Antitumor Immunity In Mice Research Articles

A bispecific CD40 agonistic antibody allowing for antibody-peptide conjugate formation to enable cancer-specific peptide delivery, resulting in improved T proliferation and anti-tumor immunity in mice

Current antibody-based immunotherapy depends on tumor antigen shedding for proper T cell priming. Here we select a novel human CD40 agonistic drug candidate and generate a bispecific antibody, herein named BiA9*2_HF, that allows for rapid antibody-peptide conjugate formation. The format is designed to facilitate peptide antigen delivery to CD40 expressing cells combined with simultaneous CD40 agonistic activity. In vivo, the selected bispecific antibody BiA9*2_HF loaded with peptide cargos induces improved antigen-specific proliferation of CD8+ (10-15 fold) and CD4+ T cells (2-7 fold) over control in draining lymph nodes. In both virus-induced and neoantigen-based mouse tumor models, BiA9*2_HF demonstrates therapeutic efficacy and elevated safety profile, with complete tumor clearance, as well as measured abscopal impact on tumor growth. The BiA9*2_HF drug candidate can thus be utilized to tailor immunotherapeutics for cancer patients.

TMED4 facilitates Treg suppressive function via ROS homeostasis in tumor and autoimmune mouse models.

Endoplasmic reticulum stress (ERS) plays crucial roles in maintaining regulatory T cells (Treg) stability and function, yet the underlying mechanism remains largely unexplored. Here we demonstrate that ERS-related protein transmembrane p24 trafficking protein 4 (TMED4) Treg-specific knockout (Tmed4ΔTreg) mice contain more Treg cells with impaired Foxp3 stability, Treg signature and suppressive activity, which leads to T cell hyperactivation, exacerbated inflammatory phenotype and boosted anti-tumor immunity in mice. Mechanistically, loss of Tmed4 causes defects in ERS and nuclear factor erythroid 2-related factor 2 (NRF2)-related antioxidant response, which results in excessive reactive oxygen species (ROS) that reduces Foxp3 stability and suppressive function of Treg cells in an IRE1α-XBP1 axis-dependent manner. The abnormalities can be effectively rescued by ROS scavenger, NRF2 inducer or forcible expression of IRE1α. Moreover, TMED4 suppresses IRE1α proteosome degradation via the ER-associated degradation (ERAD) system including BIP. Our study reveals that TMED4 maintains Treg cell stability and suppressive function through IRE1α-dependent ROS and the NRF2-related antioxidant response.

CD94 deficiency or blockade unleashes the anti-tumor immunity in mice and humanized murine models

NKG2 family members have emerged as promising targets in tumor immunotherapy. CD94 can dimerize with both inhibitory and activating NKG2 proteins, while the overall effect and value of targeting CD94 on anti-tumor immunity are unclear. Here, it is shown that the expression of CD94 is upregulated on tumor-infiltrating natural killer (NK) cells and CD8+ T cells, and is related to their exhausted characteristics. Tumor-bearing CD94 knockout (CD94-KO) mice exhibit delayed tumor growth, decreased lung metastases, and prolonged survival. Single cell RNA-seq reveal a remodeled tumor microenvironment in CD94-KO mice, with a reduction in immunosuppressive cells and an increase in anti-tumor immune cells. Moreover, NK cells and CD8+ T cells become proliferative and strongly tumoricidal in CD94-KO mice, thus contributing to the tumor inhibition effect of CD94 deficiency. Treatment with a humanized anti-CD94 blocking antibody (h15C10) alone, in tumor-bearing humanized mouse, delays tumor progression, and improves the therapeutic efficacy of PD-L1 blockade through combination therapy. Our study indicates that CD94 may work as a candidate target in checkpoint immunotherapy.

