bluuebunny/crossref_metadata_2025 huggingface dataset:
High-content screening assay for compounds that enhance or inhibit leukocyte superoxide production to identify drugs for treating immune-mediated diseases
Authors: Wu Szu-Yu Szu , Shieh Chi-Chang K
Published: January 2023

Reactive oxygen species (ROS) are produced by cells to play important roles in the effector and regulatory functions of immune cells. Our previous studies revealed that ROS act as negative regulators in IL-1β-mediated inflammation. Enhanced severity of inflammation in immune-mediated arthritis was noted when leukocyte NADPH oxidase (NOX2), which mediates the production of ROS, was defective. We hence hypothesized that drugs that modulate the leukocyte ROS production may regulate the pathogenicity of immune-mediated arthritis. Here, we devised a high-content image assay using the dye H2DCF-DA for identifying LOPAC 1280 library compounds that may facilitate or suppress superoxide production in differentiated HL-60 (dHL-60) cells. ROS levels were measured after the cells were treated with phorbol 12-myristate 13-acetate (PMA) or menadione which may induce ROS production from NOX2 or mitochondria. From a screening of 640 compounds in a 3-point interpolate titration in cell-based imaging analysis, we identified 74 drugs that may enhance the ROS production and 72 compounds that may inhibit ROS production by PMA-stimulated-dHL-60 cells. The drugs that we identified included the agonist/antagonist of neurotransmitters and their receptors, ion channel blockers, serotonin antagonists, cell stress modulators and cell cycle inhibitors. To further clarified our hypothesis, we treated NOX2-deficient mice with menadione, which tended to increase the oxidant stress in the joint tissue. The severity of the immune-mediated arthritic joint was significantly suppressed after the treatment. This novel screening method hence may be used to identify potential drugs for treating redox-related inflammatory diseases.
Angiotensin-Converting Enzyme Inhibitor (ACEI) suppresses the expression of the MHC molecules in mucosal cells of the small intestine leading to the delayed onset of autoimmune type 1 diabetes mellitus in NOD mice
Authors: Hattori Masakazu , Ueda Aiko
Published: January 2023

Angiotensin-converting enzyme (ACE) exists in the kidney, lung and small intestine of the NOD mouse (autoimmune type 1 diabetes animal model). ACE changes Angiotensin I to Angiotensin II. ACE exists in the small intestinal epithelial cells and villi, and digests the protein antigen molecules in the small intestine. ACE plays an important role in the antigen presenting process in the mucosal immune system of the duodenum and jejunum. To examine the role of ACE in the development of type 1 diabetes of the NOD mouse, ACE-Inhibitors (ACEI): captopril (CA) or enalapril maleate (EM) was dissolved in a drinking water at the concentration of 12.5mg/ml and given daily to 28-day-old NOD females and males ad libitum (89 females and 70 males in CA, 72 and 52 in EM, 120 and 87 in water control group, respectively). The animals were screened for the development of diabetes twice a week for 7 months and subjected to the Life Table analysis. The incidence of diabetes in our NOD/Kmc colony is 83% in females and 40% in males at 210 days of age.

The EM-treated NOD mice showed a significant delay of the onset of diabetes at 120 days of age in comparison with the controls (25% vs. 45% in females, p=0.002; 0% vs. 8% in males, p=0.037). The CA treatment was ineffective to delay the onset of diabetes. The histopathological examination of the pancreata showed that the EM-treated females had more islets without lymphocytic infiltration than the CA-treated or the water-control females.

The mRNA expression of MHC K and A genes was suppressed in the duodenum and jejunum of the NOD mice by EM- or CA-water for 14 days. Flow cytometric analysis showed suppression of the MHC K and I-A expression in the pancreatic lymphocytes of the EM- and CA-treated mice in comparison with the control mice.
Defining the role for the gut microbiome in the clinical efficacy of sulfasalazine therapy for IBD associated spondyloarthritis
Authors: Lima Svetlana Ferreira , Longman Randy , Rupert Amanda , Viladomiu Monica , Marderstein Andrew , Putzel Gregory , Funez-dePagnier Gabriela , Woo Viola , Metz Maeva L , Scherl Ellen J , Chou Lance
Published: January 2023

Spondyloarthritis (SpA) is the most common extra-intestinal manifestation of inflammatory bowel disease (IBD). Sulfasalazine (SAS) is one of the earliest medications used in IBD and its efficacy in spondyloarthritis is thought to depend on its antibacterial properties. Therefore, our study aims to diagnostically evaluate the role for the fecal microbiome in clinical response to SAS and identify microbial and immunologic therapeutic targets associated with clinical response.

