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SF1: IL-26 protein or mRNA expression in human cell lines and clinical samples; SF2: Single-cell profiling of CD4+ TH17 cells in TNBC patients; SF3: Confirmation of IL-26 knockdown in MDA-MB-231 cells; SF4: In vitro proliferation and in vivo metastasis of IL-26 knockdown MDA-MB-231 cells; SF5: Gene expression profiling of IL-26 knockdown MDA-MB-231 tumors; SF6: IL-26 knockdown in SUM159 and MDA-MB-468 cells and in vitro phenotyping; SF7: Western blot confirmation of IL-26 knock-in E0771 cells and IL-26 upregulation after growth in vivo; SF8: IL-26 knock-in E0771 tumors progress more rapidly than control in an immune competent setting; SF9: Stat3 activation by IL-26 is dependent on human IL10RB and IL20RA; SF10: DNA-bound IL-26 elicits inflammatory signaling in human and mouse immune cells; SF11: Confirmation of mouse neutrophil enrichment; SF12: Confirmation of Anti-Ly6G antibody depletion of mouse neutrophils; SF13: Inflammatory cytokine expression in MDA-MB-231 parental and MDA-MB-231 LM2 lung-metastatic subclone; SF14: CRISPR knockdown of IL-6, IL-8, and Cxcl1 in MDA-MB-231 LM2 CRISPR 3x cell lines/lung metastases and correlation of CRISPR 3x primary tumor volume with metastasis; SF15: IL-26 signature and Neutrophil enrichment score are significantly correlated in METABRIC dataset; SF16: T cell ELISPOT response to IL-26 vaccination in vivo.

Significant hits identified in CRISPR screen

CRISPR screen results

IL26 is a unique amphipathic member of the IL10 family of cytokines that participates in inflammatory signaling through a canonical receptor pathway. It also directly binds DNA to facilitate cellular transduction and intracellular inflammatory signaling. Although IL26 has almost no described role in cancer, our in vivo screen of inflammatory and cytokine pathway genes revealed IL26 to be one of the most significant inflammatory mediators of mammary engraftment and lung metastatic growth in triple-negative breast cancer (TNBC). Examination of human breast cancers demonstrated elevated IL26 transcripts in TNBC specimens, specifically in tumor cells as well as in Th17 CD4+ T cells within clinical TNBC specimens. IL26 did not have an autocrine effect on human TNBC cells, but rather its effect on engraftment and growth in vivo required neutrophils. IL26 enhanced mouse-derived DNA induction of inflammatory cytokines, which were collectively important for mammary and metastatic lung engraftment. To neutralize this effect, we developed a novel IL26 vaccine to stimulate antibody production and suppress IL26-enhanced engraftment in vivo, suggesting that targeting this inflammatory amplifier could be a unique means to control cancer-promoting inflammation in TNBC and other autoimmune diseases. Thus, we identified IL26 as a novel key modulator of TNBC metastasis and a potential therapeutic target in TNBC as well as other diseases reliant upon IL26-mediated inflammatory stimulation.Significance:These findings identify IL26 as a unique, clinically relevant, inflammatory amplifier that enhances TNBC engraftment and dissemination in association with neutrophils, which has potential as a therapeutic target.

Monoclonal antibodies (mAb) are a major component of cancer therapy. In this review, we summarize the different therapeutic mAbs that have been successfully developed against various tumor-expressed antigens and examine our current understanding of their different mechanisms of antitumor action. These mechanisms of action (MOA) largely center on the stimulation of different innate immune effector processes, which appear to be principally responsible for the efficacy of most unconjugated mAb therapies against cancer. This is evident in studies of mAbs targeting antigens for hematologic cancers, with emerging data also demonstrating the critical nature of innate immune-mediated mechanisms in the efficacy of anti-HER2 mAbs against solid HER2+ cancers. Although HER2-targeted mAbs were originally described as inhibitors of HER2-mediated signaling, multiple studies have since demonstrated these mAbs function largely through their engagement with Fc receptors to activate innate immune effector functions as well as complement activity. Next-generation mAbs are capitalizing on these MOAs through improvements to enhance Fc-activity, although regulation of these mechanisms may vary in different tumor microenvironments. In addition, novel antibody-drug conjugates have emerged as an important means to activate different MOAs. Although many unknowns remain, an improved understanding of these immunologic MOAs will be essential for the future of mAb therapy and cancer immunotherapy.