Activated STING-containing R-EVs from iPSC-derived MSCs promote antitumor immunity

We recently revealed that activated STING is secreted into RAB22A-induced extracellular vesicles (R-EVs) and promotes antitumor immunity in cancer cells. Whether mesenchymal stem cell (MSC)-derived R-EVs containing activated STING can be used as a novel antitumor immunotherapy remains unclear, as MSC-derived EVs are promising cell-free therapeutics due to their superior biocompatibility and safety, as well as low immunogenicity. Here, we report that induced pluripotent stem cell (iPSC)-derived MSCs can generate R-EVs with a size and mechanism of formation that are similar to those of R-EVs produced from cancer cells. Furthermore, these MSC-derived R-EVs containing activated STING induced IFNβ expression in recipient THP-1 monocytes and antitumor immunity in mice. Our findings reveal that the use of MSC-derived R-EVs containing activated STING is a promising cell-free strategy for antitumor immunity.

Abstract 5263: The impact of MRTX1133 on anti-tumor immunity in lung cancer with KRASG12D mutation

Abstract Background: To date, there have been no clinical molecules that are selective for KRASG12D mutant tumors. Although immune checkpoint inhibitors (ICIs) have improved the survival for patients with lung cancer, inferior efficacy of ICIs has been reported in KRASG12D mutant NSCLC. MRTX1133 is a selective KRASG12D inhibitor, which has been investigated in early clinical studies. In this study, we aimed to reveal the immune status using preclinical murine model of KRASG12D mutated lung cancer. In addition, we investigated whether MRTX1133 effects the anti-tumor immunity in mice bearing lung cancer with KRASG12D. Method: CCSP-rtTA/TetO-Cre mice were crossed with LSL-KRASG12D knock-in mice to obtain CCSP-rtTA/TetO-Cre/LSL-KRASG12D conditional mutant mice (KRASG12D mice). These mice were fed an autoclaved rodent diet supplemented with 0.0625% doxycycline when the mice were about 8~12 weeks old. Mice were treated with 10 mg/kg MRTX1133 in vehicle once daily by intraperitoneal injection for 4 or 8 days. After administration of MRTX1133, the lungs and spleens were harvested, and single cell suspensions were labeled with fluorophore-conjugated antibodies for FACS analyses. Results: More regulatory T cells (Tregs) infiltrated the tumors of lung of mice treated with doxycycline for a longer period of time. The expression of immune checkpoint molecules (ICMs), including PD-L1, PD-1, TIGIT, TIM-3, LAG-3, were also upregulated on CD4+ and CD8+ T cells in the KRASG12D mice. Treatment with MRTX1133 strongly inhibited KRASG12D tumor progression, reduced the percentage of Tregs and downregulated the expression of PD-1 on CD4+ and CD8+ T cells infiltrating in lung cancers. The expression of the other ICMs remained high after MRTX1133 administration. In addition, MRTX1133 tended to increase the percentage of tumor-specific CD8+ effector T cells infiltration in lung tumors. Conclusion: T cells were exhausted in mice with KRASG12D mutant lung cancer. MRTX1133 demonstrates the therapeutic efficacy in preclinical murine KRASG12D mutated lung cancer model. This efficacy is more likely to relate not only signal inhibition but also enhances the anti- tumor immune responses. Our findings could aid in developing an alternative immunotherapy strategy in patients with KRASG12D mutant lung cancer. Citation Format: Kunihiro Shono, Satoshi Watanabe, Takaaki Masuda, Ryo Suzuki, Tomoya Wakabayashi, Tomoki Sekiya, Susumu Tanaka, Aya Ohtsubo, Tomohiro Tanaka, Koichiro Nozaki, Rie Kondo, Yu Saida, Satoshi Hokari, Naohiro Yanagimura, Masashi Arita, Riuko Ohashi, Kenjiro Shima, Yosuke Kimura, Nobumasa Aoki, Yasuyoshi Ohshima, Toshiyuki Koya, Toshiaki Kikuchi. The impact of MRTX1133 on anti-tumor immunity in lung cancer with KRASG12D mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5263.

CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1-positive regulator for tumor immune evasion.

