarian tumor cells showed high levels of expression viral glycoproteins B and D and were highly phagocyted by dendritic cells (DCs). Interestingly, increased phagocytosis of tumor-infected cells by DCs was impaired by heparin, and anti-HSV glycoproteins B and D indicating that viral infection enhance adhesive interactions between DCs and tumor apoptotic bodies. Moreover, HSV-1716 infected cells expressed high levels of heat shock proteins 70 and GRP94, molecules that have been reported to induce maturation of DCs, increase cross-presentation of antigens and promote antitumor immune response. After phagocytosis of tumor-infected cells, DCs acquired a mature status in vitro and in vivo, upregulated the expression of costimulatory molecule and increased migration towards MIP-3β. Furthermore, HSV-1716 oncolytic treatment markedly reduced vascular endothelial growth factor (VEGF) levels in tumor-bearing animals thus abrogating tumor immunosuppressive milieu. These mechanisms may account for the highly enhanced antitumoral immune responses observed in HSV-1716 treated animals. Oncolytic treatment induced a significantly higher frequency of tumor-reactive IFN-γ producing cells, and induced a robust tumor infiltration by T cells. These results indicate that oncolytic therapy with HSV-1716 facilitates antitumor immune responses.

Critical Role of Dendritic Cells in Mouse Mammary Tumor Virus In Vivo Infection

ABSTRACT Mouse mammary tumor virus (MMTV) is a milk-transmitted betaretrovirus that causes mammary tumors in mice. Although mammary epithelial cells are the ultimate targets of MMTV, the virus utilizes components of the host immune system to establish infection. Previous studies indicated that dendritic cells play a role in MMTV infection. Here we show that dendritic cells are the first cells to be infected by MMTV in vivo and that they are capable of producing infectious virus that can be transmitted to other cell types. Moreover, upon contact with the virus, dendritic cells became more mature and migrated in response to the chemokine macrophage inflammatory protein 3β. Finally, we demonstrate that targeted ablation of dendritic cells in vivo dramatically attenuated MMTV infection. These data indicate that MMTV infection of dendritic cells is critical to initial propagation of the virus in vivo.

Preparation of apoptotic tumor cells with replication-incompetent HSV augments the efficacy of dendritic cell vaccines.

The use of dendritic cells (DCs) loaded with apoptotic tumor cells is an attractive approach to tumor vaccination in the absence of well-characterized tumor antigens. Apoptotic tumor cells are a convenient source of polyvalent tumor antigen, but may induce only weak immunization. We tested the role of replication-incompetent recombinant herpes simplex virus (HSV) d106 lacking all immediate early genes except ICP0 in the generation of apoptotic cells for tumor vaccination, using ID8-VEGF, a syngeneic mouse model of ovarian carcinoma expressing high levels of VEGF, and TC-1, a human papillomavirus (HPV) 16 E6- and E7-transformed adenocarcinoma. HSVd106 killed tumor cells by apoptosis. Tumor cells infected by HSVd106 were engulfed more avidly by immature DCs, and induced DC maturation more efficiently than tumor cells killed by ultraviolet B (UVB) radiation. HSVd106 infection induced stronger upregulation of heat shock protein (Hsp) 70 and glucose-related protein (GRP) 94 than UVB in cells undergoing apoptosis. Immunization of mice with DCs loaded with HSVd106-killed cells elicited stronger antitumor T-cell response, including tumor-reactive interferon-gamma-secreting and cytotoxic T cells, and resulted in significantly stronger delay in tumor growth than immunization with DCs loaded with UVB-killed tumor cells. Moreover, in the TC-1 model, a protective effect of vaccination (40% tumor free animals) was observed only after immunization with DCs loaded with HSVd106-killed cells. Thus, the use of replication-incompetent HSV strains lacking immediate early genes except ICP0 offers possible advantages in the preparation of whole tumor cell antigen for DC-based tumor vaccination.

