Attenuated Dengue virus PV001-DV induces oncolytic tumor cell death and potent immune responses.

BACKGROUND: Viral therapies developed for cancer treatment have classically prioritized direct oncolytic effects over their immune activating properties. However, recent clinical insights have challenged this longstanding prioritization and have shifted the focus to more immune-based mechanisms. Through the potential utilization of novel, inherently immune-stimulating, oncotropic viruses there is a therapeutic opportunity to improve anti-tumor outcomes through virus-mediated immune activation. PV001-DV is an attenuated strain of Dengue virus (DEN-1 #45AZ5) with a favorable clinical safety profile that also maintains the potent immune stimulatory properties characterstic of Dengue virus infection. METHODS: In this study, we utilized in vitro tumor killing and immune multiplex assays to examine the anti-tumor effects of PV001-DV as a potential novel cancer immunotherapy. RESULTS: In vitro assays demonstrated that PV001-DV possesses the ability to directly kill human melanoma cells lines as well as patient melanoma tissue ex vivo. Importantly, further work demonstrated that, when patient peripheral blood mononuclear cells (PBMCs) were exposed to PV001-DV, a substantial induction in the production of apoptotic factors and immunostimulatory cytokines was detected. When tumor cells were cultured with the resulting soluble mediators from these PBMCs, rapid cell death of melanoma and breast cancer cell lines was observed. These soluble mediators also increased dengue virus binding ligands and immune checkpoint receptor, PD-L1 expression. CONCLUSIONS: The direct in vitro tumor-killing and immune-mediated tumor cytotoxicity facilitated by PV001-DV contributes support of its upcoming clinical evaluation in patients with advanced melanoma who have failed prior therapy.

Combination oncolytic virus, radiation therapy, and immune checkpoint inhibitor treatment in anti-PD-1-refractory cancer.

BACKGROUND: Immunotherapies are becoming front-line treatments for many advanced cancers, and combinations of two or more therapies are beginning to be investigated. Based on their individual antitumor capabilities, we sought to determine whether combination oncolytic virus (OV) and radiation therapy (RT) may improve cancer outcomes. METHODS: To investigate the activity of this combination therapy, we used in vitro mouse and human cancer cell lines as well as a mouse model of skin cancer. After initial results, we further included immune checkpoint blockade, whose addition constituted a triple combination immunotherapy. RESULTS: Our findings demonstrate that OV and RT reduce tumor growth via conversion of immunologically 'cold' tumors to 'hot', via a CD8+ T cell-dependent and IL-1α-dependent mechanism that is associated with increased PD-1/PD-L1 expression, and the triple combination of OV, RT, and PD-1 checkpoint inhibition impedes tumor growth and prolongs survival. Further, we describe the response of a PD-1-refractory patient with cutaneous squamous cell carcinoma who received the triple combination of OV, RT, and immune checkpoint inhibitor (ICI), and went on to experience unexpected, prolonged control and survival. He remains off-treatment and is without evidence of progression for >44 months since study entry. CONCLUSIONS: Effective systemic antitumor immune response is rarely elicited by a single therapy. In a skin cancer mouse model, we demonstrate improved outcomes with combination OV, RT, and ICI treatment, which is associated with mechanisms involving augmented CD8+ T cell infiltration and IL-1α expression. We report tumor reduction and prolonged survival of a patient with skin cancer treated with combination OV, RT, and ICI. Overall, our data provide strong rationale for combining OV, RT, and ICI for treatment of patients with ICI-refractory skin and potentially other cancers.

Intratumoral Influenza Vaccine Administration Attenuates Breast Cancer Growth and Restructures the Tumor Microenvironment through Sialic Acid Binding of Vaccine Hemagglutinin.

Breast cancer continues to have a high disease burden worldwide and presents an urgent need for novel therapeutic strategies to improve outcomes. The influenza vaccine offers a unique approach to enhance the anti-tumor immune response in patients with breast cancer. Our study explores the intratumoral use of the influenza vaccine in a triple-negative 4T1 mouse model of breast cancer. We show that the influenza vaccine attenuated tumor growth using a three-dose intratumoral regimen. More importantly, prior vaccination did not alter this improved anti-tumor response. Furthermore, we characterized the effect that the influenza vaccine has on the tumor microenvironment and the underlying mechanisms of action. We established that the vaccine facilitated favorable shifts in restructuring the tumor microenvironment. Additionally, we show that the vaccine's ability to bind sialic acid residues, which have been implicated in having oncogenic functions, emerged as a key mechanism of action. Influenza hemagglutinin demonstrated binding ability to breast cancer cells through sialic acid expression. When administered intratumorally, the influenza vaccine offers a promising therapeutic strategy for breast cancer patients by reshaping the tumor microenvironment and modestly suppressing tumor growth. Its interaction with sialic acids has implications for effective therapeutic application and future research.

