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Background: Stimulator of Interferon Genes (STING) is a dsDNA sensor that triggers type I inflammatory responses. Recent data from our group and others support the therapeutic efficacy of STING agonists applied intratumorally or systemically in a range of murine tumor models, with treatment benefits associated with tumor vascular normalization and improved immune cell recruitment and function within the tumor microenvironment (TME). However, such interventions are rarely curative and STING agonism coordinately upregulates expression of immunoregulatory interferon-stimulated genes (ISGs) including Arg2, Cox2, Isg15, Nos2, and Pdl1 that may limit treatment benefits. We hypothesized that combined treatment of melanoma-bearing mice with STING agonist ADU-S100 together with antagonists of regulatory ISGs would result in improved control of tumor growth vs. treatment with ADUS100 alone. Methods: Mice bearing either B16 (BRAFWTPTENWT) or BPR20 (BRAFV600EPTEN-/-) melanomas were treated with STING agonist ADU-S100 plus various inhibitors of ARG2, COX2, NOS2, PD-L1, or ISG15. Tumor growth control and changes in the TME were evaluated for combination treatment vs ADU-S100 monotherapy by tumor areameasurements and flow cytometry/transcriptional profiling, respectively. Results: In the B16 melanoma model, we noted improved antitumor efficacy only when ADU-S100 was combined with neutralizing/blocking antibodies against PD-L1 or ISG15, but not inhibitors of ARG2, COX2, or NOS2. Conversely, in the BPR20 melanoma model, improved tumor growth control vs. ADU-S100 monotherapy was only observed when combining ADU-S100 with ARG2i, COX2i, and NOS2i, but not anti-PD-L1 or anti-ISG15. Immune changes in the TME associated with improved treatment outcomes were subtle but included increases in proinflammatory innate immune cells and activated CD8+CD69+ T cells and varied between the two tumor models. Conclusions: These data suggest contextual differences in the relative contributions of individual regulatory ISGs that serve to operationally limit the anti-tumor efficacy of STING agonists which should be considered in future design of novel combination protocols for optimal treatment benefit. [ABSTRACT FROM AUTHOR]

The concept of oncolytic virus (OV)-mediated cancer therapy has been shifted from an operational virotherapy paradigm to an immunotherapy. OVs often induce immunogenic cell death (ICD) of cancer cells, and they may interact directly with immune cells as well to prime antitumor immunity. We and others have developed a number of strategies to further stimulate antitumor immunity and to productively modulate the tumor microenvironment (TME) for potent and sustained antitumor immune cell activity. First, OVs have been engineered or combined with other ICD inducers to promote more effective T cell cross-priming, and in many cases, the breaking of functional immune tolerance. Second, OVs may be armed to express Th1-stimulatory cytokines/chemokines or costimulators to recruit and sustain the potent antitumor immunity into the TME to focus their therapeutic activity within the sites of disease. Third, combinations of OV with immunomodulatory drugs or antibodies that recondition the TME have proven to be highly promising in early studies. Fourth, combinations of OVs with other immunotherapeutic regimens (such as prime-boost cancer vaccines, CAR T cells; armed with bispecific T-cell engagers) have also yielded promising preliminary findings. Finally, OVs have been combined with immune checkpoint blockade, with robust antitumor efficacy being observed in pilot evaluations. Despite some expected hurdles for the rapid translation of OV-based stateof- the-art protocols, we believe that a cohort of these novel approaches will join the repertoire of standard cancer treatment options in the near future. [ABSTRACT FROM AUTHOR]