Direct vaccination with tumor cells killed with ICP4-deficient HSVd120 elicits effective antitumor immunity

We tested whether tumor cells were killed by replication-incompetent recombinant herpes simplex virus (HSV) d120 lacking immediate early gene ICP4 and whether HSVd120-killed tumor cells could be used directly for tumor vaccination. Vaccine efficacy was tested in TC-1, a murine adenocarcinoma transformed with HPV16 E6 and E7, and ID8-Vegf, a murine epithelial ovarian cancer model. HSVd120 killed tumor cells by apoptosis. Tumor cells infected by HSVd120 were engulfed more avidly by immature DCs and induced DC maturation more efficiently than tumor cells killed by ultraviolet B (UVB) radiation. HSVd120 infection induced stronger upregulation of GRP94 than UVB in cells undergoing apoptosis. Immunization of mice with HSVd120-killed cells elicited stronger antitumor T cell response, including tumor reactive interferon-gamma secreting and cytotoxic T cells, and resulted in significantly stronger delay in tumor growth than immunization with UVB-killed tumor cells. Thus, the use of replication-incompetent HSV strains lacking ICP4 offers possible advantages in the preparation of whole tumor cell antigen for direct tumor vaccination.

Toll-Like Receptor 3 Is Critical for Coxsackievirus B4-Induced Type 1 Diabetes in Female NOD Mice

Group B coxsackieviruses (CVBs) are involved in triggering some cases of type 1 diabetes mellitus (T1DM). However, the molecular mechanism(s) responsible for this remain elusive. Toll-like receptor 3 (TLR3), a receptor that recognizes viral double-stranded RNA, is hypothesized to play a role in virus-induced T1DM, although this hypothesis is yet to be substantiated. The objective of this study was to directly investigate the role of TLR3 in CVB-triggered T1DM in nonobese diabetic (NOD) mice, a mouse model of human T1DM that is widely used to study both spontaneous autoimmune and viral-induced T1DM. As such, we infected female wild-type (TLR3(+/+)) and TLR3 knockout (TLR3(-/-)) NOD mice with CVB4 and compared the incidence of diabetes in CVB4-infected mice with that of uninfected counterparts. We also evaluated the islets of uninfected and CVB4-infected wild-type and TLR3 knockout NOD mice by immunohistochemistry and insulitis scoring. TLR3 knockout mice were markedly protected from CVB4-induced diabetes compared with CVB4-infected wild-type mice. CVB4-induced T-lymphocyte-mediated insulitis was also significantly less severe in TLR3 knockout mice compared with wild-type mice. No differences in insulitis were observed between uninfected animals, either wild-type or TLR3 knockout mice. These data demonstrate for the first time that TLR3 is 1) critical for CVB4-induced T1DM, and 2) modulates CVB4-induced insulitis in genetically prone NOD mice.

Intraperitoneal Oncolytic and Tumor Vaccination Therapy with Replication-Competent Recombinant Virus: The Herpes Paradigm

The biological therapy of tumors using live viruses was first proposed a century ago but was abandoned due to potential virulence of wild-type strains. Thanks to advances in recombinant technology, replication-restricted strains have been genetically engineered, which replicate selectively within tumor cells. Examples include replication-competent mutants of herpes simplex virus (HSV), adenovirus, vesicular stomatitis virus, reovirus and measles virus. Replication-restricted oncolytic viruses are able to propagate selectively within solid tumor nodules exerting direct antitumor activity by killing infected tumor cells at the completion of a replicative cycle. In the process, they generate an intratumoral inflammatory response, which under the appropriate circumstances, may trigger the activation of an adaptive antitumor immune response, a process that has been named in situ tumor vaccination. Recombinant HSV may offer distinct advantages in oncolytic therapy of epithelial tumors. HSV is highly infectious to tumors of epithelial origin, resulting in high efficacy, there is considerable redundancy in HSV receptors, which makes the loss of HSV receptors by tumors due to mutations less likely and potent anti-herpetic drugs are commercially available, which may be used clinically to control undesired side effects. Herewith we describe the use of oncolytic viral therapy against intraperitoneal malignancies with special emphasis on oncolytic herpes simplex virus. We review the preclinical evidence on the efficacy and safety of intraperitoneal applications of HSV and discuss the rationale for its use for oncolytic therapy and in situ tumor vaccination of intraperitoneal tumors.