We have longitudinally followed IBD-SpA patients subjected to SAS therapy. Clinical data, including validated IBD and joint disease activity scores, and fecal samples from 19 patients were collected at baseline and at week 2 and 12 after SAS initiation. Metagenomic sequencing was used to define the effect of SAS on the IBD-SpA fecal microbiome and to evaluate its relationship with joint symptoms improvement. Gnotobiotic mouse models were used to test the sufficiency of the SAS effect observed in patients.

Fecal microbiome of SAS-responders was distinct from that of non-responders and 6 pre-treatment microbial markers (including Faecalibacterium prausnitzii) predicted SAS-response (AUC: 0.9). SPF mice and germ-free mice colonized with patient microbiota revealed that SAS selectively reduced mucosal-associated bacteria. Gnotobiotic mouse models revealed a critical role for A. muciniphila in modulating mucosal inflammation.

Our study reveals the ability of SAS to selectively target mucosal-associated bacteria and modulate the inflammatory impact of IBD-SpA associated pathobionts. This study highlights the potential use of microbial-based diagnostic tools to improve drug efficacy and therapeutic strategies for IBD-SpA.
Macrophage migration inhibitory factor-2 of Wuchereria bancrofti suppressed the DSS-induced colitis in a mouse model by promoting alternative activation of peritoneal macrophages and increasing the number of Treg cells in the colon
Authors: Chauhan Nikhil , Khatri Vishal , Prasanna Kumar S B , Malendez Valeria , Kalyanasundaram Ramaswamy
Published: January 2023

In this study, we cloned the homologue of macrophage migration inhibitory factor (MIF) secreted by the human filarial parasite Wuchereria bancrofti (rWbaMIF-2) and evaluated its potential in ameliorating inflammation in a dextran sulfate sodium salt (DSS)-induced colitis model. DSS administration resulted in substantial loss of body weight along with bloody diarrhea, severe inflammation in the colon and reduced length of the colon in just under 7 days. Treatment with rWbaMIF-2 reversed the clinical symptoms in DSS administered mice, characterized by no blood in the stools, minimal inflammation on the colon wall and the mice maintained normal colon length. rWbaMIF-2 treatment downregulated TNF-α, IL-6, IFN-γ, IL-1β, IL-17A, and NOS2 genes in the colon tissue and upregulated the percentage of IL-10 producing Treg and B1 cells in the colon and peritoneal cavity. Thus, one of the mechanisms by which rWbaMIF-2 induce its anti-inflammatory effect appeared to be by promoting the infiltration of IL-10 producing Treg cells into the colon. Further analysis showed alternative activation of F4/80+ large peritoneal macrophages (LPMs) with increased expression of arginase (p≤0.05) and reduced expression of iNOS, MPO activity, TNF-α, IL-6 and TLR4 in LPMs following rWbaMIF-2 treatment. Thus, for the first time, we demonstrate here that rWbaMIF-2 treatment can activate LMP macrophages alternatively and promote infiltration of Treg cells into the colon to suppress inflammation in a colitis model. These studies also demonstrate the potential of developing rWbaMIF-2 as a small molecule therapeutic agent for colitis and other inflammatory conditions. Studies funded by NIH RO1 AI116441.
Metabolite changes and anti-inflammatory effects of Astragalus membranaceus Radix by fermentation with Aspergilus strains
Authors: Lee Sung Hyen , Park Shin-Young , Lee Da-Bin , Lee Eun-Jung , Song Bit-Na , Park Bo-Ram , Choi Ji-Ho , Lee Eun-Byeol
Published: January 2023

Astragalus membranaceus (AM) is an edible medicinal resource in Korea, which has been traditionally used for cardioprotective, anti-inflammatory and longevity effects. This study was conducted to investigate metabolic changes and anti-inflammatory effects of AM by fermentation.

AM was fermented with three microorganisms of Aspergilus strains isolated from Nuruk at 30□ for 6 days (FAM). The metabolites changes in pre and post fermentation of AM were analyzed by LC-MS. The pro-inflammatory factors such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and nitric oxide (NO) were measured in lipopolysaccharide (LPS)-induced RAW 264.7 cell line.