Project objective: Despite the recent revolution in immune checkpoint inhibitors (ICIs), only modest improvement in overall survival and likely caused by not enough potent cellular immunity among BC patients. Our lab has been focus on inducing cellular immunity against HER2+ BC through vaccination against the tumor-associated antigen HER2. Approximately 20 years ago, we performed an experimental pilot study by administrating HER2 peptide and recombinant protein pulsed dendritic cells (DC vaccine) to six patients with refractory HER2+ advanced or metastatic (stage II (≥ 6 +LN), III, or stage IV) BC. We followed the patients on 2019 found that all of the six patients were still alive, 18 years after vaccination. Their blood sample were analyzed with cytometry by time-of-flight (CyTOF) and found there is a significantly increased presence of CD27 expressing memory T cells in response to HER2 peptide stimulation. Recent report on the SARS-CoV2 mRNA vaccine also suggested that CD27 expressing memory T cells plays a critical role in long-lasting cellular immunity against SARS-CoV2 infection. Therefore, we hypothesized that CD27 plays a critical role in cellular immunity against BC, and the stimulation of CD27 expressing T cells with mAb targeting CD27 significantly increase the cellular immunity triggered by vaccination against tumor-associated antigen. Results: We recapitulate the rise of CD27+ antigen specific T cells among the vaccinated patients using a transgenic mouse model expressing human CD27. When combined the adenoviral-vector based HER2 (Ad-HER2) vaccination with a single dose of human aCD27 antibody (Varlilumab), we found there is a robust increase in the HER2 specific T cells compared to vaccination alone, especially CD27+CD44+ memory CD4 T cells, even after 120 days post vaccination. Using an ICI-insensitive syngeneic HER2+ BC models, we found 50% of mice in the combination group of aCD27 antibody plus Ad-HER2 showed total tumor regression by the end of study. When combined with anti-PD1 antibody, the combination of AdHER2 and Varlilumab leads to total tumor regression in 90% of tumor bearing mice with syngeneic HER2+ BC, indicating that the vaccination against tumor associated antigen HER2 plus anti-CD27 antibody sensitized ICI-insensitive HER2+ BC toward ICI. Conclusions: Our data demonstrates that the administration of anti-CD27 antibody significantly increase the long term presence of CD27+ antigen specific memory T cells after vaccination against tumor associated antigen HER2. As consequence, combination of anti-CD27 with HER2 sensitized the immune unresponsive breast cancer toward anti-PD1 antibody. Our study suggests that the vaccination against tumor-associated antigen with mAb targeting CD27 leads to the robust cellular immunity, which is required for successful ICIs against breast cancer. Citation Format: Bin-Jin Hwang, Erika Crosby, Timothy Trotter, Li-Chung Tsao, Tao Wang, Xiao Yang, Herbert Kim Lyerly, Zachary Hartman. Sensitizing breast cancers to immune checkpoint inhibitors through CD27 agonism and vaccination against tumor-associated antigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 696.

Background: T-DXd is a new antibody-drug conjugate targeting HER2 that has demonstrated superior clinical efficacy over previous HER2-targeting agent Trastuzumab emtansine (T-DM1) in patients with advanced-stage HER2+ breast cancer (BC). T-Dxd consists of the anti-HER2 antibody Trastuzumab conjugated to Dxd (a topoisomerase-I inhibitor). While the HER2-mediated internalization of Dxd leads to direct and bystander tumor cell killing, it is unclear whether T-Dxd retains Fc-mediated induction of immunity and how these responses may interact with Dxd-mediated tumor cell killing. Therefore, in this study we investigated tumor-specific innate and adaptive immune responses elicited upon T-Dxd treatment versus other HER2-targeting agents. Method: We first compared immune responses of T-Dxd versus T-DM1 or Trastuzumab on macrophage activation after co-culture with HER2+ BC cells. We next utilized JEDI T cells to model tumor-antigen presentation by those macrophages. We also investigated the therapeutic effect of T-Dxd in an endogenous HER2 transgenic mouse model, and measured HER2-specific adaptive immune responses, in combination with immune checkpoint blockades. Results: We found that T-Dxd retains the capability to engage with FCGRs and promote ADCP of HER2+ BC cells by macrophages, with comparable efficacy to Trastuzumab. Interestingly, T-Dxd mediated ADCP combined with Dxd-mediated immunogenic cell death promoted superior macrophage activation over Trastuzumab and T-DM1, resulting in increased expression of antigen presentation genes, chemokines, and interferon-stimulated genes. In co-culture experiments with JEDI T cells, macrophages that have phagocytosed eGFP-expressing tumor cells after T-Dxd treatment showed superior activation of antigen-specific effector T cells. In vivo, T-Dxd demonstrated exceptional tumor growth inhibition over Trastuzumab, accompanied by increased HER2-specific adaptive T cell responses. Lastly, T-Dxd therapy showed synergistic antitumor activity when combined with immune checkpoint blockade of the CD47/SIRPA axis. Conclusion: T-Dxd retains antibody-mediated antitumor mechanisms of Trastuzumab such as FCGR activation and ADCP of HER2+ tumor cells. Importantly, this is synergized with T-Dxd induced immunogenic tumor cell death, which results in enhanced macrophage activation upon tumor cell phagocytosis. This result in enhanced tumor antigen presentation and activation of adaptive immunity in vitro and in preclinical mouse models, which may illustrate T-Dxd’s superior and long-lasting clinical efficacy in HER2+ BC patients. Combination therapy of T-Dxd with CD47/SIRPA checkpoint blockade resulted in synergistic therapeutic benefits in our mouse model studies, and further clinical research with this combination is warranted. Citation Format: Li-Chung Tsao, Zachary Conrad Hartman. Trastuzumab-deruxtecan (T-Dxd) induces superior macrophage/DC activation, antigen-presentation and anti-tumor adaptive immune responses, which can be augmented by CD47/SIRPA blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2944.