Tracking fluorescently labeled IL-15 and anti-PD-1 in the tumor microenvironment and draining lymph nodes.

Understanding the dynamics of the tumor microenvironment (TME) has become vital in discovering new targets for effective immunotherapies and enhancing current treatments. However, localization and distribution of immune cells and treatment biomolecules are poorly characterized to date. In this study, a murine Luminal B mammary adenocarcinoma model received a combinatorial treatment of fluorescently labeled anti-PD-1-Cy3 and IL-15 complex-Cy5 injected interperitoneally and intratumorally, respectively. Fluorescent labeling allowed for the visualization of the distribution of IL-15 complexes and anti-PD-1, as well as their localization to immune cells in the TME and tumor-draining lymph node. Using fluorescent microscopy and light sheet microscopy of whole-clarified tumors and draining lymph nodes, the localization of IL-15 complexes was found to be distributed around the periphery of the tumor at 4 h post injection and medially located at the center of the tumor at 24 h post injection, corresponding with high densities of CD8 cells in the tumor present at 48 h and 72 h post injection. Anti-PD-1 was distributed around the perimeter of the tumor and colocalized to IL-15 in the draining lymph nodes 24 h post injection. Colocalization of IL-15 was also established with NK cells, CD8+ T cells, and macrophages. This study develops a novel method to spatiotemporally track fluorescently labeled immunotherapeutic biomolecules in vivo, with implications for optimizing and further understanding the pharmacokinetics of clinical immunotherapies.

Bacterial-Based Cancer Therapy (BBCT): Recent Advances, Current Challenges, and Future Prospects for Cancer Immunotherapy.

Currently approximately 10 million people die each year due to cancer, and cancer is the cause of every sixth death worldwide. Tremendous efforts and progress have been made towards finding a cure for cancer. However, numerous challenges have been faced due to adverse effects of chemotherapy, radiotherapy, and alternative cancer therapies, including toxicity to non-cancerous cells, the inability of drugs to reach deep tumor tissue, and the persistent problem of increasing drug resistance in tumor cells. These challenges have increased the demand for the development of alternative approaches with greater selectivity and effectiveness against tumor cells. Cancer immunotherapy has made significant advancements towards eliminating cancer. Our understanding of cancer-directed immune responses and the mechanisms through which immune cells invade tumors have extensively helped us in the development of new therapies. Among immunotherapies, the application of bacteria and bacterial-based products has promising potential to be used as treatments that combat cancer. Bacterial targeting of tumors has been developed as a unique therapeutic option that meets the ongoing challenges of cancer treatment. In comparison with other cancer therapeutics, bacterial-based therapies have capabilities for suppressing cancer. Bacteria are known to accumulate and proliferate in the tumor microenvironment and initiate antitumor immune responses. We are currently well-informed regarding various methods by which bacteria can be manipulated by simple genetic engineering or synthetic bioengineering to induce the production of anti-cancer drugs. Further, bacterial-based cancer therapy (BBCT) can be either used as a monotherapy or in combination with other anticancer therapies for better clinical outcomes. Here, we review recent advances, current challenges, and prospects of bacteria and bacterial products in the development of BBCTs.

Intratumoral injection of the seasonal flu shot converts immunologically cold tumors to hot and serves as an immunotherapy for cancer.