The effective accumulation and interaction of mature dendritic cells (DCs) and naïve T cells within lymph nodes (LNs), which are driven by the CCR7-CCL19/CCL21 chemokine axis, are critical for the induction of adaptive T-cell immunity. human natural killer (NK) cells activated by interleukin (IL)-18 exhibit a unique 'helper' activity in promoting productive Dc-T cell interactions, inducing Dc maturation and shifting DC-primed T-cell responses toward a TH1 polarization. here, we demonstrate that such IL-18-activated 'helper' NK cells uniquely stimulate DCs to produce high levels of CCL19 through tumor necrosis factor α (TNFα) and interferon γ (IFNγ), a process that relies on secondary NK-cell activation by additional inflammatory signals including IFNα, IL-15, IL-12 and IL-2. DCs activated by helper NK cells not only promote the efficient CCR7-mediated recruitment of naïve cD8+ T cells, but also stimulate their expansion and expression of granzyme B. Using an ex vivo explant culture system based on LNs isolated from colorectal cancer patients, we found that CCL19 is upregulated in human tumor-associated lymphoid tissues treated with helper NK cell-stimulating factors. Our findings demonstrate the ability of 2 signal-activated helper NK cells to promote the production of the DC- and naïve/memory T cell-attracting chemokine CCL19 in LNs, and provide a rationale for the therapeutic application of IL-18-containing 'combinatorial adjuvants' to facilitate the induction of antitumor immune responses. [ABSTRACT FROM AUTHOR]

IDO2 is a newly discovered enzyme with 43 % similarity to classical IDO (IDO1) protein and shares the same critical catalytic residues. IDO1 catalyzes the initial and rate-limiting step in the degradation of tryptophan and is a key enzyme in mediating tumor immune tolerance via arrest of T cell proliferation. The role of IDO2 in human T cell immunity remains controversial. Here, we demonstrate that similar to IDO1, IDO2 also degrades tryptophan into kynurenine and is inhibited more efficiently by Levo-1-methyl tryptophan (L-1MT), an IDO1 competitive inhibitor, than by dextro-methyl tryptophan (D-1MT). Although IDO2 enzyme activity is weaker than IDO1, it is less sensitive to 1-MT inhibition than IDO1. Moreover, our results indicate that human CD4 and CD8 T cell proliferation was inhibited by IDO2, but both L-1MT and D-1MT could not reverse IDO2-mediated arrest of cell proliferation, even at high concentrations. These data indicate that IDO2 is an inhibitory mechanism in human T cell proliferation and support efforts to develop more effective IDO1 and IDO2 inhibitors in order to overcome IDO-mediated immune tolerance. [ABSTRACT FROM AUTHOR]

Stimulation of double-stranded (ds)RNA receptors can increase the effectiveness of cancer vaccines, but the underlying mechanisms are not completely elucidated. In this study, we sought to determine critical roles of host IFN-α and IFN-γ pathways in the enhanced therapeutic efficacy mediated by peptide vaccines and polyinosinic-polycytidylic acid [poly(I:C)] stabilized by lysine and carboxymethylcellulose (poly-ICLC) in the murine central nervous system (CNS) GL261 glioma. C57BL/6-background wild type (WT), IFN- α receptor- 1 ( IFN- αR1)−/− or IFN- γ−/− mice bearing syngeneic CNS GL261 glioma received subcutaneous (s.c.) vaccinations with synthetic peptides encoding CTL epitopes with or without intramuscular (i.m.) injections of poly-ICLC. The combinational treatment induced a robust transcription of CXCL10 in the glioma site. Blockade of CXCL10 with a specific monoclonal antibody (mAb) abrogated the efficient CNS homing of antigen-specific type-1 CTL (Tc1). Both IFN- αR−/− and IFN- γ−/− hosts failed to up-regulate the CXCL10 mRNA and recruit Tc1 cells to the tumor site, indicating non-redundant roles of type-1 and type-2 IFNs in the effects of poly-ICLC-assisted vaccines. The efficient trafficking of Tc1 also required Tc1-derived IFN-γ. Our data point to critical roles of the host-IFN-α and IFN-γ pathways in the modulation of CNS glioma microenvironment, and the therapeutic effectiveness of poly-ICLC-assisted glioma vaccines. [ABSTRACT FROM AUTHOR]