Background: The CD47-SIRPα axis is an innate immune checkpoint that primarily regulates myeloid cells. In the context of cancer, tumor cells upregulate CD47 to engage SIRPα inhibitory signaling to block phagocytosis. Thus, inhibition of this axis may enhance myeloid cell phagocytosis and adaptive immune responses against cancer. While our previous studies identified that CD47 mAbs enhance HER2-specific mAb therapy (Tsao et al., 2019), clinical trials have revealed that CD47-targeting mAbs are associated with severe side effects of anemia and thrombocytopenia in patients. We thus hypothesized that targeting SIRPα, which is more restrictively expressed on myeloid cells, may achieve similar but less toxic treatment effects. Methods: To ascertain the impact of SIRPα inhibition against different molecular subtypes of breast cancer, we utilized and validated a novel SIRPα KO mouse, along with SIRPα mAbs in our previously utilized models of Triple-Negative Breast Cancer (TNBC) and HER2+ Breast Cancer (Crosby et al., 2018; Tsao et al., 2019). Using both in vitro and in vivo tumor models, we tested the therapeutic impact of SIRPα inhibition/blockade combined with immunogenic chemotherapy (doxorubicin) in TNBC or combined with HER2-specific mAbs in HER2+ Breast Cancer (HER2+ BC). Results: After validating the loss of SIRPα in KO mice, we found that E0771 TNBC cells had reduced rates of engraftment in SIRPα KO mice compared to controls, but comparable rates of growth once tumors were established. We then treated E0771 engrafted SIRPα KO and wildtype mice with doxorubicin. This treatment elicited tumor rejection in the majority of SIRPα KO mice (70% of injected tumors), compared to only 10% rejection in SIRPα WT mice. To study tumor antigen-specific immunity, we expressed different forms of OVA (secreted, cytoplasmic, membrane) in tumor cells. We found that membrane expressed OVA led to universal tumor rejection (10/10 mice) with high levels of OVA-specific Abs, in contrast to non-membrane expressed forms of OVA. This suggested the importance of tumor-specific Abs in eliciting tumor rejection. To specifically test the antitumor effect of tumor-specific antibodies in the context of SIRPα blockade, we evaluated the impact of a HER2 mAb (Trastuzumab) on HER2+ BC cells. We found enhanced phagocytosis and antitumor effects by SIRPα KO macrophages in comparison to wild-type macrophages, suggesting the potential of targeting SIRPα along with the use of standard-of-care mAb. Conclusion: Our study demonstrates that SIRPα loss can prevent tumor engraftment but may not have a strong effect against established tumors without secondary immune stimulation. However, addition of immunogenic cell death-eliciting chemotherapy or tumor-specific Abs promote tumor phagocytosis and stimulate anti-tumor immunity. These results support further investigation of SIRPα-targeting combination therapies for cancer. Citation Format: Xingru Ma, Zachary Hartman, Li-Chung Tsao, Tao Wang, Cong-Xiao Liu, Xiao Yang, Gangjun Lei, Junping Wei. Inhibition of CD47-SIRPα axis to enhance immunity against different molecular subtypes of breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5067.

IL26 is a unique amphipathic member of the IL10 family of cytokines that participates in inflammatory signaling through a canonical receptor pathway. It also directly binds DNA to facilitate cellular transduction and intracellular inflammatory signaling. Although IL26 has almost no described role in cancer, our in vivo screen of inflammatory and cytokine pathway genes revealed IL26 to be one of the most significant inflammatory mediators of mammary engraftment and lung metastatic growth in triple-negative breast cancer (TNBC). Examination of human breast cancers demonstrated elevated IL26 transcripts in TNBC specimens, specifically in tumor cells as well as in Th17 CD4+ T cells within clinical TNBC specimens. IL26 did not have an autocrine effect on human TNBC cells, but rather its effect on engraftment and growth in vivo required neutrophils. IL26 enhanced mouse-derived DNA induction of inflammatory cytokines, which were collectively important for mammary and metastatic lung engraftment. To neutralize this effect, we developed a novel IL26 vaccine to stimulate antibody production and suppress IL26-enhanced engraftment in vivo, suggesting that targeting this inflammatory amplifier could be a unique means to control cancer-promoting inflammation in TNBC and other autoimmune diseases. Thus, we identified IL26 as a novel key modulator of TNBC metastasis and a potential therapeutic target in TNBC as well as other diseases reliant upon IL26-mediated inflammatory stimulation. Significance: These findings identify IL26 as a unique, clinically relevant, inflammatory amplifier that enhances TNBC engraftment and dissemination in association with neutrophils, which has potential as a therapeutic target.

Two HER2-specific mAbs, trastuzumab and pertuzumab (T+P), combined with chemotherapy comprise standard-of-care treatment for advanced HER2+ breast cancers (BC). While this antibody combination is highly effective, its synergistic mechanism-of-action (MOA) remains incompletely understood. Past studies have suggested that the synergy underlying this combination occurs through the different mechanisms elicited by these antibodies, with pertuzumab suppressing HER2 heterodimerization and trastuzumab inducing antitumor immunity. However, in vivo evidence for this synergy is lacking. In this study, we found that the therapeutic efficacy elicited by their combination occurs through their joint ability to activate the classical complement pathway, resulting in both complement-dependent cytotoxicity and complement-dependent cellular phagocytosis of HER2+ tumors. We also demonstrate that tumor C1q expression is positively associated with survival outcome in HER2+ BC patients and that complement regulators CD55 and CD59 were inversely correlated with outcome, suggesting the clinical importance of complement activity. Accordingly, inhibition of C1q in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant antitumor MOA for T+P therapy that may be functionally enhanced to potentially augment clinical therapeutic efficacy.