As a result, the 28 identified metabolites in AM between pre and post fermentation process were detected in this study. The data revealed that the levels of sucrose and astraglaloside IV and calycosin decreased throughout the process while LPC and huangqiyenin B levels increased as expected and were the highest among the metabolites after the fermentation. The FAM reduced IL-1β, IL-6, TNF-α and NO production without cytotoxicity. The results suggest that the fermentation process with Aspergilus strains can increase the functional metabolites and control the inflammatory factors in FAM. We expect the biotransformation technique of flavonoids in Astragalus membranaceus Radix may be used in the field of a healthy food industry.
Anti-CD19 CAR-T cell therapy for Systemic Lupus Erythematosus in a mouse disease model
Authors: Jin Xuexiao , Lu Linrong
Published: January 2023

Dysregulated B cell activation plays pivotal roles in systemic lupus erythematosus (SLE), which makes B-cell-depletion a potential strategy for SLE treatment. The clinical success of anti-CD19 chimeric antigen receptor (CAR) T cells in treating B-cell malignancies has attracted the attention of researchers. In this study, we aimed to investigate the feasibility of applying anti-CD19 CAR-T cell therapy into SLE treatment in a mouse disease model. We have constructed murine anti-CD19 CARs with either CD28 or 4-1BB as the intracellular costimulatory motif and evaluated the therapeutic functions of corresponding CAR-T cells by infusing them into MRL-lpr mice. We found that adoptive transfer of anti-CD19 CAR-T cells was able to achieve sustained B-cell-depletion in MRL-lpr mice. More importantly, transferring syngeneic anti-CD19 CAR-T cells can not only prevent disease pathogenesis before the onset of disease symptoms but also have substantial therapeutic benefits in disease treatment at a later stage after disease progression. Comparing to CAR-T cells with CD28 as a costimulatory motif, CAR-T cells with 4-1BB costimulatory motif work better for mouse SLE treatment. In summary, our study claimed that anti-CD19 CAR-T cell therapy worked effectively in treating murine SLE, indicating its potential in treating human patients in clinic.
BAFF-R: A new target for depletion of pathogenic B cells in experimental lupus
Authors: Neeli Indira , Jha Kumar Abhiram , Moore Charles Edward , Marion Tony , Radic Marko
Published: January 2023

Lupus is an autoimmune disease in which autoantibodies with distinct specificities may arise. However, which autoantibodies contribute to specific manifestations in this multi-system disorder is unknown. We recently demonstrated that anti-CD19 CAR T cell therapy effectively depletes CD19+ B-cells and reduces anti-DNA antibodies in autoimmune mice, resulting in improved health and increased life span. Here, we analyzed autoantibody specificities in MRL/lpr mice both before and after 4 months of anti-CD19 CAR T cell treatment. Although antibodies to cardiolipin and histones decreased in parallel with anti-dsDNA in CAR T cell treated mice, autoantibodies to a ribonucleoprotein (RNP) autoantigen with a speckled nuclear distribution remained present. This result argues that anti-RNP IgG are unlikely to contribute to nephritis and skin inflammation, which markedly improved in the anti-CD19 CAR T cell treated mice.

The anti-CD19 CAR T cell treated mice were also used to identify potential B cell surface receptors that could serve as CAR T cell targets on kidney-infiltrating B cells. Kidney RNA transcripts, as measured by RT-PCR, showed only minor changes for CD11c, CXCR4, and BCMA between CAR T-treated and control mice, whereas the expression of BAFF-R decreased proportionally with CD19 in treated mice. Thus, the construction and testing of anti-BAFF-R CARs in mice may provide a useful step toward a potential new synergistic therapy for difficult to treat lupus in patients.