Abstract: Ex vivo generation and antigen loading of dendritic cells (DCs) from cancer patients helps to bypass the dysfunction of endogenous DCs. It also allows to control the process of DC maturation and to imprint in maturing DCs several functions essential for induction of effective forms of cancer immunity. Recent reports from several groups including ours demonstrate that distinct conditions of DC generation and maturation can prime DCs for preferential interaction with different (effector versus regulatory) subsets of immune cells. Moreover, differentially-generated DCs have been shown to imprint different effector mechanisms in CD4+ and CD8+ T cells (delivery of “signal three”) and to induce their different homing properties (delivery of “signal four”). These developments allow for selective induction of tumor-specific T cells with desirable effector functions and tumor-relevant homing properties and to direct the desirable types of immune cells to tumors. [Copyright &y& Elsevier]

While multiple pathways of dendritic cell (DC) maturation result in transient production of IL-12, fully mature DCs show reduced ability to produce IL-12p70 upon a subsequent interaction with Ag-specific T cells, limiting their in vivo performance as vaccines. Such “DC exhaustion” can be prevented by the presence of IFNγ during the maturation of human DCs (type-1-polarization), resulting in improved induction of tumor-specific Th1 and CTL responses in vitro. Here, we show that type-1 polarization of mouse DCs strongly enhances their ability to induce CTL responses against a model tumor antigen, OVA, in vivo, promoting the induction of protective immunity against OVA-expressing EG7 lymphoma. Interestingly, in contrast to the human system, the induction of mouse DC1s requires the participation of IL-4, a nominal Th2-inducing cytokine. The current data help to explain the previously reported Th1-driving and anti-tumor activities of IL-4, and demonstrate that type-1 polarization increases in vivo activity of DC-based vaccines. [ABSTRACT FROM AUTHOR]

Abstract: Recent reports demonstrate that natural killer (NK) cells and dendritic cells (DC) support each other''s activity in a positive feedback. We observed that activated NK cells induce the maturation of DCs into stable type-1 polarized DCs (DC1), characterized by up to 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. DC1 induction depends on NK cell-produced IFN-γ and TNF-α, with a possible involvement of additional factors. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumor-related suppressive factors, and show strongly enhanced ability to induce Th1 and CTL responses. In analogy to resting T cells, the induction of “helper” function of NK cells relies on a two-signal activation paradigm. While NKG2D-dependent tumor cell recognition is sufficient to induce the cytotoxic “effector” function of NK cells, the induction of “NK cell help” requires additional signals from type-1 IFNs, products of virally-infected cells, or from IL-2. Compared to non-polarized DCs currently-used in clinical trials, DC1s act as superior inducers of anti-cancer CTL responses during in vitro sensitization. The current data provides rationale for the clinical use of DC1s in cancer and chronic infections (such as HIV), as a new generation DC-based vaccines, uniquely combining fully mature DC status with an elevated, rather than “exhausted” ability to produce bioactive IL-12p70. We are currently implementing stage I/II clinical trials, testing the effectiveness of DC1s induced by NK cells or by NK cell-related factors, as therapeutic vaccines against melanoma. [Copyright &y& Elsevier]