Background: The majority of colorectal carcinomas (CRCs) are insensitive to anti-PD-1/PD-L1 immune checkpoint inhibitor (ICI) antibodies. While there are many causes for ICI insensitivity, recent studies suggest that the suppression of innate immunity through a loss of innate immune adaptor expression could be a root cause of this insensitivity and an important factor in the evolution of tumor immunosuppression. Methods: We interrogated mitochondrial antiviral signaling gene (MAVS) expression through bioinformatics analyses of multiple datasets to validate its suppression in clinical samples. We then engineered MAVS expressing tumor cells and tested its ability to elicit innate and adaptive anti-tumor immunity using both in vitro and in vivo approaches, which we then confirmed using MAVS expressing viral vectors. Finally, we observed that MAVS stimulated PD-L1expression in different types of tumor cells and then assessed the combination of PD-L1 ICI antibodies with MAVS tumor expression in vivo. Results: MAVS was significantly downregulated in CRCs, but its re-expression could stimulate broad cellular interferon-related responses, in both murine and patient-derived CRCs. In vivo, local MAVS expression elicited significant anti-tumor responses in both immune-sensitive and insensitive CRC models, through the stimulation of an interferon responsive axis that elicited tumor antigen-specific adaptive immunity. Critically, we found that tumor-intrinsic MAVS expression triggered systemic adaptive immune responses that enabled abscopal CD8+ T cell cytotoxicity against distant CRCs. As MAVS also induced PD-L1 expression, we further found synergistic anti-tumor responses in combination with anti-PD-L1 ICIs. Conclusion: These data demonstrate that intratumoral MAVS expression results in local and systemic tumor antigen-specific T cell responses, which could be combined with PD-L1 ICI to permit effective anti-tumor immunotherapy in ICI resistant cancers. We are further exploring to overcome the ICI insensitivity of other types of solid tumor, with MAVS-delivery strategies other than viral vectors. Citation Format: Bin-Jin Hwang, Li-Chung Tsao, Chaitanya Acharya, Timothy Trotter, Pankaj Agarwal, Tao Wang, Junping Wei, Xiao-Yi Yang, Gang-jun Lei, Takuya Osada, Herbert Kim Lyerly, Michael A. Morse, Zachary Hartman. Sensitizing immune unresponsive colorectal cancers to immune checkpoint inhibitors through MAVS overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3531.

BackgroundThe majority of colorectal carcinomas (CRCs) are insensitive to programmed death protein-1/programmed death-ligand 1 (anti-PD-1/PD-L1) immune checkpoint inhibitor (ICI) antibodies. While there are many causes for ICI insensitivity, recent studies suggest that suppression of innate immune gene expression in tumor cells could be a root cause of this insensitivity and an important factor in the evolution of tumor immunosuppression.MethodsWe first assessed the reduction of mitochondrial antiviral signaling gene (MAVS) and related RIG-I pathway gene expression in several patient RNA expression datasets. We then engineered MAVS expressing tumor cells and tested their ability to elicit innate and adaptive anti-tumor immunity using both in vitro and in vivo approaches, which we then confirmed using MAVS expressing viral vectors. Finally, we observed that MAVS stimulated PD-L1 expression in multiple cell types and then assessed the combination of PD-L1 ICI antibodies with MAVS tumor expression in vivo.ResultsMAVS was significantly downregulated in CRCs, but its re-expression could stimulate broad cellular interferon-related responses, in both murine and patient-derived CRCs. In vivo, local MAVS expression elicited significant anti-tumor responses in both immune-sensitive and insensitive CRC models, through the stimulation of an interferon responsive axis that provoked tumor antigen-specific adaptive immunity. Critically, we found that tumor-intrinsic MAVS expression triggered systemic adaptive immune responses that enabled abscopal CD8 +T cell cytotoxicity against distant CRCs. As MAVS also induced PD-L1 expression, we further found synergistic anti-tumor responses in combination with anti-PD-L1 ICIs.ConclusionThese data demonstrate that intratumoral MAVS expression results in local and systemic tumor antigen-specific T cell responses, which could be combined with PD-L1 ICI to permit effective anti-tumor immunotherapy in ICI resistant cancers.

The HER2-specific monoclonal antibody (mAb), trastuzumab, has been the mainstay of therapy for HER2(+) breast cancer (BC) for approximately 20 years. However, its therapeutic mechanism of action (MOA) remains unclear, with antitumor responses to trastuzumab remaining heterogeneous and metastatic HER2(+) BC remaining incurable. Consequently, understanding its MOA could enable rational strategies to enhance its efficacy. Using both murine and human versions of trastuzumab, we found its antitumor activity dependent on Fcγ receptor stimulation of tumor-associated macrophages (TAMs) and antibody-dependent cellular phagocytosis (ADCP), but not cellular cytotoxicity (ADCC). Trastuzumab also stimulated TAM activation and expansion, but did not require adaptive immunity, natural killer cells, and/or neutrophils. Moreover, inhibition of the innate immune ADCP checkpoint, CD47, significantly enhanced trastuzumab-mediated ADCP and TAM expansion and activation, resulting in the emergence of a unique hyperphagocytic macrophage population, improved antitumor responses, and prolonged survival. In addition, we found that tumor-associated CD47 expression was inversely associated with survival in HER2(+) BC patients and that human HER2(+) BC xenografts treated with trastuzumab plus CD47 inhibition underwent complete tumor regression. Collectively, our study identifies trastuzumab-mediated ADCP as an important antitumor MOA that may be clinically enabled by CD47 blockade to augment therapeutic efficacy.