Regulation of tumoral PD-L1 expression is critical to advancing our understanding of tumor immune evasion and the improvement of existing antitumor immunotherapies. Herein, we describe a CRISPR-based screening platform and identified ATXN3 as a positive regulator for PD-L1 transcription. TCGA database analysis revealed a positive correlation between ATXN3 and CD274 in more than 80% of human cancers. ATXN3-induced Pd-l1 transcription was promoted by tumor microenvironmental factors, including the inflammatory cytokine IFN-γ and hypoxia, through protection of their downstream transcription factors IRF1, STAT3, and HIF-2α. Moreover, ATXN3 functioned as a deubiquitinase of the AP-1 transcription factor JunB, indicating that ATNX3 promotes PD-L1 expression through multiple pathways. Targeted deletion of ATXN3 in cancer cells largely abolished IFN-γ- and hypoxia-induced PD-L1 expression and consequently enhanced antitumor immunity in mice, and these effects were partially reversed by PD-L1 reconstitution. Furthermore, tumoral ATXN3 suppression improved the preclinical efficacy of checkpoint blockade antitumor immunotherapy. Importantly, ATXN3 expression was increased in human lung adenocarcinoma and melanoma, and its levels were positively correlated with PD-L1 as well as its transcription factors IRF1 and HIF-2α. Collectively, our study identifies what we believe to be a previously unknown deubiquitinase, ATXN3, as a positive regulator for PD-L1 transcription and provides a rationale for targeting ATXN3 to sensitize checkpoint blockade antitumor immunotherapy.

Comparative evaluation of antitumor effects of TNF superfamily costimulatory ligands delivered by mesenchymal stem cells

Stimulation of costimulatory receptors serves as an alternative immunotherapeutic strategy other than checkpoint inhibition. However, systemic administration of the agonistic antibodies is associated with severe toxicities, which is one of the major obstacles for their clinical application. This study aimed to develop a mesenchymal stem cell (MSC)-based system for tumor-targeted delivery of TNF superfamily ligands and assess their potential in enhancing antitumor immunity. Here we established an MSC-based system for tumor-targeted delivery of TNF superfamily ligands, including TNFSF4, 9 and 18. The TNFSF receptors (TNFRSFs) were evaluated in mouse models and patient samples for lung and colorectal cancers. TNFRSFs were all expressed at various levels on tumor-infiltrated lymphocytes, with TNFRSF18 being the most prevalent receptor. Human umbilical cord-derived MSCs expressing these costimulatory ligands (MSC-TNFSFs) effectively activated lymphocytes in vitro and elicited antitumor immunity in mice. TNFSF4 showed the least antitumor efficacy in both LLC1 and CT26 tumor models. MSC-TNFSF9 showed the most potent tumor-inhibiting effect in the LLC1 tumor model, while MSCs expressing TNFSF18 in combination with CXCL9 most significantly repressed CT26 tumor growth. Overall, TNFSF9 and TNFSF18 exhibited stronger lymphocyte-stimulating and antitumor activities than TNFSF4. Our study provides evidence that antitumor effects of agonism of different costimulatory receptors may vary in different tumor types and presents a promising approach for targeted delivery of TNF superfamily costimulatory ligands to avoid the systemic toxicities and side effects associated with immune agonist antibodies.

Sini Decoction Inhibits Tumor Progression and Enhances the Anti-Tumor Immune Response in a Murine Model of Colon Cancer.

Sini decoction (SND) is a widely used Traditional Chinese Medicine (TCM). The reports of SND application in colorectal cancer (CRC) is limited. The objective of this study is to investigate the anti-tumor activity of SND in the treatmeant of CRC. SND was analyzed using high-performance liquid chromatography. A CRC metastasis model was established using murine CT-26 cells. Whole-body fluorescence imaging was used to observe CRC liver metastasis. Liver morphology was determined using hematoxylin-eosin staining. Cytokine mRNA expression (interleukin-2 (IL-2), interleukin-10 (IL-10), interferon-gamma (IFN-γ), and tumor necrosis factor beta (TNF-β)) were determined using real-time reverse transcription polymerase chain reaction. Spectral flow cytometry was used to detect mouse tumor immune subgroups. Databases were used to find potential target genes of SND. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to identify potential signaling pathways of target genes. SND suppressed CRC liver metastasis and alleviated liver injury in vivo. After SND treatment, IL-2 and IFN-γ were upregulated, whereas IL-10 and TGF-β were downregulated. Moreover, CD3+, CD8+T cells, natural killer T cells, and macrophages increased significantly after SND treatment, while CD4+CD25+T cells decreased significantly. Importantly, increasing the aconite concentration had a better anti-tumor effect. Fifty-50 compounds in SND were screened, and 611 potential target genes were identified. Functional analyses showed that the genes were associated with the PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, and HIF-1 signaling pathway. SND exerts anti-tumor activity by inhibiting tumor progression and enhancing antitumor immunity in mice, suggesting its application to prevent and treat CRC.