Simple Summary: The clinical effectiveness of Bacillus Calmette-Guérin (BCG) is limited to patients with early stages of bladder cancer (BlCa) and its effects are often transient. To understand the mechanisms limiting the effectiveness of BCG, we evaluated its impact on the human BlCa tumor microenvironment (TME) and the feasibility of its pharmacologic modulation. We observed that BCG non-selectively induces both CTL-attracting chemokines and Treg/MDSC attractants and suppressive factors in human BlCa tissue explants, in a mechanism involving NFκB-induced PGE2 synthesis and EP4 signaling. In contrast to non-selective impact of NFκB blockade on BCG-induced inflammation, the PGE2 antagonism selectively enhanced the BCG-driven production of CTL attractants but eliminated the induction of Treg/MDSC attractants and suppressive factors, enhancing the CTL migration but reducing Treg attraction to BCG-treated BlCa. Since intratumoral CTL accumulation predicts long term patient outcomes and the effectiveness of cancer immunotherapies, our data indicates the feasibility of targeting the PGE2-chemokine interplay to enhance the therapeutic effects of BCG. Bacillus Calmette-Guérin (BCG) is commonly used in the immunotherapy of bladder cancer (BlCa) but its effectiveness is limited to only a fraction of patients. To identify the factors that regulate the response of human BlCa tumor microenvironment (TME) to BCG, we used the ex vivo whole-tissue explant model. The levels of COX2 in the BCG-activated explants closely correlated with the local production of Treg- and MDSCS attractants and suppressive factors, while the baseline COX2 levels did not have predictive value. Accordingly, we observed that BCG induced high levels of MDSC- and Treg-attracting chemokines (CCL22, CXCL8, CXCL12) and suppressive factors (IDO1, IL-10, NOS2). These undesirable effects were associated with the nuclear translocation of phosphorylated NFκB, induction of COX2, the key enzyme controlling PGE2 synthesis, and elevation of a PGE2 receptor, EP4. While NFκB blockade suppressed both the desirable and undesirable components of BCG-driven inflammation, the inhibitors of PGE2 synthesis (Celecoxib or Indomethacin) or signaling (EP4-selective blocker, ARY-007), selectively eliminated the induction of MDSC/Treg attractants and immunosuppressive factors but enhanced the production of CTL attractants, CCL5, CXCL9 and CXCL10. PGE2 blockade allowed for the selectively enhanced migration of CTLs to the BCG-treated BlCa samples and eliminated the enhanced migration of Tregs. Since the balance between the CTLs and suppressive cells in the TME predicts the outcomes in patients with BlCa and other diseases, our data help to elucidate the mechanisms which limit the effectiveness of BCG therapies and identify new targets to enhance their therapeutic effects. [ABSTRACT FROM AUTHOR]

Simple Summary: Those with a history of colorectal cancer benefit from aspirin following completion of treatment, however, it is believed that this benefit is restricted to patients who have tumors with certain mutations. It is not known whether taking aspirin during active treatment helps patients or whether it depends on tumor genetics in this setting. We investigated 147 patients who underwent chemoradiation for rectal cancer at our institution, including 42 who were taking aspirin at the time of chemoradiation. Concurrent aspirin use was associated with significantly improved tumor control and survival. Genetic analysis of the tumor did not indicate that these benefits were restricted to certain molecular conditions. Interestingly, aspirin was associated with more advantageous immune response in tumor specimens. Based on these findings, the addition of aspirin to chemoradiation should be considered for rectal cancer patients, independent of molecular conditions. Background: The benefit of aspirin in rectal cancer during chemoradiation therapy (CRT) and the factors affecting its efficacy are not well characterized. We compared the outcomes of rectal patients undergoing neoadjuvant CRT based on aspirin use. Methods: Patients undergoing CRT for rectal cancer from 2010 to 2018 were evaluated. Aspirin use was determined by medication list prior to treatment. RNA sequencing and subsequent gene set enrichment analysis was performed on surgically resected specimens. Results: 147 patients underwent neoadjuvant CRT with a median follow-up of 38.2 months. Forty-two patients were taking aspirin prior to CRT. Aspirin users had significantly less local and distant progression, and improved progression-free and overall survival. On RNA-sequencing, neither PI3KCA nor KRAS mutational status were associated with the benefit of aspirin use or tumor downstaging. PTGS2/COX2 expression trended lower in aspirin users, but not with tumor response. Aspirin use was associated with increases of M1 macrophages, plasma cells, CD8+ T cells, and reduction of M2 macrophages in the resected tumor. Conclusions: Concurrent aspirin use during neoadjuvant CRT was associated with improved local and distant tumor control leading to significantly improved survival. Neither mutations in KRAS or PI3CKA, nor the levels of COX-2 expression at the time of resection of the residual tumor were predictive of these aspirin benefits. [ABSTRACT FROM AUTHOR]

