Jason C. L. Spurrell

CD8 T Cell-Mediated Killing of Cryptococcus neoformans Requires Granulysin and Is Dependent on CD4 T Cells and IL-15

Granulysin is located in the acidic granules of cytotoxic T cells. Although the purified protein has antimicrobial activity against a broad spectrum of microbial pathogens, direct evidence for granulysin-mediated cytotoxicity has heretofore been lacking. Studies were performed to examine the regulation and activity of granulysin expressed by CD8 T cells using Cryptococcus neoformans, which is one of the most common opportunistic pathogens of AIDS patients. IL-15-activated CD8 T cells acquired anticryptococcal activity, which correlated with the up-regulation of granulysin. When granules containing granulysin were depleted using SrCl2, or when the gene was silenced using 21-nt small interfering RNA duplexes, the antifungal effect of CD8 T cells was abrogated. Concanamycin A and EGTA did not affect the antifungal effect, suggesting that the activity of granulysin was perforin independent. Following stimulation by the C. neoformans mitogen, CD8 T cells expressed granulysin and acquired antifungal activity. This activity required CD4 T cells and was dependent upon accessory cells. Furthermore, IL-15 was both necessary and sufficient for granulysin up-regulation in CD8 T cells. These observations are most consistent with a mechanism whereby C. neoformans mitogen is presented to CD4 T cells, which in turn activate accessory cells. The resultant IL-15 activates CD8 T cells to express granulysin, which is responsible for antifungal activity.

Activation of p38 and ERK Signaling during Adenovirus Vector Cell Entry Lead to Expression of the C-X-C Chemokine IP-10

ABSTRACT The use of adenovirus vectors for human gene therapy is limited by potent inflammatory responses that result in significant morbidity. In kidney-derived epithelial cells (REC), activation of extracellular signal-regulated kinase 1/2 (ERK) and p38 kinase (p38) pathways occurred within 20 min of transduction with the serotype 5 adenovirus vector AdCMVβgal. Inhibition of ERK and p38 with U0126 and SB203580, respectively, reduced the expression of IP-10 mRNA following transduction with AdCMVβgal. To determine the role of the coxsackievirus-adenovirus receptor (CAR) or αv integrins in the activation of ERK and p38 and the expression of IP-10, REC cells were transduced with the fiber-modified and RGD-deleted adenovirus vectors AdL.F(RAEK-HA) and AdL.PB(HA), respectively. Compared with the wild-type capsid vector Ad5Luc, transduction with AdL.F(RAEK-HA) and AdL.PB(HA) resulted in reduced ERK-p38 activation and less IP-10 mRNA expression. The decreased IP-10 expression induced by the tropism-modified vectors was due to diminished transduction, since increasing multiplicity of infection resulted in increased IP-10 expression. Inhibition of adenovirus penetration with bafilomycin A1 or ammonium chloride attenuated the activation of ERK-p38 and IP-10 mRNA expression following infection, suggesting that endosomal escape was required to trigger these pathways. In vivo, direct inhibition of ERK and p38 signaling pathways inhibited adenovirus vector-induced IP-10 expression in mouse liver 1 h following transduction. These results demonstrate the importance of signaling via ERK and p38 in the early host response to adenovirus vectors and will permit the development of novel strategies to improve the safety and efficacy of these agents in human gene therapy.

Primary Dendritic Cells Phagocytose Cryptococcus neoformans via Mannose Receptors and Fcγ Receptor II for Presentation to T Lymphocytes

ABSTRACT Different “professional” antigen-presenting cells (APC) have unique characteristics that favor or restrict presentation of microbial antigens to T cells, depending on the organism. Cryptococcus neoformans is a pathogenic yeast that presents unique challenges to APC, including its large size, its rigid cell wall, and its ability to stimulate T cells as a mitogen. T-cell proliferation in response to the C. neoformans mitogen (CnM) requires phagocytosis and processing of the organisms by accessory cells prior to presentation of CnM to T cells. Because of the requirement for uptake of the organism and more limited costimulatory requirements of mitogens, macrophages might be the most likely cellular source for the accessory cell. However, the present study demonstrates that a transiently adherent cell that was CD3−, CD14−, CD19−, CD56−, HLA-DR+, and CD83+ with a dendritic morphology, rather than monocyte-derived or tissue (alveolar) macrophages, was the most efficient APC for presentation of CnM. A large number of these cells bound and internalized the organism, and only a small number of dendritic cells were required for presentation of the mitogen to T cells. Further, the mannose receptor and Fcγ receptor II were required for presentation of C. neoformans, as blocking either of these receptors abrogated both uptake of C. neoformans and lymphocyte proliferation in response to CnM. These studies demonstrate the surprising fact that dendritic cells are the most efficient accessory cells for CnM.