Background Regulatory T cells (Tregs) suppress T-cell immune activation and human immunodeficiency virus type 1 (HIV-1) replication, but the role of Tregs in HIV-1 reservoir persistence is poorly defined. Methods Tregs were depleted by denileukin diftitox in humanized mice with chronic HIV-1 infection. Viral replication in lineage cells was determined by p24 expression. Levels of HIV-1 RNA and DNA in human cells, as well as replication-competent-virus-producing cells, were measured to quantified viral replication and reservoirs. Results Treg depletion resulted in a blip of HIV-1 replication in T cells but not in myeloid cells. The major activated reservoir cells were memory CD4+ T cells in vivo. Interestingly, the transient activation of viral replication led to HIV-1 reservoir reduction after viremia resuppression, as indicated by the quantity of HIV-1 DNA and replication-competent-virus-producing cells. Furthermore, we demonstrated that Tregs use cyclic adenosine monophosphate (cAMP)-dependent protein kinase A pathway to inhibit HIV-1 activation and replication in resting conventional T cells in vitro. Conclusion Tregs suppress HIV-1 replication in T cells and contribute to HIV-1 reservoir persistence. cAMP produced in Tregs is involved in their suppression of viral gene activation and expression. Treg depletion combined with combination antiretroviral therapy provides a novel strategy for HIV-1 cure.

Despite the efficient suppression of HIV-1 replication that can be achieved with combined antiretroviral therapy (cART), low levels of type I interferon (IFN-I) signaling persist in some individuals. This sustained signaling may impede immune recovery and foster viral persistence. Here we report studies using a monoclonal antibody to block IFN-α/β receptor (IFNAR) signaling in humanized mice (hu-mice) that were persistently infected with HIV-1. We discovered that effective cART restored the number of human immune cells in HIV-1-infected hu-mice but did not rescue their immune hyperactivation and dysfunction. IFNAR blockade fully reversed HIV-1-induced immune hyperactivation and rescued anti-HIV-1 immune responses in T cells from HIV-1-infected hu-mice. Finally, we found that IFNAR blockade in the presence of cART reduced the size of HIV-1 reservoirs in lymphoid tissues and delayed HIV-1 rebound after cART cessation in the HIV-1-infected hu-mice. We conclude that low levels of IFN-I signaling contribute to HIV-1-associated immune dysfunction and foster HIV-1 persistence in cART-treated hosts. Our results suggest that blocking IFNAR may provide a potential strategy to enhance immune recovery and reduce HIV-1 reservoirs in individuals with sustained elevations in IFN-I signaling during suppressive cART.

Plasmacytoid dendritic cells (pDCs) are the major source of type I interferons (IFN-I) in rapid response to viral infections, with constitutive expression of interferon regulatory factor 7 (IRF7). HIV-1 expresses several accessory proteins to counteract specific IFN-induced host restriction factors. As one abundant virion-associated protein, HIV-1 Vpr remains enigmatic in enhancing HIV-1 infection via unclear mechanisms. Here we report that Vpr impaired IFN-I induction in pDCs to enhance HIV-1 replication in CD4+ T cells. Blockade of IFN-I signaling abrogated the effect of Vpr on HIV-1 replication. Virion-associated Vpr suppressed IFN-I induction in pDC by TLR7 agonists. Modulation of IFN-I induction by Vpr was genetically dependent on its activity of TET2 degradation. We further demonstrate that Vpr-mediated TET2 degradation reduced expression of IRF7 in pDCs. Finally, degradation of TET2 in pDCs by Vpr reduced the demethylation level of the IRF7 promoter via CXXC5-dependent recruitment. We conclude that HIV-1 Vpr functions to promote HIV-1 replication by suppressing TET2-dependent IRF7 expression and IFN-I induction in pDCs. The Vpr-TET2-IRF7 axis provides a novel therapeutic target to control HIV-1 infection.

SummaryUnderstanding the negative regulators of antiviral immune responses will be critical for advancing immune-modulated antiviral strategies. NLRX1, an NLR protein that negatively regulates innate immunity, was previously identified in an unbiased siRNA screen as required for HIV infection. We find that NLRX1 depletion results in impaired nuclear import of HIV-1 DNA in human monocytic cells. Additionally, NLRX1 was observed to reduce type-I interferon (IFN-I) and cytokines in response to HIV-1 reverse-transcribed DNA. NLRX1 sequesters the DNA-sensing adaptor STING from interaction with TANK-binding kinase 1 (TBK1), which is a requisite for IFN-1 induction in response to DNA. NLRX1-deficient cells generate an amplified STING-dependent host response to cytosolic DNA, c-di-GMP, cGAMP, HIV-1, and DNA viruses. Accordingly, Nlrx1−/− mice infected with DNA viruses exhibit enhanced innate immunity and reduced viral load. Thus, NLRX1 is a negative regulator of the host innate immune response to HIV-1 and DNA viruses.