Research was supported by the Lupus Research Alliance and the UTHSC Maturation Award.
Chloroquine modulates inflammatory autoimmune responses through Nurr1 in autoimmune diseases
Authors: Park Tae-Yoon , Kim Kwang-Soo
Published: January 2023

For over a half-century the anti-malarial drug chloroquine (CQ) has been used as a therapeutic agent, alone or in combination, to treat autoimmune diseases. However, neither the underlying mechanism(s) of action nor their molecular target(s) are well defined. The orphan nuclear receptor Nurr1 (also known as NR4A2) is an essential transcription factor affecting the development and maintenance of midbrain dopaminergic neurons. In this study, using in vitro T cell differentiation models, we demonstrate that CQ activates TREG cell differentiation and induces Foxp3 gene expression in a Nurr1-dependent manner. Remarkably, CQ appears to induce Nurr1 function by two distinct mechanisms: firstly, by direct binding to Nurr1’s ligand-binding domain and promoting its transcriptional activity and secondly by upregulation of Nurr1 expression through the CREB signaling pathway. In contrast, CQ suppressed gene expression and differentiation of pathogenic TH17 cells. Importantly, using a valid animal model of inflammatory bowel disease (IBD), we demonstrated that CQ promotes Foxp3 expression and differentiation of TREG cells in a Nurr1-dependent manner, leading to significant improvement of IBD-related symptoms. Taken together, these data suggest that CQ ameliorates autoimmune diseases via regulating Nurr1 function/expression and that Nurr1 is a promising target for developing effective therapeutics of human inflammatory autoimmune diseases.
CRS free T cell therapy: T cell membrane anchored and tumor targeted IL12 (ATTIL12)-T cell therapy eliminates large tumors, overcomes heterogeneity, and avoids adverse effects
Authors: Hu Jiemiao , Zhang Wendong , Xia Xueqing , Yang Qing , Chen Yuhui , Fowlkes Natalie W , Livingston John Andrew , Zhang Zhongting , Mahadeo Kris M , Gill Jonathan B , Dotti Gianpietro , Kopetz Scott , Gorlick Richard , Li Shulin
Published: January 2023

Adoptive transfer of genetic engineered T cells becomes an effective approach in treating hematologic malignancies, but shows poor responses against solid tumors due to multifactorial reasons. Interleukin-12 exhibits robust T cell activating and anti-tumor efficacy. However, its clinical application was impeded due to the lethal adverse effects. We demonstrated previously that tumor targeted IL-12 (ttIL12) gene therapy avoids the toxicity of wildtype IL12 and effectively eliminate tumor metastasis. In this study, we developed a second generation of ttIL12— membrane-anchored ttIL-12 (attIL12)—to modify naive T cells, CD19CAR-T cells and OT1 T cells. This attIL12-modified T cells silent the toxicity of both IL12 and CAR-T cells associated cytokine release syndrome (CRS). Surprisingly, this ATTIL12-T cell therapy is able to eliminate multiple types of tumors with either homo- or hetero-generous environment or large size. We are in full speed to prepare a phase I clinical trial.
Engineering Chimeric Antigen Receptor T cells Against Immune Checkpoint Inhibitors PD-1/PD-L1 for Treating Pancreatic Cancer
Authors: Liu Chao Lien , Fan Ming Huei , Miao Carol H. , Liao Yi Jen
Published: January 2023

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a 5-year survival rate of 6%. Major obstacles to successful treatment of pancreatic cancer are the immunosuppressive tumor microenvironment (TME) and antigenic complexity or heterogeneity. Programmed death-ligand 1 (PD-L1) is expressed on PDAC and immunosuppressed cells within the TME, providing suitable immunotherapy targets. We applied a chimeric antigen receptor (CAR) strategy to target immune checkpoint PD-1/PD-L1 interactions. Lentiviral vectors were used to express the extracellular domain of human PD-1 (PD1ACR) or the scFv region of aPD-L1 (PDL1CAR) that bind to PD-L1, and each was fused to intracellular signaling domains containing CD3 zeta, CD28, and 4-1BB (CD137). Both engineered CAR T cells recognized and eliminated PD-L1-overexpressing CFPAC1 cells efficiently at approximately 80% in vitro. Adoptive transfer of both CAR T cells enhanced T-cell persistence and induced specific regression of established CFPAC1 cancer >80% in a xenograft model. Ki67 expression in tumors decreased, whereas proinflammatory cytokines/chemokines increased in CAR T-cell-treated mouse sera. PD1ACR and PDL1CAR obtained a similar therapeutic efficacy. Thus, these armed third-generation PD-L1-targeted CAR T cells confer antitumor activity and ability to combat T-cell exhaustion, providing a potentially new and innovative CAR T cell immunotherapy against pancreatic cancers.
Modular Antigen-Specific T Cell Suppression Assay for In Vitro Drug Screening
Authors: Kleiman Eden , Sierra Gloria , Daftarian Pirouz
Published: January 2023