Reprogramming the tumor microenvironment to increase immune-mediated responses is currently of intense interest. Patients with immune-infiltrated "hot" tumors demonstrate higher treatment response rates and improved survival. However, only the minority of tumors are hot, and a limited proportion of patients benefit from immunotherapies. Innovative approaches that make tumors hot can have immediate impact particularly if they repurpose drugs with additional cancer-unrelated benefits. The seasonal influenza vaccine is recommended for all persons over 6 mo without prohibitive contraindications, including most cancer patients. Here, we report that unadjuvanted seasonal influenza vaccination via intratumoral, but not intramuscular, injection converts "cold" tumors to hot, generates systemic CD8+ T cell-mediated antitumor immunity, and sensitizes resistant tumors to checkpoint blockade. Importantly, intratumoral vaccination also provides protection against subsequent active influenza virus lung infection. Surprisingly, a squalene-based adjuvanted vaccine maintains intratumoral regulatory B cells and fails to improve antitumor responses, even while protecting against active influenza virus lung infection. Adjuvant removal, B cell depletion, or IL-10 blockade recovers its antitumor effectiveness. Our findings propose that antipathogen vaccines may be utilized for both infection prevention and repurposing as a cancer immunotherapy.

IL-15 Complexes Induce Migration of Resting Memory CD8 T Cells into Mucosal Tissues.

IL-15 is an essential cytokine known to promote T cell survival and activate the effector function of memory phenotype CD8 T cells. Blocking IL-15 signals also significantly impacts tissue-specific effector and memory CD8 T cell formation. In this study, we demonstrate that IL-15 influences the generation of memory CD8 T cells by first promoting their accumulation into mucosal tissues and second by sustaining expression of Bcl-6 and T-bet. We show that the mechanism for this recruitment is largely dependent on mammalian target of rapamycin and its subsequent inactivation of FoxO1. Last, we show that IL-15 complexes delivered locally to mucosal tissues without reinfection is an effective strategy to enhance establishment of tissue resident memory CD8 T cells within mucosal tissues. This study provides mechanistic insight into how IL-15 controls the generation of memory CD8 T cells and influences their trafficking and ability to take up residence within peripheral tissues.

Non-oncogenic Acute Viral Infections Disrupt Anti-cancer Responses and Lead to Accelerated Cancer-Specific Host Death.

In light of increased cancer prevalence and cancer-specific deaths in patients with infections, we investigated whether infections alter anti-tumor immune responses. We report that acute influenza infection of the lung promotes distal melanoma growth in the dermis and leads to accelerated cancer-specific host death. Furthermore, we show that during influenza infection, anti-melanoma CD8+ T cells are shunted from the tumor to the infection site, where they express high levels of the inhibitory receptor programmed cell death protein 1 (PD-1). Immunotherapy to block PD-1 reverses this loss of anti-tumor CD8+ T cells from the tumor and decreases infection-induced tumor growth. Our findings show that acute non-oncogenic infection can promote cancer growth, raising concerns regarding acute viral illness sequelae. They also suggest an unexpected role for PD-1 blockade in cancer immunotherapy and provide insight into the immune response when faced with concomitant challenges.

Adoptive T-cell transfer combined with a single low dose of total body irradiation eradicates breast tumors.

The major objectives of tumor vaccination are to induce the regression of established tumors and to favor the establishment of long-lasting tumor-specific immunity, capable of protecting the host from relapse. Immunotherapeutic strategies such as the administration of tumor-associated antigenic peptides offer one means to boost preexisting antitumor CD8+ T cell immunity. Our recent work reveals that established breast tumors are rejected and tumor recurrence prevented when low-dose irradiation is combined with the adoptive transfer of Mammaglobin A epitope-specific CD8+ T cells.

Adoptive transfer of Mammaglobin-A epitope specific CD8 T cells combined with a single low dose of total body irradiation eradicates breast tumors.

Adoptive T cell therapy has proven to be beneficial in a number of tumor systems by targeting the relevant tumor antigen. The tumor antigen targeted in our model is Mammaglobin-A, expressed by approximately 80% of human breast tumors. Here we evaluated the use of adoptively transferred Mammaglobin-A specific CD8 T cells in combination with low dose irradiation to induce breast tumor rejection and prevent relapse. We show Mammaglobin-A specific CD8 T cells generated by DNA vaccination with all epitopes (Mammaglobin-A2.1, A2.2, A2.4 and A2.6) and full-length DNA in vivo resulted in heterogeneous T cell populations consisting of both effector and central memory CD8 T cell subsets. Adoptive transfer of spleen cells from all Mammaglobin-A2 immunized mice into tumor-bearing SCID/beige mice induced tumor regression but this anti-tumor response was not sustained long-term. Additionally, we demonstrate that only the adoptive transfer of Mammaglobin-A2 specific CD8 T cells in combination with a single low dose of irradiation prevents tumors from recurring. More importantly we show that this single dose of irradiation results in the down regulation of the macrophage scavenger receptor 1 on dendritic cells within the tumor and reduces lipid uptake by tumor resident dendritic cells potentially enabling the dendritic cells to present tumor antigen more efficiently and aid in tumor clearance. These data reveal the potential for adoptive transfer combined with a single low dose of total body irradiation as a suitable therapy for the treatment of established breast tumors and the prevention of tumor recurrence.

NKG2D signaling on CD8⁺ T cells represses T-bet and rescues CD4-unhelped CD8⁺ T cell memory recall but not effector responses.

CD4-unhelped CD8(+) T cells are functionally defective T cells primed in the absence of CD4(+) T cell help. Given the co-stimulatory role of natural-killer group 2, member D protein (NKG2D) on CD8(+) T cells, we investigated its ability to rescue these immunologically impotent cells. We demonstrate that augmented co-stimulation through NKG2D during priming paradoxically rescues memory, but not effector, CD8(+) T cell responses. NKG2D-mediated rescue is characterized by reversal of elevated transcription factor T-box expressed in T cells (T-bet) expression and recovery of interleukin-2 and interferon-γ production and cytolytic responses. Rescue is abrogated in CD8(+) T cells lacking NKG2D. Augmented co-stimulation through NKG2D confers a high rate of survival to mice lacking CD4(+) T cells in a CD4-dependent influenza model and rescues HIV-specific CD8(+) T cell responses from CD4-deficient HIV-positive donors. These findings demonstrate that augmented co-stimulation through NKG2D is effective in rescuing CD4-unhelped CD8(+) T cells from their pathophysiological fate and may provide therapeutic benefits.

The CD8+ memory T-cell state of readiness is actively maintained and reversible.

The ability of the adaptive immune system to respond rapidly and robustly upon repeated antigen exposure is known as immunologic memory, and it is thought that acquisition of memory T-cell function is an irreversible differentiation event. In this study, we report that many phenotypic and functional characteristics of antigen-specific CD8 memory T cells are lost when they are deprived of contact with dendritic cells. Under these circumstances, memory T cells reverted from G(1) to the G(0) cell-cycle state and responded to stimulation like naive T cells, as assessed by proliferation, dependence upon costimulation, and interferon-gamma production, without losing cell surface markers associated with memory. The memory state was maintained by signaling via members of the tumor necrosis factor receptor superfamily, CD27 and 4-1BB. Foxo1, a transcription factor involved in T-cell quiescence, was reduced in memory cells, and stimulation of naive CD8 cells via CD27 caused Foxo1 to be phosphorylated and emigrate from the nucleus in a phosphatidylinositol-3 kinase-dependent manner. Consistent with these results, maintenance of G(1) in vivo was compromised in antigen-specific memory T cells in vesicular stomatitis virus-infected CD27-deficient mice. Therefore, sustaining the functional phenotype of T memory cells requires active signaling and maintenance.

Cutting edge: migration to nonlymphoid tissues results in functional conversion of central to effector memory CD8 T cells.

Memory CD8 T cells, essential for defense against intracellular pathogens, are heterogeneous with respect to phenotype and function. Constitutively lytic effector memory cells primarily reside in nonlymphoid tissues, whereas secondary lymphoid tissues contain functionally quiescent central memory cells. However, the mechanism by which functionally distinct memory populations are maintained is unknown. In this study, we show that resting CD8 memory cells modified their functional abilities upon entry into nonlymphoid tissues, as exemplified by the induction of granzyme B and lytic activity. Contemporaneously, the costimulator CD27 was down-regulated. These findings hold important implications for memory cell lineage development and tissue-specific immunity.

Fully functional memory CD8 T cells in the absence of CD4 T cells.

The role of CD4 T cells in providing help to CD8 T cells in primary and secondary responses to infection remains controversial. Using recombinant strains of virus and bacteria expressing the same Ag, we determined the requirement for CD4 T cells in endogenous CD8 T cell responses to infection with vesicular stomatitis virus and Listeria monocytogenes (LM). Depletion of CD4 T cells had no effect on the frequency of primary or secondary vesicular stomatitis virus-specific CD8 T cells in either lymphoid or nonlymphoid tissues. In contrast, the primary LM-specific CD8 T cell response was CD4 T cell dependent. Surprisingly, the LM-specific CD8 T cell recall response was also CD4 T cell dependent, which correlated with a requirement for CD40/CD40L interactions. However, concomitant inhibition of CD40L and CD4 T cell removal revealed that these pathways may be operating independently. Importantly, despite the absence of CD4 T cells during the recall response or throughout the entire response, CD8 memory T cells were functional effectors and proliferated equivalently to their "helped" counterparts. These data call into question the contention that CD4 T cells condition memory CD8 T cells during the primary response and indicate that the principal role of CD4 T cells in generating CD8 memory cells after infection is augmentation of proliferation or survival through costimulatory signals.

Activated primary and memory CD8 T cells migrate to nonlymphoid tissues regardless of site of activation or tissue of origin.

Following activation within secondary lymphoid tissue, CD8 T cells must migrate to targets, such as infected self tissue, allografts, and tumors, to mediate contact-dependent effector functions. To test whether the pattern of migration of activated CD8 T cells was dependent on the site of Ag encounter, we examined the distribution of mouse Ag-specific CD8 T cells following local challenges. Our findings indicated that activated CD8 T cells migrated pervasively to all nonlymphoid organs irrespective of the site of initial Ag engagement. Using an adoptive transfer system, migration of nonlymphoid memory cells was also examined. Although some limited preference for the tissue of origin was noted, transferred CD8 memory T cells from various nonlymphoid tissues migrated promiscuously, except to the intestinal mucosa, supporting the concept that distinct memory pools may exist. However, regardless of the tissue of origin, reactivation of transferred memory cells resulted in widespread dissemination of new effector cells. These data indicated that recently activated primary or memory CD8 T cells were transiently endowed with the ability to traffic to all nonlymphoid organs, while memory cell trafficking was more restricted. These observations will help refine our understanding of effector and memory CD8 T cell migration patterns.

Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation, cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells.

Cross-presentation of cell-bound Ags from established, solid tumors to CD8 cells is efficient and likely to have a role in determining host response to tumor. A number of investigators have predicted that when tumor Ags are derived from apoptotic cells either no response, due to Ag "sequestration," or CD8 cross-tolerance would ensue. Because the crucial issue of whether this happens in vivo has never been addressed, we induced apoptosis of established hemagglutinin (HA)-transfected AB1 tumors in BALB/c mice using the apoptosis-inducing reagent gemcitabine. This shrank the tumor by approximately 80%. This induction of apoptosis increased cross-presentation of HA to CD8 cells yet neither gross deletion nor functional tolerance of HA-specific CD8 cells were observed, based on tetramer analysis, proliferation of specific CD8 T cells, and in vivo CTL activity. Interestingly, apoptosis primed the host for a strong antitumor response to a second, virus-generated HA-specific signal in that administration of an HA-expressing virus after gemcitabine administration markedly decreased tumor growth compared with viral administration without gemcitabine. Thus tumor cell apoptosis in vivo neither sequesters tumor Ags nor cross-tolerizes tumor-specific CD8 cells. This observation has fundamental consequences for the development of tumor immunotherapy protocols and for understanding T cell reactivity to tumors and the in vivo immune responses to apoptotic cells.

Tissue-level regulation of Th1 and Th2 primary and memory CD4 T cells in response to Listeria infection.

Ag-specific Th1 and Th2 cytokine-producing CD4 T cells were quantitated in secondary lymphoid and tertiary tissues following oral Listeria monocytogenes infection. Although the response to Listeria was previously believed to be predominantly Th1 like, CD4 T cells producing IL-4 or IL-5 comprised a substantial proportion of the overall primary and memory response. The frequency of IFN-gamma-, IL-4-, or IL-5-producing primary effector or memory CD4 T cells was significantly higher in lung, liver, and intestinal lamina propria (LP) as compared with spleen and lymph node. However, maximum numbers of IL-4- and IL-5-producing cells were detected in the LP several days after the peak of the Th1 response, and IL-5 production was skewed toward the mucosal tissues. Remarkably, the recall response resulted in sustained Th1 and Th2 responses in tertiary, but not lymphoid tissues and long-term retention of Th1 and Th2 memory cells in equal proportions in the LP. Finally, CD40 ligand was essential for induction of IFN-gamma in the spleen and LP, but not in the liver and lung, while the IL-4 response required CD40 ligand only in the spleen. Therefore, the rules governing the effector phenotype, and the overall magnitude of the CD4 response, are regulated at the level of individual tissues.