MRNA-based Vaccines Targeting the T-cell Epitope-rich Domain of Epstein Barr Virus Latent Proteins Elicit Robust Anti-Tumor Immunity in Mice.

Epstein-Barr virus (EBV) is associated with various malignancies and infects >90% of the global population. EBV latent proteins are expressed in numerous EBV-associated cancers and contribute to carcinogenesis, making them critical therapeutic targets for these cancers. Thus, this study aims to develop mRNA-based therapeutic vaccines that express the T-cell-epitope-rich domain of truncated latent proteins of EBV, including truncatedlatent membrane protein 2A (Trunc-LMP2A), truncated EBV nuclear antigen 1 (Trunc-EBNA1), and Trunc-EBNA3A. The vaccines effectively activate both cellular and humoral immunity in mice and show promising results in suppressing tumor progression and improving survival time in tumor-bearing mice. Furthermore, it is observed that the truncated forms of the antigens, Trunc-LMP2A, Trunc-EBNA1, and Trunc-EBNA3A, are more effective than full-length antigens in activating antigen-specific immune responses. In summary, the findings demonstrate the effectiveness of mRNA-based therapeutic vaccines targeting the T-cell-epitope-rich domain of EBV latent proteins and providing new treatment options for EBV-associated cancers.

SUN1/2 controls macrophage polarization via modulating nuclear size and stiffness

Alteration of the size and stiffness of the nucleus triggered by environmental cues are thought to be important for eukaryotic cell fate and function. However, it remains unclear how context-dependent nuclear remodeling occurs and reprograms gene expression. Here we identify the nuclear envelope proteins SUN1/2 as mechano-regulators of the nucleus during M1 polarization of the macrophage. Specifically, we show that LPS treatment decreases the protein levels of SUN1/2 in a CK2-βTrCP-dependent manner to shrink and soften the nucleus, therefore altering the chromatin accessibility for M1-associated gene expression. Notably, the transmembrane helix of SUN1/2 is solely required and sufficient for the nuclear mechano-remodeling. Consistently, SUN1/2 depletion in macrophages facilitates their phagocytosis, tissue infiltration, and proinflammatory cytokine production, thereby boosting the antitumor immunity in mice. Thus, our study demonstrates that, in response to inflammatory cues, SUN1/2 proteins act as mechano-regulators to remodel the nucleus and chromatin for M1 polarization of the macrophage.

Combination therapy of HIFα inhibitors and Treg depletion strengthen the anti-tumor immunity in mice.

Hypoxia-inducible factor 1 alpha (HIF1α), under hypoxic conditions, is known to play an oxygen sensor stabilizing role by exerting context- and cell-dependent stimulatory and inhibitory functions in immune cells. Nevertheless, how HIF1α regulates T cell differentiation and functions in tumor settings has not been elucidated. Herein, we demonstrated that T-cell-specific deletion of HIF1α improves the inflammatory potential and memory phenotype of CD8+ T cells. We validated that T cell-specific HIF1α ablation reduced the B16 melanomas development with the indication of ameliorated antitumor immune response with enhanced IFN-γ+ CD8+ T cells despite the increase in the Foxp3+ regulatory T-cell population. This was further verified by treating tumor-bearing mice with a HIF1α inhibitor. Results indicated that HIF1α inhibitor also recapitulates HIF1α ablation effects by declining tumor growth and enhancing the memory and inflammatory potential of CD8+ T cells. Furthermore, a combination of Treg inhibitor with HIF1α inhibitor can substantially reduce tumor size. Collectively, these findings highlight the notable roles of HIF1α in distinct CD8+ T-cell subsets. This study suggests the significant implications for enhancing the potential of T cell-based antitumor immunity by combining HIF1α and Tregs inhibitors.

USP2 promotes tumor immune evasion via deubiquitination and stabilization of PD-L1.

The abnormal upregulation of programmed death ligand-1 (PD-L1) on tumor cells impedes T-cell mediated cytotoxicity through PD-1 engagement, and further exploring the mechanisms regulation of PD-L1 in cancers may enhance the clinical efficacy of PD-L1 blockade. Here, using single-guide RNAs (sgRNAs) screening system, we identify ubiquitin-specific processing protease 2 (USP2) as a novel regulator of PD-L1 stabilization for tumor immune evasion. USP2 directly interacts with and increases PD-L1 abundance in colorectal and prostate cancer cells. Our results show that Thr288, Arg292 and Asp293 at USP2 control its binding to PD-L1 through deconjugating the K48-linked polyubiquitination at lysine 270 of PD-L1. Depletion of USP2 causes endoplasmic reticulum (ER)-associated degradation of PD-L1, thus attenuates PD-L1/PD-1 interaction and sensitizes cancer cells to T cell-mediated killing. Meanwhile, USP2 ablation-induced PD-L1 clearance enhances antitumor immunity in mice via increasing CD8+ T cells infiltration and reducing immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), whereas PD-L1 overexpression reverses the tumor growth suppression by USP2 silencing. USP2-depletion combination with anti-PD-1 also exhibits a synergistic anti-tumor effect. Furthermore, analysis of clinical tissue samples indicates that USP2 is positively associated with PD-L1 expression in cancer. Collectively, our data reveal a crucial roleof USP2 for controlling PD-L1 stabilization in tumor cells, and highlight USP2 as a potential therapeutic target for cancer immunotherapy.

Targeting macrophage Syk enhances responses to immune checkpoint blockade and radiotherapy in high-risk neuroblastoma.

Neuroblastoma (NB) is considered an immunologically cold tumor and is usually less responsive to immune checkpoint blockade (ICB). Tumor-associated macrophages (TAMs) are highly infiltrated in NB tumors and promote immune escape and resistance to ICB. Hence therapeutic strategies targeting immunosuppressive TAMs can improve responses to ICB in NB. We recently discovered that spleen tyrosine kinase (Syk) reprograms TAMs toward an immunostimulatory phenotype and enhances T-cell responses in the lung adenocarcinoma model. Here we investigated if Syk is an immune-oncology target in NB and tested whether a novel immunotherapeutic approach utilizing Syk inhibitor together with radiation and ICB could provide a durable anti-tumor immune response in an MYCN amplified murine model of NB. Myeloid Syk KO mice and syngeneic MYCN-amplified cell lines were used to elucidate the effect of myeloid Syk on the NB tumor microenvironment (TME). In addition, the effect of Syk inhibitor, R788, on anti-tumor immunity alone or in combination with anti-PDL1 mAb and radiation was also determined in murine NB models. The underlying mechanism of action of this novel therapeutic combination was also investigated. Herein, we report that Syk is a marker of NB-associated macrophages and plays a crucial role in promoting immunosuppression in the NB TME. We found that the blockade of Syk in NB-bearing mice markedly impairs tumor growth. This effect is facilitated by macrophages that become immunogenic in the absence of Syk, skewing the suppressive TME towards immunostimulation and activating anti-tumor immune responses. Moreover, combining FDA-approved Syk inhibitor, R788 (fostamatinib) along with anti-PDL1 mAb provides a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in mice bearing small tumors (50 mm3) but not larger tumors (250 mm3). However, combining radiation to R788 and anti-PDL1 mAb prolongs the survival of mice bearing large NB9464 tumors. Collectively, our findings demonstrate the central role of macrophage Syk in NB progression and demonstrate that Syk blockade can "reeducate" TAMs towards immunostimulatory phenotype, leading to enhanced T cell responses. These findings further support the clinical evaluation of fostamatinib alone or with radiation and ICB, as a novel therapeutic intervention in neuroblastoma.