T cell exhaustion (TEX) describes a T cell differentiation continuum of hierarchical progressive diminution of effector function. Many factors have been attributed to this including expression of multiple co-inhibitory receptors, altered transcription factor expression, epigenetic rewiring and dysregulated metabolism. Antigen persistence is necessary for driving TEX maintenance in both chronic viral infection and cancer settings. In addition to persistent antigen exposure, tumor-infiltrating lymphocytes (TILs) within the tumor microenvironment (TME) encounter immunosuppressive metabolic byproducts, suppressive cytokines, hypoxia and cellular debris which converge to suppress T cell function. These suppressed T cells are incapable of mounting an optimal anti-tumor response due to dampened proliferation, metabolic fitness and/or cytolytic capability. Our team has previously deployed usage of the recall antigen assay to screen immune checkpoint inhibitor (ICI) drug candidates. We have shown that in some donor PBMCs, ICI drugs such as pembrolizumab are able to boost antigen-specific T cell recall. We adapted this antigen recall assay to study TME-mediated T cell suppression. We show application of this TME-based recall assay by addition of adenosine which suppresses antigen-specific T cell expansion without altering T cell viability. Interestingly, T cell suppression was partially reversed with addition of CMV lysate. This assay is modular in that TME metabolites can be used alone or in combination, with the potential to screen for donors who are in vitro “responders” and “non-responders” to various drug classes.
The Force Awakens: Illuminating the Role of Kynurenine in Cancer Progression and Treatment
Authors: Ashoura Norah E , Dekker Joseph , Triplett Todd A , Garrison Kendra , Blazeck John , Karamitros Christos , Lamb Candice , Tanno Yuri , Ehrlich Lauren Ilyse Richie , Zhang Michelle , Manfredi Mark G. , Stone Everett , Georgiou George
Published: January 2023

Cancer is the second leading cause of death in the United States, with an estimated 40% of all Americans expected to be diagnosed with cancer in their lifetime. Despite progress in treatment options, major obstacles in current therapies must be overcome to limit their harmful side effects on patients. To evade immune clearance, many cancers elevate tryptophan (Trp) catabolism in the tumor microenvironment (TME) by upregulating the enzyme indoleamine 2,3-dioxygenase (IDO). As a result, cancer cells (1) monopolize extracellular tryptophan and (2) release L-kynurenine into the TME, an independent immune signal. This change in metabolism generates immune suppression in the TME, but whether the cause arises from Trp depletion or the accumulation of the IDO product kynurenine (L-Kyn) remains highly controversial. Kynurenine is known to induce immunosuppressive phenotypes through aryl hydrocarbon receptor (AhR) activation. However, the functional role of this binding is still poorly understood, despite an explosion of interest and information in the field. Our strategy uses the enzyme kynureninase (KynU) to degrade kynurenine into non-toxic products, thereby restoring anti-tumor immunity without harming the tryptophan metabolism of healthy cells. Moreover, this work aims to determine kynurenine’s molecular effect on T cells to better understand why its depletion successfully relieves tumor burden.
Antifungals improve anti-PD1 efficacy in a murine breast cancer model
Authors: Kershaw Kathleen , Henson Regina M. , Abrajan Brandon , Choi Rachel Y. , Underhill David M. , Shiao Stephen L.
Published: January 2023

Every year over 200,000 North American women are diagnosed with breast cancer, which is the second leading cause of cancer death among women. The introduction of effective immunotherapies has revolutionized patient care in many cancers, including breast, but only a subset of patients exhibit clinical response. Factors determining a patient’s response to immunotherapy is an area of intense research. The microbiome is one potential contributing factor. The human intestinal microbiome is comprised largely of bacteria, but also harbors fungi, viruses and archaea that undoubtedly have significant biological functions. Recent reports have detailed the association of specific strains of bacteria with positive response to PD-1/PDL1 therapy in several cancers however, little is known regarding the involvement of commensal fungi. Previous work in our lab found a high diversity of fungi associated with the human gut, which interact with the immune system and influence the severity of gastrointestinal inflammation and allergic airway disease. Using a syngeneic murine model of triple negative breast cancer, we found that antifungal treatment sensitizes previously resistant tumors to anti-PD1 therapy, leading to a significant decrease in growth rate as well as increased survival compared to anti-PD-1 treatment alone. Antifungal treatment altered the microbiome composition as evidenced by 16s and ITS1 sequencing of fecal pellets, as well as altered the tumor immune compartment to promote anti-tumor activity when combined with anti-PD1. These data suggest fungi have a role in shaping the immune-tumor microenvironment and provide insight into how perturbation of the microbiome might improve response to immunotherapy.
Determining the mechanism of the immune response to histotripsy ablation of pancreatic cancer
Authors: Hendricks Alissa Danielle , Brock Rebecca Michaela , Gannon Jessica , Zeher Allison , Sereno Jacqueline , Vlaisavljevich Eli , Allen Irving C
Published: January 2023

Histotripsy is a novel, non-thermal, image guided ablation modality that can rapidly kill cells in a targeted region with millimeter precision. Previous work established that there is an immune response to histotripsy ablation, however a mechanism behind this response has not been established. For the current study, we utilized an in vitro model, Pan02 (pancreatic adenocarcinoma), transfected with a known antigen (hemaglutanin/HA), and verified findings in vivo. Cells were ablated at therapeutic and sub-therapeutic doses to determine the release of damage associated molecular pattern (DAMP). For both treatments, gel electrophoresis confirmed the release of damaged DNA, and western blots verified the release of intact HMGB1 and HA proteins. To determine the efficacy of the released DNA to stimulate an immune response, we exposed naïve macrophages to isolated DNA from the treatment supernatant and showed activation by 24 hours. Additionally, to show the potential for a systemic immune response, complete supernatant was given to naïve dendritic cells, that were in turn co-cultured with naïve T cells. CD4+ and CD8+ T cell activation was confirmed with IL-2 and INFg ELISAs as well as flow cytometry. To verify these findings, mice with Pan02 tumors were treated, and demonstrated similar DAMP and antigen release. Further, changes in immune cell populations within the tumor were consistent, with increased macrophages and dendritic cells 24 hours and 7 days and in T cell populations at 7 and 14 days after treatment. These results show the role of specific DAMPs and immune cell populations in responding to histotripsy ablation. Overall, these results indicate a robust immunomodulatory response to histotripsy tumor ablation.
NKG2A inhibits the anti-tumor CD8 T cell immune response elicited by radiotherapy
Authors: Battaglia Nicholas Griffin , Caldon Johnathan J. , Okoshi Ethan N. , Gerber Scott A. , Lord Edith M.
Published: January 2023

More than half of patients with solid tumor malignancies undergo treatment with radiotherapy (RT). In addition to causing direct tumor cell death, RT results in release of tumor antigen and damage associated molecular patterns that elicit a CD8 T cell and IFN-g dependent anti-tumor immune response. To further bolster this response, RT has been combined with immune checkpoint inhibitors such as anti-PD-1 and anti-CTLA-4. Despite a modest increase in survival when these therapies are used alone or together in conjunction with RT, many patients fail to respond to treatment, suggesting other mechanisms of immune suppression exist in the tumor microenvironment (TME). Using syngeneic B16 F10 melanoma and C38 colorectal adenocarcinoma models, we have studied the role of the inhibitory receptor NKG2A. We have observed that NKG2A is expressed only on tumor infiltrating lymphocytes (TILs) and expression of this receptor does not change with RT. Furthermore, RT increases intratumoral expression of the ligand for NKG2A, Qa-1b, as does IFN-g stimulation in vitro. Blockade of NKG2A alone through use of B16 F10 cells lacking Qa-1b expression or blocking antibodies did not significantly increase survival of mice treated with RT. Further analysis revealed that among CD8 TILs, only a minority of cells express NKG2A alone, with most TILs co-expressing NKG2A and PD-1 or PD-1 alone. Thus, we combined anti-NKG2A and anti-PD-1 blockade and observed increased survival in mice treated with RT, whereas either therapy alone was ineffective. These results suggest that NKG2A blockade could be combined with RT and existing immunotherapies clinically to improve patient response. Supported by awards T32AI007285 from the NIAID and R01CA028332 from the NCI.
Tumor cell-intrinsic PD-L1 signals promote DNA damage responses that mediate resistance to Chk1 and PARP inhibitors in vivo
Authors: Kornepati Anand V , Zhang Deyi , Padron Alvaro S , