bluuebunny/crossref_metadata_2025 huggingface dataset:
Consumption of the fish oil high fat diet uncouples obesity and tumor growth through induction of reactive oxygen species in pro-tumor macrophages
Authors: Li Bing , Jin Rong , Hao Jiaqing
Published: January 2023

It is clear that obesity is associated with increased risk of many types of cancer. However, obesity can be induced by various high fat diets (HFDs) from different fat sources. It remains unknown whether fatty acid composition in different HFDs influences obesity-associated tumor development. Herein we report that consumption of either a cocoa butter or fish oil HFD induced similar obesity in mouse models. While obesity induced by the cocoa butter HFD was associated with accelerated tumor growth, consumption of the fish oil HFD uncoupled obesity from increased tumor growth and exhibited a decrease in pro-tumor macrophages. Interestingly, compared to FA components in both HFDs, n-3 FAs rich in the fish oil HFD induced significant production of reactive oxygen species (ROS) and macrophage cell death. Moreover, we found that A-FABP expression in the pro-tumor macrophages facilitated n-3 FA intracellular transportation and mitochondrial FA oxidation. A-FABP deficiency diminished n-3 FA-mediated ROS production and macrophage cell death both in vitro and in vivo. Altogether, our results demonstrate a novel mechanism by which n-3 FAs induce ROS-mediated pro-tumor macrophage cell death in an A-FABP dependent manner. This study provides mechanistic insights into dietary supplementation with fish oil for cancer prevention and puts forwards a new concept that not all HFDs leading to obesity are tumorigenic.
Increasing AMPK activity supports enhanced oxidative metabolism, proliferation, and in vitro recovery of human CD4+ T cells
Authors: Braverman Erica , Dobbs Andrea , Monlish Darlene , Brown Rebecca Audrey , Byersdorfer Craig A
Published: January 2023

Background

In adoptive immunotherapy, treatment success correlates with the in vivo expansion and subsequent persistence of transferred cells. In T cells, survival and memory formation are both enhanced when cells exhibit higher levels of oxidative metabolism. AMP-activated protein kinase (AMPK) is a master regulator of metabolism, promoting oxidative phosphorylation when nutrients are scarce. We hypothesized that enhanced AMPK signaling would improve T cell therapeutic potential by increasing their oxidative potential and prolonging their persistence.

Results

Jurkat and primary human T cells were transduced with a lentiviral construct encoding either AMPKγ2 or an empty vector. All AMPKγ2 transduced cells showed increased activation of the AMPK pathway, with AMPK-transduced Jurkat T cells exhibiting increased baseline and maximal oxygen consumption as measured on a Seahorse Metabolic Analyzer. Furthermore, they were able to maintain this enhanced oxidative metabolism despite prolonged nutrient starvation. In addition, AMPK-transduced primary human T cells increased their mitochondrial density and improved their in vitro proliferation, with significantly increased recovery of CD4+ T cells.

Discussion

Increasing AMPK activity by over-expressing AMPKγ2 increases the in vitro oxidative potential, proliferation, and recovery of human T cells, particularly CD4+ T cells. These changes are all attractive features for subsequent adoptive immunotherapy. Further work will evaluate the mechanism underlying the phenotype and metabolome of AMPK-transduced cells and will assess their metabolic activity and persistence in vivo.
Role of heat shock factor 1 (HSF1) on activation-induced metabolic reprogramming of T cells
Authors: Pandya Bhaumik D , Moskophidis Demetrius , Mivechi Nahid F
Published: January 2023

Heat Shock Factor 1 (HSF1) is transcriptionally activated in response to a variety of environmental stressors. Data from our laboratory indicates that genetic inactivation of HSF1 significantly delays tumorigenesis in various tumor models. Increasing evidence supports the paradigm that modulating the metabolic bioenergetics of T cells i.e., inhibiting glycolytic flux or enforcing fatty acid oxidation, enhances the formation of more efficient anti tumor CD8+ T-cells. Since, HSF1 is involved in cellular metabolism, in this study, we explored the strategies to manipulate (or reprogram) the metabolic pathways used by the immune system to improve anti-tumor immunity. We observed that deletion of HSF1 profoundly affects the metabolic reprogramming of naive CD8+ T cells upon anti-CD3/CD28 stimulation in vitro. Furthermore, HSF1-deficient T cells show reduced oxygen consumption rate and extracellular acidification rate upon activation. These effects correspond to delayed T-cell receptor signaling and slower activation of naive T cells preventing them from moving towards early exhaustion stage resulting in improved longevity. In addition, we detected delayed initiation of colon cancer MC38 tumors that was implanted into a T-cell specific HSF1 deficient mice compared to wild-type mice. Finally, we confirmed that the delay in tumorigenesis is mediated through CD8 response since treatment of above tumor bearing mice with anti-CD8 antibody reversed the suppressive effects of HSF1 deficient CD8 T cells on tumor growth. Taken together, HSF1 can be a potential therapeutic target to overcome the limitations with existing cancer therapies and further studies will be directed towards exploring impact of HSF1 on checkpoint blockade.
The role of CD36 in macrophage lipid metabolism and function in tumor microenvironment
Authors: Xu Ziyan Zoe , Xu Shihao , Kuhlmann Alexandra , Kaech Susan M
Published: January 2023

Macrophages are a highly plastic population of innate immunity with diverse functions in homeostasis. Despite their relative abundance in tumor microenvironment (TME), the exact function of tumor-associated macrophages (TAMs) and how they are regulated remain largely unknown. Macrophages also play key roles in lipid metabolism, disruption of which can result in pathologies such as atherosclerosis. Interestingly, TME has also been characterized as a lipid-rich context, we are interested in understanding how lipids in TME affect TAMs’ pro- or anti-tumor function.

Here, we firstly characterized the lipid metabolic phenotype of TAMs in a mouse melanoma model. We found that up-regulation of lipid storage and uptake is associated with the most tolerogenic TAM subset marked by high level of F4/80. We also found high expression of scavenger receptor CD36 on F4/80hi TAMs which mediates uptake of oxidized LDL (oxLDL) from TME. Using germ line knockout (Cd36−/−) and myeloid-specific deficiency (Cd36 flox/flox x Csf1r-Cre) mouse models, we observed that uptake of oxLDL by TAMs was significantly blocked, along with rescued immunosuppressive phenotype characterized by decreased expression of PD-L1, CD206 and elevated secretion of TNFa, which together contribute to slower tumor growth. We further conducted single-cell RNA sequencing on Cd36−/− and wild-type TAMs, and found several pathways as potential mechanisms for CD36-mediated pro-tumor phenotype.

In conclusion, we discovered that high expression of CD36 on TAMs mediates uptake of oxLDL and contribute to pro-tumor function. Our data suggests that metabolites in TME may play crucial roles in metabolic and functional reprogramming of tumor-infiltrating immune populations.
BCG immunotherapy response associated immune transcriptomic and spatial immune profiles of non-muscle invasive bladder tumors
Authors: Chenard Stephen , Ren Runhan , Jackson Chelsea , Xu Amanda , Berman David , Siemens D. Robert , Koti Madhuri
Published: January 2023

Bladder cancer is a management-intensive disease that leads to ~200, 000 deaths worldwide every year. Upon initial diagnosis, approximately 75% of cases are categorized as non-muscle-invasive bladder cancer (NMIBC). Adjuvant Bacillus Calmette-Guérin (BCG) immunotherapy is the gold standard treatment for high risk NMIBC patients. Despite being used for over 40 years, many BCG patients exhibit tumor recurrence, a subset of which display progression to a muscle-invasive form of the disease that requires undesired bladder removal surgery.

We recently demonstrated that the interferon response induced by BCG could be further complemented via synergistic activation of the Stimulator of Interferon Genes (STING) pathway. Towards clinical translation of this combinatorial immunomodulation and in order to determine immune biomarkers associated with response to BCG, we retrospectively investigated the immune transcriptomic profiles of tumors from BCG responders and non-responders. Interestingly, tumors from BCG non-responders showed a higher immune cell abundance and higher immune checkpoint gene expression.

To further independently validate the transcriptomic findings at protein levels, we conducted spatial immune profiling of 12 immune cell markers (Ki-67+, CD8+ proliferating T cells, FoxP3+ T regulatory cells, CD79+ B cells, PD-1, PD-L1 and IDO1 immune checkpoints, CD68+ and CD163+ M1 and M2 macrophages) in a cohort of 571 NMIBC tumors. Preliminary findings showed that tumors from BCG non-responders are characterized by infiltration of immunosuppressive T regulatory cells. Findings from this study will have the potential to guide future immune biomarker guided immunomodulatory treatment combinations.
Intradermal delivery of a combination of immunomodulatory antibodies can promote rejection of hyperplastic skin in a mouse model of actinic keratosis
Authors: Wright Quentin , Minnie Simone , Leggatt Graham R
Published: January 2023

Effective immunotherapy strategies for cutaneous squamous cell carcinoma (SCC) and its precancerous lesion, actinic keratosis (AK), is an emerging area of interest in skin cancer research. Presently, clinical trials are focusing on systemic administration of immune checkpoint inhibitors and agonistic antibodies in advanced SCC. However, few studies have addressed the intradermal route of administration for antibody therapies, in either early stage SCC or AK.

In this study, we examined the effects of intradermal antibody therapy in combination with a topical TLR7/8 agonist, imiquimod, in a mouse model of precancerous skin. Using intradermal administration of anti-4-1BB and anti-PD-1 antibodies in combination with topical administration of imiquimod, precancerous skin grafted onto syngeneic recipients underwent a significant reduction in graft size relative to anti-PD-1/imiquimod and isotype antibody/sorbelene control cream groups. Interestingly, flow cytometry analysis of the anti-4-1bb/PD-1/ imiquimod and isotype/control cream groups, 14 days after cessation of therapy administration, revealed a distinct CD8 T cell subpopulation bearing hallmarks of T cell exhaustion (EOMES, PD-1, TIGIT and Tim-3 positive cells) in the spleen of the treatment group.

We conclude that intradermal and topical therapy combinations can induce favourable outcomes in the treatment of skin precancers.
T-cell Enriching Therapy using combination of IL-12 gene electrotransfer and anti-PD-1 attenuates the resistance of anti-PD1 in preclinical metastatic cancer
Authors: Shi Guilan , Scott Megan , Lundberg Cathryn , Heller Richard
Published: January 2023

Immune checkpoint blockades, such as anti-PD-1, have demonstrated significant clinical benefits. However, both primary and acquired resistance has become major obstacles. Limited immune cell infiltration into tumors and expression of PD-1 are two reasons leading to resistance to PD-1 inhibition. Our laboratory has demonstrated that expression of plasmid IL-12 delivered intraumorally can increase immune cell infiltration as well as potentially increase levels of PD-1. In this current study, we used the intraperitoneal metastatic B16F10 tumor model and report that IL-12 gene electrotransfer (IL-12 GET) combined with anti-PD-1 induced long-lived CD8+PD-1− T cells. Strikingly, the effector cytolytic activity of CTLs was expanded and maintained for up to 60 days, indicating that these cells do not differentiate into a memory functional phenotype. In vivo imaging results, illuminate a lower flux signal in B16.F10 intraperitoneal metastases following the combination treatment. Simultaneously, the primary subcutaneous tumors are fully regressed in most of these mice (80%). Expression of CD44+ CD62L− on CD8 CTLs increased with time, suggesting that the memory response was induced at around 120 days. Depletion experiments revealed that CD8+ T cells and CD4+ T cells were required for tumor regression. Our results revealed that this treatment not only boosted CD3+ T-cell infiltration into tumor mass but also upregulated the production of proinflammatory cytokines such as IFN-γ as well as chemokines CXCL9, CXCL10 and CXCL11, promoting accumulation of T cells to the tumor microenvironment and enhancing the effector functions of infiltrated T cells and simultaneously, attenuate the resistant of immune checkpoint blockade of anti-PD1.
Optimal Treatment Regimen of Using Chemo-Immunotherapy for Solid Tumors
Authors: Chaturvedi Pallavi , George Varghese , Liu Bai , Zhu Xiaoyun , Wang Meng , Rhode Peter , Wong Hing C
Published: January 2023

Chemotherapeutic agents not only cause apoptosis and necrosis of cancer cells but also induce cellular senescence (TIS), resulting in a persistent cell population that re-enters the cell cycle with stemness characteristics via the senescence-associated secretory phenotype. These senescent cancer cells, therefore, seed disease relapses if they are not effectively eliminated. TIS cancer cells lack HLA expression and cannot be targeted by T cells. Herein, we show that chemotherapy-induced senescent B16F10 cells express the NK ligands Rae1e and Ulbp1, and are effectively killed by mouse NK cells in vitro. Docetaxel treatment also induced senescent B16F10 cells in vivo. We have previously shown that the TGF-β trap/IL-15 fusion protein, aka HCW9218, effectively activates NK cells in vivo. Therefore, we treated B16F10 tumor bearing mice with docetaxel and then a single dose of HCW9218 in combination with the ADCC antibody TA99. This treatment regimen exhibited potent antitumor activity compared to docetaxel alone. End point analysis showed that HCW9218-treated tumors have lower mitotic activity, reduced expression of senescence markers, and increased frequencies of infiltrating CD8 T cells. We further showed in this model that HCW9218 treatment followed by anti-PD-L1 therapy was more effective than anti-PD-L1 therapy followed by HCW9218. These results suggest that the optimal sequence of cancer treatment is: (1) standard-of-care chemotherapy to control tumor burden and induce cellular senescence, (2) immunotherapy for removal of senescent cells and enhanced tumor infiltration of CD8+ T cells and (3) checkpoint blockade to maintain infiltrated T cell effector function to enhance therapeutic responses and treatment durability.
Glutamate receptor signaling is critical for T cell function and antitumor activity
Authors: Shanker Anil , de Aquino Maria Teresa Prudente , Hodo Thomas W , Uzhachenko Roman
Published: January 2023

The interaction between the nervous system and immune system has sparked interest in recent years. Emerging evidence shows an intricate neuroimmune network. We were intrigued by the expression of various neurotransmitter receptors on T lymphocytes. Specifically, following TCR stimulation, glutamate receptors (GluR) were significantly upregulated on both CD4+ and CD8+ T cells, with a peak at 48 h. Concomitant with the upregulation of activation molecules CD69, CD25 and CD44, proliferating CD8+ T cells presented higher levels of GluA3 and mGluR1 when compared with resting cells. By blocking group I metabotropic glutamate receptors and AMPA receptors through antagonists, we show that CD8+ T cells have a delayed activation but their viability is not altered. However, GluR blockade affected CD8+ T cell proliferation and their ability to kill tumor cells in vivo or target cells in vitro. While the frequency of GluR+CD8+ T cells ranges around 30% of total CD8+ T population, the effect of blocking glutamate signaling through receptor antagonists is a dominant effect and it is mediated by transient reduction in protein phosphorylation of TCR mediated pathways as well as calcium modulation. Overall, data suggest that glutamate receptors may have a stimulatory effect on T cell activation and glutamate agonists may boost T cell response in an immunosuppressive setting such as cancer.
Artemisinin improves the efficiency of anti-PD-L1 therapy in T-cell lymphoma
Authors: Yan Dehong , Zhang Mengqi , Adeshakin Adeleye O. , Wan Xiaochun
Published: January 2023

Although immune checkpoint inhibitors targeting PD-1/PD-L1 pathway have obtained favorable results in T-cell lymphoma clinical trials, the majority of T-cell lymphoma patients appear to be minimally sensitive to PD-1/PD-L1 blockade. The malaria drug artemisinin has previously been identified as a potent anti-lymphoma drug and was tested as a combination treatment with anti-PD-L1 antibody in T-cell lymphoma. In this study, we found that artemisinin caused ferroptosis by up-regulating ROS levels and increasing misfolded protein in T-cell lymphoma via down-regulating ERK pathway. As expected, artemisinin or anti-PD-L1 monoclonal antibody therapy alone could inhibit EL4 T-cell lymphoma growth in Balb/c nude mice. Interestingly, combination therapy with artemisinin greatly improved the anti-lymphoma efficiency of anti-PD-L1 monoclonal antibody. These results provide further rationale for the combination therapy of artemisinin with anti-PD-L1 antibody in preclinical settings.

This work was supported by the National Natural Science Foundation of China (Grant 81501356 and Grant 81373112), the Shenzhen Basic Science Research Project (Grants JCYJ20170413153158716 and JCYJ20170818164619194), Nanshan pilot team project (LHTD20160004), Start-up funding (CYZZ20180307154657923), Guangdong Provincial Research Award for Thousand Talents Program Scholars.
IL-37 suppresses hepatocellular carcinoma development through T cell activation and angiogenesis suppression
Authors: Zhu Ying , MEI YU , Liu Haiyan
Published: January 2023

IL-37, a relatively newly identified IL-1 family cytokine, has been shown to play an important immune regulatory role in inflammatory diseases, autoimmune diseases and carcinogenesis. IL-37 has been demonstrated to recruiting NK cells, stimulating CD4+ T cells, as well as inhibiting tumor angiogenesis to suppress tumor growth in various tumor models. In this current study, we found that IL-37 could suppress hepatocellular carcinoma (HCC) development in murine orthotopic HCC model and DEN-induced HCC model. Further study revealed that IL-37 could enhance the T cell killing capacity. This anti-tumor effect was fully abrogated in NOD-SCID mice. Furthermore, IL-37 could inhibit tumor angiogenesis in murine HCC model. In vitro study revealed that IL-37 treatment directly promoted HUVECs migration and tubule formation capacity, indicating IL-37 is a pro-angiogenic factor. However, the supernatant from IL-37 over-expression HCC cell line promoted HUVECs apoptosis and impaired the migration and tubule formation capacity of HUVECs. Microarray data suggested IL-37 affected the pro- and anti-angiogenic factors expression pattern from tumor cells. The pro-angiogenic factors ANGPT1, CYP1B1, FN1, MMP2, MCAM, PIK3CG were downregulated by IL-37, while the anti-angiogenic factor ANGPT1 was upregulated. These results indicated IL-37 could affect angiogenesis in both direct and indirect ways. Overall, our results demonstrated that IL-37 could suppress HCC development through T cell activation and angiogenesis suppression.
The role of the SWI/SNF complex in the tumor immune microenvironment
Authors: Chaudhri Apoorvi , Wang Yunfei , Shi Leilei , Singh Anand , Rai Kunal , Hwu Patrick
Published: January 2023

Remodeling of the chromatin is a regulatory mechanism in order to close or open the chromatin and mediate gene regulation. The SWI/SNF complex is evolutionary conserved and uses the energy from ATP to remodel the chromatin. This complex is mutated in about twenty percent of all human cancers. The subunits of the complex are known to have a tumor suppressor function, however this understanding can be debated. Studies have shown that in the event that one of the members is mutated, residual members of the complex can have aberrant activity more towards an oncogenic phenotype. The tumor suppressor or the oncogenic potential of this complex is context dependent and needs a deeper understanding. In the setting of a tumor, recent research has shown that, inhibition of the subunits of this complex can enhance the ability of the immune system to kill the tumor owing to their oncogenic potential. Here we discuss and analyze the various BAF, PBAF and the core catalytic subunits of the complex. Using both in-silico and wet lab approaches, we relate the functioning of the subunits to affect the tumor infiltrating immune cell subsets. Using data sets, we correlate the subunits with immune based therapy response. While each of the subunits play diverse roles, together we highlight the importance of this complex to shape the tumor immune landscape.
Formyl peptide receptor (FPR) agonists promote immune evasion independent of functional Ncf1 derived ROS
Authors: Bonner Michael Y. , Olofsson Peter , Holmdahl Rikard
Published: January 2023

Previous studies have shown that in both the absence of the Ncf1 gene or in the presence of a loss-of-function SNP, mice have a lower tumor burden when compared to wildtype animals. Here, we investigate a formyl peptide receptor (FPR) agonist in this setting and demonstrate that it promotes tumor immune evasion through an immune-Ncf1/ROS-dependent mechanism.

We observed an increased colonization of B16F10 tumor cells in the lungs of FPR-agonist-treated wildtype C57BL6N mice compared to vehicle-control-treated mice. A significant reduction in tumor burden was expectedly seen in the Ncf1*/* mutant strain compared to the wildtype. However, FPR-agonist treatment did not promote tumor growth in Ncf1*/* treated mice when compared to vehicle-control-treated Ncf1*/* mice. These results suggest that FPR agonists can promote tumor evasion by acting through FPRs on immune cells in a NOX2/ROS-independent fashion and in a fashion independent of FPRs on the tumor.
Host- versus cell-dependent effects of β-Arrestin 1 expression in prostate tumorigenesis
Authors: Adekoya Timothy Oluseyi , Smith Nikia , Richardson Micheler
Published: January 2023

Prostate cancer (PCa) constitutes a serious health challenge and remains one of the main causes of cancer-related death among men. The more aggressive form of the disease is androgen independent resulting in lack of response to androgen deprivation therapy. The scaffold proteins β-arrestin 1 and 2 (βarr1 and βarr2) which are known to mediate G protein-coupled receptors (GPCRs) desensitization and internalization, were also shown to modulate prostate tumorigenesis. βarr1 is overexpressed (~4 fold) in PCa cells relative to βarr2. In this study, we investigate the effect of β-arrestins expression in prostate tumorigenesis using in-vivo xenografts and autochthonous transgenic adenocarcinoma mouse prostate (TRAMP) model deficient in β-arrestins expression. Depletion of βarr1 in TRAMP mice (TRAMP/βarr1−/−) increased PCa growth and decreased overall survival, relative to TRAMP and TRAMP/βarr2−/− animals. Tumors from TRAMP/βarr1−/− mice displayed increase androgen receptor (AR), but decrease prostate specific antigen (PSA) expression. In contrast, depletion of βarr1 in MDA PCa 2b cells (MDA PCa 2b-βarr1−/−) decreased cell proliferation, wound closure and anchorage independent cell growth compared to wild type MDA PCa 2b and MDA PCa 2b-βarr2−/− cells. Nude mice xenografts of MDA PCa 2b-βarr1−/− cells also showed significant decrease in tumor growth. Interestingly, tumor lysates from MDA PCa 2b-βarr1−/− xenografts exhibited decrease AR and increase PSA expression, relative to control tumors. Altogether, the data indicate that βarr1 modulates AR/PSA signaling to regulate prostate cancer aggressiveness. However, the effect of βarr1 is context-sensitive depending on its expression in the host relative to the tumor cells.
REPERTOIRE OF CIRCULATING ANTIBODIES CORRESPONDING TO MOLECULAR SUBTYPES OF BREAST CANCER
Authors: Chapoval Andrei I , Podlesnykh Stepan V , Kolosova Evgenia A , Shcherbakov Dmitry N , Shoikhet Jakov N , Lazarev Alexander F , Vikhlyanov Igor V , Sinkina Tatiyana V , Johnston Stephen A , Ilyichev Alexander A
Published: January 2023

Changes in the repertoire of circulating antibodies is a promising biomarker for various diseases. We have developed microarrays containing more than thousands of peptides with random amino acid sequences that can be used to analyze the entire repertoire of circulating antibodies. Using these peptide microarrays, we have shown that in the blood of patients diagnosed with breast cancer (BC) antibodies interacting with certain peptides are found. Moreover, circulating antibodies of patients with different molecular subtypes of BC (luminal A, luminal B and Basal-like) interact with different, but overlapping, panels of peptides. We have selected 634 peptides from 120K peptides presented on microarrays that specifically interact with circulating antibodies of blood plasma from patients with various subtypes of BC. This panel of peptides that specifically interact with the blood plasma immunoglobulins of patients with various molecular BC subtypes can be used for the development of non-invasive diagnostic test systems. Microchips containing thousands of peptides can be used as a tool for molecular screening populations at risk for various diseases. Thus, peptide microarrays can be used to create a new tool for studying the repertoire of circulating antibodies for different diseases evaluation, as well as for studying and evaluating protein-protein interactions.
Plasticity and Polarization of Human NK Cells in the Tumor Microenvironment
Authors: Moreno Nieves Uriel Y , Tay Joshua , Saumyaa Saumyaa , Mundy David , Sunwoo John B.
Published: January 2023

The presence and function of innate lymphoid cells (ILC), including natural killer (NK) cells, within human tumors has been poorly characterized. Here, we have assessed the heterogeneity of NK and ILC populations by single-cell RNA sequencing (scRNAseq) of hundreds of individual NK cells and ILCs within human head and neck squamous cell carcinoma (HNSCC), matched lymph node metastases, matched peripheral blood, and blood from healthy donors. Fresh tumor specimens and blood were obtained from 8 patients undergoing surgical resection of HNSCC. Unsupervised clustering revealed 8 different clusters of NK cells and ILC subsets. We observed significant heterogeneity, with distinct subsets showing profiles consistent with that of conventional NK cells, ILC1-like cells and ILC3-like cell. Importantly, peripheral NK cells were distinct from intratumoral NK cells. The presence of these different cell subsets within primary HNSCC tumors was confirmed by flow cytometry. Further, plasticity between the NK cell subsets was supported by in vitro and in vivo experimental data. Given the ability of NK and ILCs to polarize the immune responses through the secretion of cytokines and the ability of certain subsets to kill target cells, we hypothesize that the differences observed in NK and ILC populations and their plasticity may result in different immune responses, and may influence clinical outcomes following therapy.
Transcriptome network analysis identifies co-expressed CXCL13, CXCR5, and associated genes contributes to the aggressive phenotype of TNBC
Authors: Dill Courtney Denise , Dammer Eric , Griffen Tiara L , Young Corey D , Carey Kaylin , Nunez Sha’Kayla , Lillard James
Published: January 2023

Triple negative breast cancer (TNBC) is an aggressive disease associated with poor prognosis. Currently, there are no targeted therapies for this disease. Standard therapy for TNBC consists of a combination of chemotherapeutic drugs. However, patients often become resistant to this intervention and ultimately succumb to this disease. Hence, understanding the cellular and molecular mechanisms associated with TNBC progression is essential to develop more effective therapies. We hypothesize co-expressed CXCL13, CXCR5, and associated genes are significantly correlated with TNBC and poor overall survival. A bioinformatic approach was used to characterize this new potential drug target for TNBC. Our patient cohort was obtained from The Cancer Genome Atlas. Weighted gene network co-expression analysis identified gene networks associated with factors influencing BrCa prognosis. An ANOVA test identified genes differentially expressed among Luminal A, Luminal B, HER-2, and TNBC tumor groups. Finally, gene ontology analysis was performed via GO-ELITE and EPIC was used to estimate the proportion of various cell types found in bulk samples. We identified seven unique gene networks that were significantly and positively correlated with TNBC. The turquoise module (M1), contained CXCL13, CXCR5, and associated genes that drive tertiary lymphoid structure formation, regulation of immune response, and immune system processes. The M1 module was also significantly and positively correlated with TNBC, but not overall survival. The genes signatures contained in the M1 module were significantly correlated to CD8+ T cell, macrophage, B cell, CD4+ T cell, cancer-associated fibroblast, and NK cell, as determined by EPIC analysis.
Neuropilin-1 Amplifies Regulatory T cell Function in Human Cancer
Authors: Chuckran Christopher A , Cillo Anthony R , Moskovitz Jessica , Overacre-Delgoffe Abigail E , Somasundaram Ashwin , Kirkwood John M , Luketich James , Pennathur Arjun , Coffman Lan , Modugno Francesmary , Edwards Robert , Schoen Robert E , Ferris Robert L , Bruno Tullia C , Vignali Dario AA
Published: January 2023

Regulatory T cells (Tregs) maintain peripheral tolerance but also contribute to cancer progression by dampening anti-tumor immunity. Knocking out neuropilin-1 (NRP1) on murine Tregs attenuates tumor growth by rendering intratumoral Tregs non-suppressive. Whereas NRP1 is constitutively expressed on all mouse Tregs, NRP1 is infrequently expressed on Tregs in the peripheral blood of healthy humans. We hypothesized that (1) NRP1 expression is upregulated in cancer patients, (2) NRP1 marks highly suppressive human Tregs, and (3) sustained Treg activation initiates NRP1 expression.

NRP1+ Tregs are enriched in tumors across numerous cancer types but largely devoid in non-cancerous inflamed and healthy tissues. Intratumoral NRP1 expression negatively correlates with disease-free survival in head and neck squamous cell carcinoma (HNSCC; p = 0.03; Hazard ratio = 5.4). Intratumoral NRP1+ Tregs upregulate a suppressive module indicated by co-expression of TIGIT, CCR8, and multiple TNF receptor super family members. Correspondingly, NRP1+ Tregs potently suppress CD8+ T cell proliferation in vitro, which can be disrupted with NRP1-specific blockade. Furthermore, Treg activation in vitro drives NRP1 expression, and sustained TCR signals are required to maintain NRP1 expression.

We conclude that NRP1+ Tregs are selectively enriched in human cancer and oppose anti-tumor immunity. Therefore, destabilizing intratumoral Tregs using NRP1 blockade may complement other T cell therapies such as anti-PD1 blockade.
Live attenuated influenza virus vaccine protects obese mice
Authors: Estrada Leonardo D , Vazquez-Pagan Ana G , Schultz-Cherry Stacey
Published: January 2023

Fifty percent of the United States population is predicted to be obese (body mass index > 30) by 2030. Obese individuals are considered a high-risk group for developing severe influenza disease. To compound this, studies have shown that inactivated influenza vaccination has decreased efficacy in this high-risk population. However, the efficacy of live-attenuated vaccines has not been investigated to date. Therefore, we sought to determine if a cold-adapted A/California/04/2009 (caCA/09) H1N1 influenza virus vaccine protected obese mice from lethal influenza virus challenge. In these studies, we demonstrate that intranasal administration of caCA/09 vaccine protected genetically and diet-induced obese (DIO) mice from a lethal challenge with wild-type influenza virus (CA/09) 4 weeks post-vaccination. Protection was not due to route of administration, as intranasal vaccination with inactivated caCA/09 virus was not protective. Unlike non-vaccinated obese mice, caCA/09 vaccinated obese mice cleared the virus and controlled viral spread, as determined by lung TCID50 and immunohistochemistry, respectively. Protection of obese mice lasts at least 17 weeks post-vaccination. Mechanistically, we found that effector memory CD4 and CD8 T cells were increased in lungs of vaccinated mice that may provide heterosubtypic protection. Indeed, the vaccinated obese mice were protected from a lethal challenge with an H5 deselected influenza virus. In conclusion, live-attenuated virus vaccination is a viable option to protect obese hosts against increased disease severity and mortality.
Immune responses to an inactivated influenza vaccine in Indigenous Australians
Authors: Hensen Luca , Rowntree Louise , Damelang Timon , Chung Amy , Wheatley Adam , Auladell Maria , Koutsakos Marios , Nguyen Oanh , Loh Liyen , Aban Malet , Hurt Aeron , Kent Stephen , Flanagan Katie , Plebanski Magdalena , Nelson Jane , Everitt Anngie , Miller Adrian , Davies Jane , Tong Steven , Kedzierska Katherine
Published: January 2023

Indigenous Australians are highly susceptible to severe influenza disease during seasonal and pandemic infections. Current antibody-based vaccines targeting humoral immunity are the most effective way to combat influenza infections but show reduced efficacy in Indigenous Australians. The underlying mechanisms are still unknown. We recruited 166 Indigenous & non-Indigenous donors to elucidate adaptive immune responses induced by the inactivated quadrivalent influenza vaccine. Participants were bled at baseline, day 7 and day 28 post-vaccination. We analysed hemagglutination inhibition titres against the vaccine and previously circulating strains, analysed IgG1 and IgG3 allotypes, evaluated total IgG glycosylation and performed Luminex assays to dissect antibody responses against viral HA, NA and NP proteins. Additionally, we assessed influenza-specific B cell responses with fluorescent HA probes, antibody-secreting cells and circulating Tfh cells. More than 15,000 datapoints were collected to compare adaptive immune responses between Indigenous and non-Indigenous donors. We found that the majority of immune readouts were comparable between the two groups. However, interestingly, we found significantly lower pre-vaccine titres and lower back-boosting to pre-pandemic H1N1 viruses in Indigenous compared to non-Indigenous donors. This might explain, at least in part, high susceptibility of Indigenous Australians to the 2009 pandemic. Our comprehensive dataset will be used to understand and improve optimal vaccine responses in Indigenous and non-Indigenous populations globally.
Combined CpG and MPL as vaccine adjuvants improve duration of protection induced by single dose influenza A virus protein vaccination
Authors: Lampe Anna T , Clark Madeline , Brown Deborah M
Published: January 2023

Despite vaccination, it is estimated more than 37 million people experienced influenza illness in the US during the 2018–19 season. Efficacy of the influenza A virus (IAV) vaccine may be improved with adjuvants that activate innate immunity. We examined the potential of combined pattern recognition receptor agonists CpG and MPL to increase vaccine induced protection. When administered with a single, low dose intramuscular hemagglutinin protein vaccine, mice receiving CpG+MPL experienced less weight loss after homologous challenge eight weeks after vaccination compared to mice receiving single agonists. However, when mice were challenged earlier after vaccination no significant improvements were observed in weight loss or viral titers, suggesting that combined CpG+MPL improves duration of protection. Despite not inducing excessive injection site damage, CpG+MPL elevated systemic inflammatory cytokines and chemokines 24 hours after vaccination compared to agonists alone. CpG and/or MPL increased monocyte derived migratory dendritic cell numbers and CD4 T cell proliferation in the draining lymph nodes (DLN) three days after vaccination. CpG+MPL also enhanced early T cell activation, measured by CD69 expression, in the DLN compared unadjuvanted vaccines. Additionally, at four weeks, mice vaccinated with CpG+MPL displayed a trend of elevated IgG2a/c antibody production compared to mice receiving single agonists. However, CD4 and CD8 T cell activation and cytokine production in the DLN after homologous challenge was not significantly impacted by CpG+MPL. Together these results suggest that CpG+MPL induces enhanced innate immune responses that contribute to extended duration of protection against homologous challenge.
The Complex of Poly-γ-Glutamic Acid and Alum Induces Cross-Protective Immunity of Pandemic H1N1 Influenza Vaccine
Authors: Nguyen Quyen Thi , Kwak Chaewon , Kim Jaemoo , Yang Jihyun , Poo Haryoung
Published: January 2023

Vaccination is the most effective strategy to protect against infectious diseases, and adjuvants are key vaccine components that improve antigen-specific immune responses. Here, we developed a new adjuvant named PGA/Alum by combining poly-γ-glutamic acid (γ-PGA) with alum and investigated its ability to enhance the immunogenicity and the cross-reactive efficacy of pandemic H1N1 (pH1N1) influenza vaccine antigen. PGA/Alum highly induced in vitro activation and antigen processing of dendritic cells, and enhanced antigen delivery to draining lymph nodes and antigen-specific immunogenicity in mice using OVA as a model antigen. Also, OVA-specific antibody production, cytotoxic T lymphocyte (CTL) activity, and antibody-dependent cellular cytotoxicity (ADCC) were greatly increased by PGA/Alum. These abilities of PGA/Alum improved the protective efficacy of pH1N1 vaccine antigen by increasing hemagglutination-inhibition titers, enhancing ADCC and CTL activity, and speeding viral clearance following homologous viral challenge. Importantly, PGA/Alum significantly enhanced the cross-protective efficacy of pH1N1 vaccine against heterologous viruses [A/Puerto Rico/8/34 (H1N1) and A/Hong Kong/1/1968 (H3N2)] by inducing cross-reactive ADCC and CTL activities. Together, our results strongly suggest that PGA/Alum may be a promising vaccine adjuvant for preventing influenza and other infectious diseases.
Photo-regulated control of cytokine signaling via bioinspired, polymer-induced latency
Authors: Dreaden Erik C , Perdue Lacey A. , Do Priscilla , Chyong Andy , Salaita Khalid , Lesinski Gregory , Porter Christopher C
Published: January 2023

Cytokine signaling is challenging to study or therapeutically modulate as the effects of these protein are often pleotropic and context-dependent. One mechanism by which cytokines with diverse effects can, however, transmit cell type-specific information is via expression in an inactive, or latent, form in which the protein is sterically shielded by a binding partner that is later displaced or degraded (e.g. TGF-β1 association with GARP or LAP). Inspired by this precision, we hypothesized that photo-reversible chemical modification could impart orthogonal specificity to cytokines not expressed in a latent state, such as IL-2, IL-15, or IL-12.

Here, we describe a strategy whereby cytokines are modified with photo-labile polymers that mimic the induction of latency while appended, then de-shield to recover protein activity in response to monochromatic light exposure, thus controlling the magnitude, duration, and location of cytokine signals. Using this strategy, we show that latent IL-2 and IL-15 activity can be photo-modulated as much as 103-fold and that polymer-induced IL-2 latency, alone, can bias affinity away from immunosuppressive CD25 binding. We further show that protein de-repression can be achieved with spatial resolution approaching that of a single immune cell and demonstrate the feasibility of transcutaneous IL-2 photoactivation using engineered tissue phantoms. We find that such modifications also prolong the circulation of IL-12 in vivo, thus obviating the need for frequent, high dosing in therapeutic settings.

Future extensions of this approach could enable multicolor, optical reprogramming of cytokine signaling networks and lead to immunotherapies that are more tissue-specific and patient-personalized.
T cells exhibit prolonged immunity against melanoma tumors expressing high, but not low affinity antigens
Authors: Rivera Reyes Amalia M , Wyatt Megan M. , Dwyer Connor J. , Knochelmann Hannah M. , Smith Aubrey S , Rangel Rivera Guillermo O , Paulos Chrystal M
Published: January 2023

We seek to identify the intrinsic properties in T cells from patients responsive or not to immunotherapy. Therapeutic efficacy is seen in patients where the immune system attacks ‘hot’ tumors due to their high mutation burden. ‘Cold’ tumors, in contrast, are not effectively recognized by T cells due to low mutation burden or expression of self-antigen. To simulate this clinical scenario, we used two different melanoma models: 1) B16F10 expressing a low affinity peptide (mgp100 = i.e. self-antigen), which represents a cold tumor or 2) B16F10 expressing a high affinity peptide (hgp100 = i.e. neoantigen), which represents a hot tumor. In these tumor-bearing mice, we infused pmel-1 CD8+ T cells expressing a TCR that recognizes gp100 peptide on B16F10 melanoma. We hypothesized that the function and persistence of adoptively transferred (ACT) cells would be increased in responsive tumors compared to unresponsive. Our findings show that pmel-1 were less exhausted and persisted more in mice bearing responsive tumors. In these mice, efficiency was observed as shrinkage of large subcutaneous tumors post ACT treatment and enhanced long-term survival. Interestingly, mice bearing hot tumors had metabolically improved pmel-1 cells, demonstrated by their ability to take up glucose and mitochondrial health. Mice that were cured from bearing responsive tumors were protected when rechallenged with both cold and hot tumors simultaneously, implicating a memory response. Herein, we underscore how tumor antigen affinity can drastically change the fate and memory of T cells.
Harnessing antiviral memory T cells for tumor immunotherapy
Authors: Rosato Pamela , Wijeyesinghe Sathi P , Ning Jianfang , Gavil Noah Veis , Wu Shaoping , Chen Clark , Vezys Vaiva , Masopust David
Published: January 2023

Overcoming the immunosuppressive tumor microenvironment remains a major impediment to successful cancer immunotherapy. Virus-specific memory T cells are positioned throughout the entire body to sense reinfection or recrudescence. Mouse models have demonstrated upon reencountering cognate antigen, these tissue resident memory T cells (TRM) induce a local immunostimulatory environment that activates and recruits innate and adaptive arms of the immune system, and we extend these functions to include recruitment of circulating antibody. Like healthy tissue, we observe that mouse and human tumors are commonly surveyed by virus-specific memory CD8+ T cells. This was seen in a range of tumor types including traditionally ‘immune-privilege’ tissues such as glioblastoma. Given the described immunostimualtory functions of antiviral TRM in healthy tissue, we tested if we could leverage antiviral CD8+ T cells in tumors as an immunotherapy. Local delivery of adjuvant-free peptide derived from previously encountered viruses successfully reactivated antiviral T cells within melanoma and glioblastoma tumors. This arrested growth of checkpoint blockade-resistant and poorly immunogenic tumors in mice. Antiviral T cell reactivation triggered antigen presentation and cytotoxic pathways within the tumor, activating T cells, dendritic cells and natural killer cells. Viral peptide treatment of ex vivo human tumors demonstrated upregulation of immune activation gene expression profiles similar to those observed in mice. Lastly, viral peptide therapy renders resistant mouse tumors susceptible to PD-L1 blockade. Thus, re-stimulating known antiviral immunity may provide a novel therapeutic approach for a broad range of tumors.
From biTEs to a novel switchable chimeric antigen receptor platform for gated tumor targeting
Authors: Bachmann Michael P , Berndt Nicole , Arndt Claudia , Hoffmann Anja , Koristka Stefanie , Loureiro Liliana , Feldmann Anja
Published: January 2023

Over the past decades we followed the idea of gated targeting of tumor cells starting with modular bispecific antibodies (bsab) followed by T cells genetically modified with modular chimeric antigen receptors (CARs). During these studies we developed a series of bispecific T cell engagers (BiTEs) and in addition the modular UniCAR platform(s). Recently for two of our BiTEs and a first UniCAR complex clinical phase 1 studies could be started. Based on these developments we now established a next generation switchable adaptor CAR platform technology termed RevCARs and characterized it with respect to functionality in vitro and in vivo, possibility of steering of CAR T cell activity (reversible and repeated switching on and off), cytokine response, pharmacokinetic, and imaging with positron emission tomography (PET) in experimental mice: In order to develop polycistronic vectors encoding for more than one CAR (e.g. one signaling CAR and one costimulatory CAR) we were forced to reduce the size of the individual CAR genes. For this purpose minimized CAR genes each using as extracellular domain just a short peptide epitope sequence instead of an antibody domain are fused in a single vector and used for transduction of T cells leading to a defined expression ratio optimally adjusted for “AND” and “OR” gated targeting. The specificity and activation of these RevCARs is mediated via bsab based targeting molecules (revTMs) directed to specific cell surface tumor antigens (TAAs). Due to the small size of revTMs RevCAR T cells can easily and repeatedly be turned on and off simply by starting or stopping of their infusion. In summary, we will present and update the current status of our preclinical and clinical work related to retargeting of T cells to tumor cells.
IL-6 fuels durable memory for Th17 cell-mediated responses to tumors
Authors: Knochelmann Hannah , Dwyer Connor , Smith Aubrey S , Wyatt Megan , Rivera Guillermo Rangel , Lesinski Gregory , Li Zihai , Rubinstein Mark , Paulos Chrystal M
Published: January 2023

Accessibility of T cell transfer therapies is hindered by cost and time required for product development. We found that shortening ex vivo expansion of Th17 cells licenses a proinflammatory cell product which induces cytokine storm with high levels of systemic IL-6 in tumor-bearing hosts. Unique from other helper subsets, briefly expanded Th17 cells eradicate large tumors in low doses and generate long-lived memory against tumor rechallenge, suggesting a benefit to the inflammatory state. As the toxicity of cytokine release is managed in patients through IL-6 blockade, we addressed the physiologic impact of IL-6 on efficacy and durability of Th17 cell therapy in this immunocompetent model. We hypothesized that peak IL-6 induced by Th17 cells was critical to their durable memory properties. To address this, we used the TRP-1 transgenic mouse model where CD4+ T cells express a TCR that recognizes TRP-1 of B16F10 melanoma. Acute IL-6 blockade post Th17 transfer did not impact the primary antitumor response, yet abrogated long-term responses by promoting tumor relapse upon secondary challenge. Mechanistically, IL-6 blockade reduced pSTAT3 and Bcl2 in transferred T cells and promoted a greater frequency of FoxP3+ Treg cells in the peripheral blood and tumor-draining lymph nodes. Finally, serum IL-6 was inversely correlated with engraftment of tumor-specific Tregs from the transferred product. Overall, short-term expanded Th17 cells uniquely induce IL-6, which promotes Th17 survival, suppresses engraftment of tumor-specific Tregs, and is critical to durable memory. This work may suggest that the universal strategy to inhibit IL-6 during cytokine release syndrome may come at the expense of long-term efficacy for cell therapy.