Background: HER2 overexpression and amplification define ~20% of breast cancers (BC) that are currently treated using HER2-specific monoclonal antibodies (mAb), such as trastuzumab. While multiple studies have demonstrated trastuzumab’s efficacy to be largely dependent upon triggering innate and adaptive immune responses, its dominant immune mechanism of therapeutic action (MOA) remains unclear. As trastuzumab’s efficacy is subverted in advanced immunosuppressive cancers, an understanding of its MOA and strategy to boost its immune effect is of critical clinical interest and scientific significance for immunologically enhancing other targeted mAb treatments. Methods: In this study, we developed a novel fully murine trastuzumab mAb (mTrastuzumab) to determine the MOA for HER2-mAb antitumor efficacy. We utilized these mAbs in multiple models that allowed us to interrogate antibody-dependent cell cytotoxicity and phagocytosis (ADCC and ADCP), as well as complement-dependent cytotoxicity (CDC) in vitro and in vivo. To determine the antitumor efficacy of these HER2 mAbs, we employed orthotopic HER2+ murine cells (transformed by HER2) in immunocompetent, FCGR KO, and immunocompetent settings, as well as utilized a unique transgenic HER2+ BC model driven by an oncogenic isoform of HER2 to best simulate an endogenous mammary tumor immune microenvironment (Turpin et al., 2016). Results: We found that mTrastuzumab significantly suppressed tumor growth and stimulated the infiltration of tumor-associated macrophages (TAMs), without affecting other immune cell types in the microenvironment. In contrast to other studies, we also found that this antitumor activity did not require adaptive immunity or NK cells, but did require activating Fc-gamma receptor engagement, implicating ADCP as the potential dominant MOA. Consistent with these findings, we further demonstrated that mTrastuzumab activated mFcγR4 and mFcγR3 signaling and elicited significant macrophage-mediated ADCP of HER2+ BC in vitro, but not ADCC or CDC. To test if enhanced ADCP could confer more effective antitumor immunity, we combined mTrastuzumab with blockade of the ADCP checkpoint, CD47 (“don’t eat me signal”), and found that this combination significantly increased mTrastuzumab-mediated ADCP in vitro without altering ADCC. Critically, we also found that this combination significantly enhanced TAM infiltration in vivo, as well as stimulated stronger antitumor efficacy and prolonged survival in highly immunosuppressive tumor microenvironments. Conclusion: Our study demonstrates the major MOA of clinically relevant HER2 mAbs requires the engagement with TAMs to elicit ADCP of tumor cells, which could be significantly enhanced by CD47 blockade. We conclude CD47 blockade could unleash the full potential of HER2 mAb therapy by abrogating the innate immunosuppressive signals on macrophages to stimulate immunity and enhance antitumor efficacy. Citation Format: Li-Chung Tsao, Herbert Kim Lyerly, Zachary Hartman. Trastuzumab therapy suppresses HER2+ breast tumor growth through inducing ADCP by tumor-associated macrophages and synergizes with CD47 checkpoint blockade [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B03.

While the use of immune checkpoint blockade (ICB) has gained significant traction as a viable therapy for cancers, many more cancers remain refractory to these immunotherapies due to their highly immunosuppressive tumor microenvironment (TME). This is perhaps best exemplified in colorectal carcinomas (CRCs) where ~90% of CRCs (non-MSI) are unresponsive to ICBs. Secondary analyses of ICB responders suggest that the presence of T cells and a functional interferon signal transduction pathway within cancer cells is likely required for PDL1 ICB efficacy, as mutations in this pathway confer resistance to PD1/PDL1 therapies. Our studies have indicated that this lack of response may be due to mutations and loss of genes that mediate the innate immune responses in tumor cells, such as MAVS (Mitochondrial Antiviral Signaling Protein), which we found were significantly reduced in colorectal cancer. Thus, we hypothesize that stimulation of the MAVS pathway would elicit strong interferon signaling capable of reversing immunosuppression to allow for successful immune-mediated tumor regression. To circumvent issues associated with single ligand stimulation, we exploited the bow-tie structure of the MAVS through its overexpression as a novel genetic means to elicit ligand free innate immune signaling in the TME. We found that overexpression of MAVS elicited robust stimulation of Type I and II interferon pathways, along with a unique interferon gene signature (through transcriptomic analysis of infected primary cells), in comparison to TLR and STING stimulated counterparts. Using MAVS inducible-expressing colorectal cell lines, we found that MAVS expression stimulated significant antitumor responses in vivo through its stimulation of adaptive tumor-specific immune responses in anti-PDL1-resistant models. Moreover, in 20% (4 out of 20) of MAVS-induced cancers, we observed complete tumor regression as well as an immunologic memory response (in 100% of regressed mice) through tumor rechallenge experiment. Utilizing MAVS expressing viral (adeno and AAV) vectors, we could demonstrate significant induction of interferon responses among different cells in the tumor microenvironment, including tumor cells, fibroblasts, as well as dendritic cells. Intralesional injection of tumors using these vectors elicited profound antitumor responses against primary tumors, as well as an abscopal effect against syngeneic implanted tumors. However, we found that MAVS expression significantly upregulated PDL1 in infected cells, suggesting that MAVS vectors may synergize with PDL1 ICB. In conclusion, our data demonstrate the importance of innate immunity activation by MAVS pathway in generating antitumor effects and immune “hot spots” in colorectal tumor microenvironment, which may synergize with PD1/PDL1 checkpoint blockade antibodies. Thus, our findings suggest that a MAVS cancer gene therapy may offer an alternative approach to activate immunity in “immunologically cold” or “T-cell excluded” tumors. This abstract is also being presented as Poster A45. Citation Format: Bin-Jin Hwang, Li-Chung Tsao, Gabriel DeLeon, Junping Wei, Xiao-Yi Yang, Gangjun Lei, Tao Wang, Michael M. Morse, Herbert Kim Lyerly, Zachary Conrad Hartman. Overexpression of MAVS stimulates antitumor immunity and significantly reduces tumor growth of immune-insensitive colorectal cancer in vivo [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR2.

HIV-1 expresses several accessory proteins to counteract host anti-viral restriction factors to facilitate viral replication and disease progression. One such protein, Vpr, has been implicated in affecting multiple cellular processes, but its mechanism remains elusive. Here we report that Vpr targets TET2 for polyubiquitylation by the VprBP-DDB1-CUL4-ROC1 E3 ligase and subsequent degradation. Genetic inactivation or Vpr-mediated degradation of TET2 enhances HIV-1 replication and substantially sustains expression of the pro-inflammatory cytokine interleukin-6 (IL-6). This process correlates with reduced recruitment of histone deacetylase 1 and 2 to the IL-6 promoter, thus enhancing its histone H3 acetylation level during resolution phase. Blocking IL-6 signaling reduced the ability of Vpr to enhance HIV-1 replication. We conclude that HIV-1 Vpr degrades TET2 to sustain IL-6 expression to enhance viral replication and disease progression. These results suggest that disrupting the Vpr-TET2-IL6 axis may prove clinically beneficial to reduce both viral replication and inflammation during HIV-1 infection.

Approximately 20% of breast cancers (BC) are defined by HER2 overexpression and treated using HER2-specific monoclonal antibodies (mAb), such as Trastuzumab. Previous studies have demonstrated Trastuzumab can limit signaling, elicit antibody dependent cell cytotoxicity (ADCC), antibody dependent phagocytosis (ADCP), and adaptive immune responses to HER2, however its primary therapeutic mechanism of action (MOA) remains unclear. As HER2 mAb efficacy is subverted in advanced cancers, understanding and boosting their MOA is of critical clinical interest and scientific significance for HER2+ BC as well as for other targeted mAbs. Using a series of fully murine Trastuzumab mAbs of different isotypes, we found that the IgG2A (high A/I) isotype was essential for tumor growth suppression, suggesting an innate immune MOA. In contrast to previous reports, our studies revealed that NK cells, neutrophils, T-cell, or B-cell populations were unnecessary for this anti-tumor MOA. However, we found that a murine IgG2A Trastuzumab (mTras) altered the immune microenvironment in vivo through neutrophil suppression (~4 fold, p4 fold, p Thus, our study demonstrates the primary MOA of a clinically relevant HER2 mAb requires engagement with TAMs to stimulate their expansion and elicit ADCP, which could be significantly enhanced by CD47-SIRPa blockade. This suggests that the strategic use of CD47-SIRPa innate blockade may allow for significant enhancement of anti-tumor immunity in combination with other cancer antigen targeting mAb therapies (targeting EGFR, CD20, etc) as a more effective strategy to stimulate anti-tumor immune responses in advanced immunosuppressive cancers. Citation Format: Li-Chung Tsao, Jun-Ping Wei, Gang-jun Lei, Tao Wang, Xiao-Yi Yang, Cong-Xiao Liu, H. Kim Lyerly, Zachary C. Hartman. Tumor macrophage-mediated antibody dependent cell phagocytosis (ADCP) is theprimary mechanism mediating HER2 mAb (Trastuzumab) anti-tumor responses which can be synergistically enhanced by CD47 innate immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3955.

Background CD4 T cell depletion during HIV-1 infection is associated with AIDS disease progression, and the HIV-1 Env protein plays an important role in the process. Together with CXCR4, CCR5 is one of the two co-receptors that interact with Env during virus entry, but the role of CCR5 in Env-induced pathogenesis is not clearly defined. We have investigated CD4 T cell depletion mechanisms caused by the Env of a highly pathogenic CXCR4/CCR5 dual-tropic HIV-1 isolate R3A. Results We report here that R3A infection induced depletion of both infected and uninfected “bystander” CD4 T cells, and treatment with CCR5 antagonist TAK-779 inhibited R3A-induced bystander CD4 T cell depletion without affecting virus replication. To further define the role of Env-CCR5 interaction, we utilized an Env-mutant of R3A, termed R3A-5/6AA, which has lost CCR5 binding capability. Importantly, R3A-5/6AA replicated to the same level as wild type R3A by using CXCR4 for viral infection. We found the loss of CCR5 interaction resulted in a significant reduction of bystander CD4 T cells death during R3A-5/6AA infection, whereas stimulation of CCR5 with MIP1-β increased bystander pathogenesis induced by R3A-5/6AA. We confirmed our findings using a humanized mouse model, where we observed similarly reduced pathogenicity of the mutant R3A-5/6AA in various lymphoid organs in vivo. Conclusion We provide the first evidence that shows CCR5 interaction with a dual-tropic HIV-1 Env played a significant role in Env-induced depletion of CD4 T cells. Electronic supplementary material The online version of this article (doi:10.1186/s12977-016-0255-z) contains supplementary material, which is available to authorized users.

While the use of immune checkpoint blockade (ICB) has gained significant traction as a viable therapy for certain cancers, many more cancers remain refractory to immunotherapies due to their highly immunosuppressive microenvironment. This is perhaps best exemplified in the largest group of cancers, colorectal carcinomas (CRCs) where ~90% of CRCs (non-microsatellite instable) are unresponsive to ICBs. Recent studies have indicated that this lack of response may be due to mutations and loss of genes that mediate the innate immune responses, such as MAVS (Mitochondrial Antiviral Signaling Protein) and STING (Stimulator of Interferon Genes). We hypothesized that delivery of active versions of these genes would elicit strong interferon signaling that could reverse the tumor immunosuppressive environment, to effectively create immune “hotspots” and allow for successful immune-mediated tumor regression. As a proof-of-concept, we developed doxycycline-inducible activated forms STING and MAVS in mouse CRC models (MC38 and CT26 lines), and found the expression of MAVS or STING resulted in robust activation of interferon-stimulated genes and other cytokines/chemokines. To determine if delivery of these genes could be achieved in vivo, we developed cell lines capable of resisting the anti-viral effect of interferon stimulation. This system allowed us to generate adenoviral vectors that express active forms of MAVS and more recently, STING. We found that Ad-MAVS strongly elicited interferon signaling in a variety of cell types and tumor lines, suggesting its effectiveness as an interferon stimulatory agent. To determine its anti-tumor effect, we intra-lesionally injected tumors and observed strongly inhibited tumor growth and enhanced T-cell infiltration in established tumor masses in vivo. This correlated with enhanced anti-tumor specific T cell responses, but not humoral responses. The induction of local and systemic immunity was confirmed in a bi-lateral treatment model that demonstrated the impact of systemic immunity by MAVS induction. Furthermore, we found that MAVS-induced tumor regression was lost in NOD/SCID mice, suggesting that the induction of tumor-specific T-cell adaptive immunity was critical to anti-tumor responses. Lastly, we found the activation of MAVS resulted in upregulated expression of PD-L1 on tumor cells, suggesting combinatorial treatments of checkpoint blockades therapy with targeted MAVS activation can result in synergistic effects. In conclusion, our data demonstrated the importance of innate immunity activation by MAVS or STING pathway in generating immune hotspots in colorectal tumor microenvironment, which promotes antitumor adaptive T cell responses and tumor growth regression. Citation Format: Li-Chung Tsao, Herbert Kim Lyerly, Zachary Hartman, Gabriel Deleon. Targeted activation of innate immune adaptors MAVS and STING promotes antitumor responses in colorectal cancer models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B66.

HIV-1 expresses several accessory proteins to counteract host anti-viral restriction factors to facilitate viral replication and disease progression. One such protein, Vpr, has been implicated in affecting multiple cellular processes, but its mechanism remains elusive. Here we report that Vpr targets TET2 for polyubiquitylation by the VprBP-DDB1-CUL4-ROC1 E3 ligase and subsequent degradation. Genetic inactivation or Vpr-mediated degradation of TET2 enhances HIV-1 replication and substantially sustains expression of the pro-inflammatory cytokine interleukin-6 (IL-6). This process correlates with reduced recruitment of histone deacetylase 1 and 2 to the IL-6 promoter, thus enhancing its histone H3 acetylation level during resolution phase. Blocking IL-6 signaling reduced the ability of Vpr to enhance HIV-1 replication. We conclude that HIV-1 Vpr degrades TET2 to sustain IL-6 expression to enhance viral replication and disease progression. These results suggest that disrupting the Vpr-TET2-IL6 axis may prove clinically beneficial to reduce both viral replication and inflammation during HIV-1 infection. HIV-1 Vpr protein counteracts host anti-viral restriction factors to facilitate viral replication and disease progression. Lv et al. demonstrate that Vpr promotes degradation of TET DNA dioxygenases. TET2 deletion or depletion enhances HIV-1 replication and sustains pro-inflammatory cytokine IL-6, while blocking IL-6 reduces Vpr's ability to enhance HIV-1 replication. © 2018 Elsevier Inc.

Microbial infections are the most common cause of death in humans. Type I interferon (IFN-I) is a key element bridging the host innate and adaptive immune response against infections. Delineating the molecular regulation network of IFN-I signaling is critical for developing novel antiviral strategy and benefiting rational therapy. Using an unbiased siRNA screen, we find NLRX1, one nucleotide-binding leucine-rich-repeat-containing protein, is a host factor that promotes an early step of HIV-1 infection. NLRX1 suppresses type-I interferon (IFN-I) and cytokines in response to HIV-1 reverse-transcribed DNA and enhances the nuclear import of HIV-1 DNA. In addition to HIV, NLRX1 also reduces STING-dependent host response to cytosolic DNA, c-di-GMP, cGAMP, and DNA virus. Mechanistically, NLRX1 associates with STING in mitochondria-associated ER membranes, and prevents STING recruiting TBK1 and activating downstream interferon signaling. By using purified recombinant proteins, we found NLRX1 interacts directly with STING. Furthermore, DNA virus infected Nlrx1−/− mice exhibited enhanced innate immunity and reduced morbidity and viral load. In summary, these findings reveal that NLRX1 is a checkpoint protein for DNA sensing adaptor STING and may represent a novel precision target for anti-viral therapy.

Regulatory T (Treg) cells play an important role in modulating immune activation and human immunodeficiency virus type 1 (HIV-1) infection. However, the role of Treg cells in HIV-1 latency establishment or maintenance in vivo remains unknown because of the lack of a robust model. We have established a humanized mouse model that supports long-term stable HIV-1 replication. When treated with combination antiretroviral therapy (cART), HIV-1 infection was stably suppressed to undetectable levels. Upon cART interruption, HIV-1 replication rebounded rapidly to pre-treatment levels. Interestingly, HIV-1 viral replication was significantly increased upon Treg cell depletion under effective cART. The HIV-1 reactivation during cART was mainly through increased gene expression from T cells. The rebound viruses during cART did not acquire drug resistance mutations. We found that most activated HIV-1 reservoir target cells were CD4 memory T cells in all lymphoid organs. These findings demonstrate that Treg cells suppress HIV-1 replication and its reactivation from latently infected T cells in vivo. Treg cell depletion provides an efficient way to reverse latency and reactivate HIV-1 reservoirs in vivo.

Wall-associated kinases comprise a sub-family of receptor-like kinases that function in plant growth and stress responses. Previous studies have shown that the rice wall-associated kinase, OsWAK25, interacts with a diverse set of proteins associated with both biotic and abiotic stress responses. Here, we show that wounding and BTH treatments induce OsWAK25 transcript expression in rice. We generated OsWAK25 overexpression lines and show that these lines exhibit a lesion mimic phenotype and enhanced expression of rice NH1 (NPR1 homolog 1), OsPAL2, PBZ1 and PR10. Furthermore, these lines show resistance to the hemibiotrophic pathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe oryzae, yet display increased susceptibility to necrotrophic fungal pathogens, Rhizoctonia solani and Cochliobolus miyabeanus.