Abstract 1098: Combination of a DNA vaccine-induced immune checkpoint blockade and radiation therapy induces anti-tumor immunity

Abstract Background: Immune checkpoint blockade (ICB) immunotherapy improves survival for many cancer patients, but those with immunologically ‘cold’ tumors do not derive benefit. Radiation therapy (RT) has been demonstrated to augment antitumor immunity in preclinical and clinical studies and may enhance response to ICB in immunologically cold tumors. We have developed DNA-based, cost-effective, and versatile ICB immunotherapies. Here, we test the use of these alone and in combination with RT as a novel treatment approach in syngeneic murine melanoma models. Materials and methods: We constructed ICB DNA vaccines targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed cell death 1 (PD-1), CTLA-4 Vax and PD-1 Vax, respectively. These were delivered to mice by intramuscular or intratumoral electroporation. The effects of combinations of ICB DNA vaccines and RT were monitored for generation of endogenous anti-CTLA-4 and anti-PD-1 antibodies, modification of tumor microenvironment (TME), and tumor response in mice bearing syngeneic B78 or B16 melanoma. Results: Intramuscular vaccination with CTLA-4 Vax and PD-1 Vax induced endogenous anti-CTLA-4 and anti-PD-1 antibodies, respectively, and the titers increased with additional vaccinations. Vaccination with CTLA-4 Vax and PD-1 Vax alone or in combination (Dual Vax) did not inhibit the murine B16 melanoma model; however, the combination of CTLA-4 Vax and RT (CTLA-4 Vax + RT) led to enhanced infiltration of the TME with effector T cells. Dual Vax combined with RT enabled systemic anti-tumor immunity in mice bearing two B16 melanoma tumors in which only one was radiated. With intra-tumoral route of vaccination, the Dual Vax + RT treatment induced a trend toward systemic anti-tumor response in the immunologically cold B78 tumor model. Conclusions: DNA vaccines that stimulate endogenous production of antibodies against immune checkpoints may serve as an alternative form of ICB. The combination of ICB DNA vaccine and RT enabled anti-tumor immunity and tumor response in preclinical models of melanoma. Citation Format: Keng-Hsueh Lan, Ying-Chun Sheng, Keng-Li Lan, KyungMann Kim, Sung-Hsin Kuo, Zachary Morris. Combination of a DNA vaccine-induced immune checkpoint blockade and radiation therapy induces anti-tumor immunity [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 1098.

Tumor Suppressor Adenomatous Polyposis Coli Sustains Dendritic Cell Tolerance through IL-10 in a β-Catenin-Dependent Manner.

Dendritic cells (DC) play important roles in balancing immunity and tolerance, in which β-catenin signaling plays an important role, yet the underlying mechanisms remain elusive. In this study, we investigated the functions of the tumor suppressor adenomatous polyposis coli (APC), also a key component of the β-catenin upstream destruction complex in DC. APC depletion in DC does not alter DC and T cell homeostasis under resting conditions. However, APC deficiency in DC leads to attenuated antitumor immunity in mice, which exhibit fewer CD8+ T cells and more Foxp3+ regulatory T cells in tumor and draining lymph nodes. Loss of APC in DC does not affect the expression levels of costimulatory molecules. However, APC-deficient DC produce more IL-10 and exhibit a higher ability of inducing regulatory T cells but a lower ability of priming CD8+ T cells, both of which can be reversed by IL-10 inhibition. Lastly, β-catenin depletion in APC-deficient DC rescues their antitumor immunity and reverses elevated IL-10 production. Taken together, our results identify that APC drives DC tolerance via the β-catenin/IL-10 axis.

PD-L1 translocation to the plasma membrane enables tumor immune evasion through MIB2 ubiquitination.

Programmed death-ligand 1 (PD-L1), a critical immune checkpoint ligand, is a transmembrane protein synthesized in the endoplasmic reticulum of tumor cells and transported to the plasma membrane to interact with programmed death 1 (PD-1) expressed on T cell surface. This interaction delivers coinhibitory signals to T cells, thereby suppressing their function and allowing evasion of antitumor immunity. Most companion or complementary diagnostic devices for assessing PD-L1 expression levels in tumor cells used in the clinic or in clinical trials require membranous staining. However, the mechanism driving PD-L1 translocation to the plasma membrane after de novo synthesis is poorly understood. Herein, we showed that mind bomb homolog 2 (MIB2) is required for PD-L1 transportation from the trans-Golgi network (TGN) to the plasma membrane of cancer cells. MIB2 deficiency led to fewer PD-L1 proteins on the tumor cell surface and promoted antitumor immunity in mice. Mechanistically, MIB2 catalyzed nonproteolytic K63-linked ubiquitination of PD-L1, facilitating PD-L1 trafficking through Ras-associated binding 8-mediated (RAB8-mediated) exocytosis from the TGN to the plasma membrane, where it bound PD-1 extrinsically to prevent tumor cell killing by T cells. Our findings demonstrate that nonproteolytic ubiquitination of PD-L1 by MIB2 is required for its transportation to the plasma membrane and tumor cell immune evasion.

МЕХАНИЗМЫ ПРОТИВООПУХОЛЕВОГО ДЕЙСТВИЯ МАЛЫХ ДОЗ РАДИАЦИИ, СВЯЗАННЫЕ С АКТИВАЦИЕЙ ЗАЩИТНЫХ СИСТЕМ КЛЕТКИ

Background: The effects of ionizing radiation (IR) involve a highly orchestrated series of events in cells, including DNA damage and repair, cell death, and changes in the level of proliferation associated with the stage of the cell cycle. A large number of existing studies in literature have examined the activity of genes and their regulators in mammalian cells in response to high doses of ionizing radiation. Although there are many studies, the research in effect of low doses of ionizing radiation remains limited. Though much progress has been made in understanding the basic principles of effects of low doses radiation on individual components of biological systems, less is known about how low doses affect target molecules and regulate the cellular networks (e.g., activation of the immune system, genes and their regulators in the phenomenon of hormesis, the formation of an adaptive response). These observations determined the purpose of the work: to investigate the activity of genes and non-coding RNAs (long non-coding RNAs and microRNAs) in various organs of mice with transplanted Lewis carcinoma after low doses radiation. Material and methods: 24 female mice C57Bl/6 were transplanted subcutaneously with Lewis carcinoma cells (105 cells in 0.2 ml of Hanks’ solution). Total 4-fold X-ray irradiation with an interval of 4 days at a dose of 0.075 Gy (0.85 Gy/min) was performed on the RUST M1 from 6 days after transplantation; the tumor size was measured daily. The mice were divided into the following groups: biocontrol, biocontrol+irradiation, tumor and tumor+irradiation. On the 19th day from the beginning of the experiment, the mice were euthanized. The expression profiles of mRNA genes, long non-coding RNAs and microRNAs controlling the response to radiation were determined in the bone marrow, thymus, spleen and tumor of mice. Results: Fractionated low doses irradiation of mice with transplanted Lewis carcinoma caused a growth decrease of implanted tumour cells compared to the similar group without irradiation. At the same time, there was an activation of oncosuppressors, and a decrease in the activity of oncogenes in the thymus and spleen of mice with tumor and irradiation. In the tumor group, without irradiation, the number of activated oncogenes prevailed over the number of inactivated ones. Conclusion: Thus, the low doses radiation exposure led to the activation of antitumor immunity in mice, which emerged in slowing tumor growth in animals and was represented in the induction of oncosuppressors and inhibition of oncogenes expression.

Abstract PR021: CCR1 expression defines pancreatic tumor associated macrophages and drives their immunosuppressive properties

Abstract The tumor microenvironment of pancreatic ductal adenocarcinoma (PDA) includes abundant fibroblasts and infiltrating immune cells, the latter largely immunosuppressive. Immunotherapy approaches have been ineffective in PDA, pointing to the need for a better understanding of the mechanisms of immunosuppression. We previously identified C-C Motif Chemokine Receptor 1 (CCR1) as overexpressed in macrophages exposed to pancreatic cancer cell conditioned medium in vitro. By single-cell RNA sequencing, we found CCR1 to be expressed by tumor associated macrophages (TAMs) and granulocytes in both human and mouse PDA. Conversely, the ligands for CCR1, C-C Motif Chemokine Ligands (CCLs), are produced by tumor associated fibroblasts. Thus, we sought to investigate the functional role of CCR1 in pancreatic cancer using a combination of genetically engineered mouse models and pharmaceutically approaches. In a first set of experiments, we generated KC;CCR1-/- mice (Ptf1a-Cre;LSL-KrasG12D;CCR1-/-) to determine the requirement of CCR1 during oncogenic KRAS induced pancreatic cancer initiation. We did not observe a difference in PanIN formation/progression in KC;CCR1-/- compared to KC mice. However, we observed increased immune infiltration, including CD8 T cells, in KC;CCR1-/- pancreata. In a second set of experiments, we then orthotopically transplanted two independent mouse pancreatic cancer cells derived from the KPC model in syngeneic CCR1 knockout hosts. We observed reduced tumor growth, which was rescued by CD8 T cell depletion, indicating an increase of anti-tumor immunity in mice lacking CCR1. Consistently, we observed elevated cytotoxic Granzyme B expression, as well as an increase of apoptotic cells in tumors harvested from Ccr1-/- mice. Through mass cytometry (CyTOF) and co-immunofluorescence staining we discovered that tumor associated macrophages from CCR1-/- mice expressed less Arginase 1 and CD206, both immunosuppressive markers, compared to macrophages in wild type tumors. In the last set of experiments, we used the CCR1 inhibitor J-113863, administered to mice following establishment of an orthotopically implanted tumor. Similar to the genetic model, CCR1 inhibition resulted in smaller tumors. Further, targeting CCR1 either genetically or using a CCR1 inhibitor synergizes with anti-PDL1 immune checkpoint blockade to reduce tumor growth. Together, our data is consistent with the notion that tumor associated macrophages lacking CCR1 expression are less immunosuppressive, consequently allowing increased CD8 T cell-mediated anti-tumor immunity. Targeting CCR1 in combination with immune checkpoint blockade improves antitumor efficacy in pancreatic cancer. Citation Format: Yaqing Zhang, Wei Yan, Kristee L. Brown, Ashely Velez-Delgado, Zeribe C. Nwosu, Katelyn Donahue, Padma Kadiyala, Sion Yang, Faith R. Avritt, Xi He, Carlos Espinoza, Marina Pasca di Magliano. CCR1 expression defines pancreatic tumor associated macrophages and drives their immunosuppressive properties [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR021.

Spermidine activates mitochondrial trifunctional protein and improves antitumor immunity in mice.

Spermidine (SPD) delays age-related pathologies in various organisms. SPD supplementation overcame the impaired immunotherapy against tumors in aged mice by increasing mitochondrial function and activating CD8+ T cells. Treatment of naïve CD8+ T cells with SPD acutely enhanced fatty acid oxidation. SPD conjugated to beads bound to the mitochondrial trifunctional protein (MTP). In the MTP complex, synthesized and purified from Escherichia coli, SPD bound to the α and β subunits of MTP with strong affinity and allosterically enhanced their enzymatic activities. T cell-specific deletion of the MTP α subunit abolished enhancement of programmed cell death protein 1 (PD-1) blockade immunotherapy by SPD, indicating that MTP is required for SPD-dependent T cell activation.

Alum-anchored intratumoral retention improves the tolerability and antitumor efficacy of type I interferon therapies

Effective antitumor immunity in mice requires activation of the type I interferon (IFN) response pathway. IFNα and IFNβ therapies have proven promising in humans, but suffer from limited efficacy and high toxicity. Intratumoral IFN retention ameliorates systemic toxicity, but given the complexity of IFN signaling, it was unclear whether long-term intratumoral retention of type I IFNs would promote or inhibit antitumor responses. To this end, we compared the efficacy of IFNα and IFNβ that exhibit either brief or sustained retention after intratumoral injection in syngeneic mouse tumor models. Significant enhancement in tumor retention, mediated by anchoring these IFNs to coinjected aluminum-hydroxide (alum) particles, greatly improved both their tolerability and efficacy. The improved efficacy of alum-anchored IFNs could be attributed to sustained pleiotropic effects on tumor cells, immune cells, and nonhematopoietic cells. Alum-anchored IFNs achieved high cure rates of B16F10 tumors upon combination with either anti-PD-1 antibody or interleukin-2. Interestingly however, these alternative combination immunotherapies yielded disparate T cell phenotypes and differential resistance to tumor rechallenge, highlighting important distinctions in adaptive memory formation for combinations of type I IFNs with other immunotherapies.