We have armed a tumor-selective oncolytic vaccinia virus (vvDD) with the chemokine (CK) CXCL11, in order to enhance its ability to attract CXCR3+antitumor CTLs and possibly NK cells to the tumor microenvironment (TME) and improve its therapeutic efficacy. As expected, vvDD-CXCL11 attracted high numbers of tumor-specific T cells to the TME in a murine AB12 mesothelioma model. Intratumoral virus-directed CXCL11 expression enhanced local numbers of CD8+CTLs and levels of granzyme B, while reducing expression of several suppressive molecules, TGF-β, COX2, and CCL22 in the TME. Unexpectedly, we observed that vvDD-CXCL11, but not parental vvDD, induced a systemic increase in tumor-specific IFNγ-producing CD8+T cells in the spleen and other lymph organs, indicating the induction of systemic antitumor immunity. This effect was associated with enhanced therapeutic efficacy and a survival benefit in tumor-bearing mice treated with vvDD-CXCL11, mediated by CD8+T cells and IFNγ, but not CD4+T cells. These results demonstrate that intratumoral expression of CXCL11, in addition to promoting local trafficking of T cells and to a lesser extent NK cells, has a novel function as a factor eliciting systemic immunity to cancer-associated antigens. Our data provide a rationale for expressing CXCL11 to enhance the therapeutic efficacy of oncolytic viruses (OVs) and cancer vaccines. [ABSTRACT FROM PUBLISHER]

Chemokine-driven interactions of immune cells are essential for effective antitumor immunity. Human natural killer (NK) cells can be primed by the interleukin (IL)-1--related proinflammatory cytokine IL-18 for unique helper activity, which promotes dendritic cell (DC) activation and DC-mediated induction of type-1 immune responses against cancer. Here, we show that such IL-18--primed "helper" NK cells produce high levels of the immature DC (iDC)--attracting chemokines CCL3 and CCL4 upon exposure to tumor cells or the additional inflammatory signals IFN-α, IL-15, IL-12, or IL-2. These "helper" NK cells potently attract iDCs in a CCR5-dependent mechanism and induce high DC production of CXCR3 and CCR5 ligands (CXCL9, CXCL10, and CCL5), facilitating the subsequent recruitment of type-1 effector CD8+ T( Teff) cells. Using cells isolated from the malignant ascites of patients with advanced ovarian cancer, we show that "helper" NK cell-inducing factors can be used to enhance local production of Teff cell-recruiting chemokines. Our findings reveal the unique chemokine expression profile of "helper" NK cells and highlight the potential for using two-signal--activated NK cells to promote homing of type-1 immune effectors to the human tumor environment. [ABSTRACT FROM AUTHOR]

Tumor infiltration with effector CD8+ T cells (Teff) predicts longer recurrence-free survival in many types of human cancer, illustrating the broad significance of Teff for effective immunosurveillance. Colorectal tumors with reduced accumulation of Teff express low levels of Teff-attracting chemokines such as CXCL10/IP10 and CCL5/RANTES. In this study, we investigated the feasibility of enhancing tumor production of Teff-attracting chemokines as a cancer therapeutic strategy using a tissue explant culture system to analyze chemokine induction in intact tumor tissues. In different tumor explants, we observed highly heterogeneous responses to IFNα or poly-I:C (a TLR3 ligand) when they were applied individually. In contrast, a combination of IFNα and poly-I:C uniformly enhanced the production of CXCL10 and CCL5 in all tumor lesions. Moreover, these effects could be optimized by the further addition of COX inhibitors. Applying this triple combination also uniformly suppressed the production of CCL22/MDC, a chemokine associated with infiltration of T regulatory cells (Treg). The Teff-enhancing effects of this treatment occurred selectively in tumor tissues, as compared with tissues derived from tumor margins. These effects relied on the increased propensity of tumor-associated cells (mostly fibroblasts and infiltrating inflammatory cells) to hyperactivate NF-κB and produce Teff -attracting chemokines in response to treatment, resulting in an enhanced ability of the treated tumors to attract Teff cells and reduced ability to attract Treg cells. Together, our findings suggest the feasibility of exploiting NF-κB hyperactivation in the tumor microenvironment to selectively enhance Teff entry into colon tumors. [ABSTRACT FROM AUTHOR]

Abstract: The development of effective vaccine strategies for intracellular bacteria, including tuberculosis, is one of the major frontiers of medical research. Our previous studies showed that dendritic cell (DC) vaccine is a promising approach for eliciting protective immunity against intracellular bacteria. However, it has been reported that standard fully mature DCs show reduced ability to produce IL-12p70 upon subsequent interaction with antigen (Ag)-specific T cells, limiting their in vivo performance for vaccines. Recently, we found that such “DC exhaustion” could be prevented by the presence of IL-4 and IFN-γ during the maturation of mouse DCs (type-1 polarization), resulting in improved induction of anti-tumor immunity in cancer. Here we show that such type-1 polarized DCs promote dramatic enhancement of protective immunity against an intracellular bacterium, Listeria monocytogenes. Murine bone marrow-derived DCs were cultured and matured with LPS, IL-4 and IFN-γ (type-1 polarized DCs), and with LPS alone (non-polarized DCs). DCs were loaded with listeriolysin O (LLO) 91-99, H2-Kd-restricted epitope of L. monocytogenes, and were injected into naïve BALB/c mice intravenously. Type-1 polarized DCs produced significantly higher levels of IL-12p70 than non-polarized DCs in vitro, and this vaccine strongly enhanced LLO 91-99-specific CD8+ T cells exhibiting epitope-specific cytotoxic activity and IFN-γ production, leading to significant induction of protective immunity against L. monocytogenes. Type-1 polarized DCs are potential candidates for enhancing protective immunity in the design of effective vaccination strategies against intracellular bacteria. [Copyright &y& Elsevier]

HLA-A2 transgenic mice bearing established HLA-A2neg B16 melanomas were effectively treated by intratumoral (i.t.) injection of syngeneic dendritic cells (DCs) transduced to express high levels of interleukin (IL)-12, resulting in CD8+ T cell-dependent antitumor protection. In this model, HLA-A2-restricted CD8+ T cells do not directly recognize tumor cells and therapeutic benefit was associated with the crosspriming of HLA-A2-restricted type-1 CD8+ T cells reactive against antigens expressed by stromal cells [i.e., pericytes and vascular endothelial cells (VEC)]. IL-12 gene therapy-induced CD8+ T cells directly recognized HLA-A2+ pericytes and VEC flow-sorted from B16 tumor lesions based on interferon (IFN)-γ secretion and translocation of the lytic granule-associated molecule CD107 to the T cell surface after coculture with these target cells. In contrast, these CD8+ T effector cells failed to recognize pericytes/VEC isolated from the kidneys of tumor-bearing HHD mice. The tumor-associated stromal antigen (TASA)-derived peptides studied are evolutionarily conserved and could be recognized by CD8+ T cells harvested from the blood of HLA-A2+ normal donors or melanoma patients after in vitro stimulation. These TASA and their derivative peptides may prove useful in vaccine formulations against solid cancers, as well as, in the immune monitoring of HLA-A2+ cancer patients receiving therapeutic interventions, such as IL-12 gene therapy. [ABSTRACT FROM AUTHOR]

In C57BL/6 (B6) mice, most herpes simplex virus (HSV)-specific CD8 T cells recognize a strongly immunodominant epitope on glycoprotein B (gB498) and can inhibit HSV type 1 (HSV-1) reactivation from latency in trigeminal ganglia (TG). However, half of the CD8 T cells retained in latently infected TG of B6 mice are not gB498 specific and have been largely ignored. The following observations from our current study indicate that these gB498-nonspecific CD8 T cells are HSV specific and may contribute to the control of HSV-1 latency. First, following corneal infection, OVA257-specific OT-1 CD8 T cells do not infiltrate the infected TG unless mice are simultaneously immunized with OVA257 peptide, and then they are not retained. Second, 30% of CD8 T cells in acutely infected TG that produce gamma interferon in response to HSV-1 stimulation directly ex vivo are gB498 nonspecific, and these cells maintain an activation phenotype during viral latency. Finally, gB498-nonspecific CD8 T cells are expanded in ex vivo cultures of latently infected TG and inhibit HSV-1 reactivation from latency in the absence of gB498-specific CD8 T cells. We conclude that many of the CD8 T cells that infiltrate and are retained in infected TG are HSV specific and potentially contribute to maintenance of HSV-1 latency. Identification of the viral proteins recognized by these cells will contribute to a better understanding of the dynamics of HSV-1 latency. [ABSTRACT FROM AUTHOR]