Lipopolysaccharide-stimulated or granulocyte-macrophage colony-stimulating factor-stimulated monocytes rapidly express biologically active IL-15 on their cell surface independent of new protein synthesis.

Although IL-15 shares many of the biological activities of IL-2, IL-2 expression is primarily under transcriptional regulation, while the mechanisms involved in the regulation of IL-15 are complex and not completely understood. In the current study, we found that CD14+ monocytes constitutively exhibit both IL-15 mRNA and protein. IL-15 protein was found stored intracellularly and stimulation of CD14+ monocytes with either LPS or GM-CSF resulted in mobilization of IL-15 stores to the plasma membrane. This rapidly induced surface expression was the result of a translocation of preformed stores, confirming that posttranslational regulatory stages limit IL-15, because it was not accompanied by an increase in IL-15 mRNA and occurred independent of de novo protein synthesis. After fixation, activated monocytes, but not resting monocytes, were found to support T cell proliferation, and this effect was abrogated by the addition of an IL-15-neutralizing Ab. The presence of preformed IL-15 stores and the ability of stimulated monocytes to mobilize these stores to their surface in an active form is a novel mechanism of regulation for IL-15.

Oncolytic Viral Therapy for Prostate Cancer: Efficacy of Reovirus as a Biological Therapeutic

Reovirus is a nonattenuated double-stranded RNA virus that exploits aberrant signaling pathways allowing selective cytotoxicity against multiple cancer histologies. The use of reovirus as a potential treatment modality for prostate cancer has not previously been described, and in this study evidence of in vitro and in vivo activity against prostate cancer was seen both in preclinical models and in six patients. The human prostate carcinoma cell lines PC-3, LN-CaP, and DU-145 exposed to replication-competent reovirus showed evidence of infection as illustrated by viral protein synthesis, cytopathic effect, and release of viral progeny. This oncolytic effect was found to be manifested through apoptosis, as DNA fragmentation, Apo 2.7 expression, Annexin V binding, and poly(ADP-ribose) polymerase cleavage were observed in live reovirus-infected cells, but not in uninfected or dead virus-treated cells. In vivo, hind flank severe combined immunodeficient/nonobese diabetic murine xenograft showed reduction in tumor size when treated with even a single intratumoral injection of reovirus. Finally, intralesional reovirus injections into a cohort of six patients with clinically organ-confined prostate cancer resulted in minimal side effects and evidence of antitumor activity. Histologic analysis after prostatectomy found a significant CD8 T-cell infiltration within the reovirus-injected areas as well as evidence of increased caspase-3 activity. These findings suggest that reovirus therapy may provide a promising novel treatment for prostate cancer and also imply a possible role for viral immune targeting of tumor.

Interleukin-15 Induces Antimicrobial Activity after Release by Cryptococcus neoformans-Stimulated Monocytes

A newly described cytokine, interleukin (IL)-15, shares many activities with IL-2; however, little is known about the stimuli for release of IL-15, and its role in antimicrobial host defense has not previously been demonstrated. This study found that Cryptococcus neoformans is a potent stimulus for the release of biologically active IL-15 from monocytes. Both IL-15 and IL-2 made significant contributions to lymphocyte proliferation and lymphocyte-mediated anticryptococcal activity to encapsulated and acapsular C. neoformans. IL-15 restored lymphocyte proliferation and anticryptococcal activity that had been abrogated by blocking IL-2. IL-15 also enhanced the anticryptococcal activity of lymphocytes but did not enhance the activity of monocytes. This suggests that IL-15 and IL-2 cooperate for lymphocyte activation and proliferation in vitro and demonstrates that IL-15 can induce antimicrobial activity. Taken together, these data suggest that microbes, and in particular C. neoformans, are an important stimulus for IL-15 and that IL-15 may have an important role in induction of antimicrobial effector mechanisms.

Phagocytosis and Protein Processing Are Required for Presentation of Cryptococcus neoformans Mitogen to T Lymphocytes

ABSTRACT In addition to eliciting antigen specific T-cell-mediated immunity,Cryptococcus neoformans possesses a mitogen (CnM) that activates naive T cells to proliferate. This mechanism of T-cell activation is accessory cell dependent and major histocompatibility complex unrestricted. CnM-induced T-cell proliferation correlates with internalization of the organism, suggesting that intracellular processing is required to liberate CnM prior to presentation to T cells. To determine whether phagocytosis and processing are required, various inhibitors of accessory cell uptake and processing were used.C. neoformans was observed within the accessory cells. Paraformaldehyde fixation of the accessory cell abrogated presentation of CnM to T cells, indicating that a dynamic accessory cell surface was required. A lysosomotropic agent abrogated the response to CnM but had no effect on a control stimulus that did not require processing. Both aspartic acid and cysteine protease inhibitors blocked effective processing of CnM, so that it was unable to stimulate T cells. Finally, an inhibitor of microfilament polymerization abrogated proliferation to CnM. These results indicate that the mitogenic activity of C. neoformans requires phagocytosis of the organism, lysosomal or endosomal processing, proteolytic activity, and microfilament polymerization and intracellular transport as a prerequisite for T-cell proliferation.

Oncolytic Reovirus and Immune Checkpoint Inhibition as a Novel Immunotherapeutic Strategy for Breast Cancer

As the current efficacy of oncolytic viruses (OVs) as monotherapy is limited, exploration of OVs as part of a broader immunotherapeutic treatment strategy for cancer is necessary. Here, we investigated the ability for immune checkpoint blockade to enhance the efficacy of oncolytic reovirus (RV) for the treatment of breast cancer (BrCa). In vitro, oncolysis and cytokine production were assessed in human and murine BrCa cell lines following RV exposure. Furthermore, RV-induced upregulation of tumor cell PD-L1 was evaluated. In vivo, the immunocompetent, syngeneic EMT6 murine model of BrCa was employed to determine therapeutic and tumor-specific immune responses following treatment with RV, anti-PD-1 antibodies or in combination. RV-mediated oncolysis and cytokine production were observed following BrCa cell infection and RV upregulated tumor cell expression of PD-L1. In vivo, RV monotherapy significantly reduced disease burden and enhanced survival in treated mice, and was further enhanced by PD-1 blockade. RV therapy increased the number of intratumoral regulatory T cells, which was reversed by the addition of PD-1 blockade. Finally, dual treatment led to the generation of a systemic adaptive anti-tumor immune response evidenced by an increase in tumor-specific IFN-γ producing CD8+ T cells, and immunity from tumor re-challenge. The combination of PD-1 blockade and RV appears to be an efficacious immunotherapeutic strategy for the treatment of BrCa, and warrants further investigation in early-phase clinical trials.

Abstract 4336: Oncolytic reovirus as a novel therapy for neuroblastoma

Background: Neuroblastoma (NB), a tumour of neurocrest progenitor cells of the sympathetic nervous system, is the most common extracranial pediatric tumour and accounts for approximately 15 percent of childhood cancer mortality. Even with surgery, radiation therapy, aggressive cytotoxic chemotherapy including autologous stem cell transplantation and more recently immunotherapy, high risk neuroblastoma has only a 30 percent predicted survival rate. Reovirus, a double-stranded RNA virus, has been shown to be effective against a myriad of cancers through its ability to preferentially lyse cancer cells with aberrant Ras pathway signaling. In this study, we investigate the potential of reovirus (serotype 3, strain Dearing) as a novel treatment for neuroblastoma and neuroblastoma tumour initiating cells (nbTIC). Experimental Design/Results: Reovirus induces dramatic cytotoxic effects at a multiplicity of infection (MOI) of 40 within 48h as assessed by WST-1 viability assays for the IMR-32, IMR-5, SK-N-AS, SK-N-SH, LAN-1, LAN-5, and SHEP human neuroblastoma cell lines in vitro. Interestingly the human neuroblastoma tumor-initiating cell lines, NB-12, NB88 and NB122 (kind gift from Dr David Kaplan) were also found to be very sensitive to reovirus-induced cytotoxicity, suggesting a role for reovirus treatment of refractory neuroblastoma. In order to further study reovirus, immunotherapy and neuroblastoma, a syngeneic, immunocompetent murine model (Neuro2a, A/J neuroblastoma model) was utilized to test reovirus-induced cytotoxicity and RV-directed immunotherapy of Neuro2A in vitro and in vivo. Preliminary data suggest that the Neuro2a cells are exquisitely sensitive to reovirus in vitro. Updated results will be presented. Conclusion: These preclinical results suggest that reovirus holds promise as a novel therapeutic for neuroblastoma and that it warrants further investigation in early phase clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4336. doi:10.1158/1538-7445.AM2011-4336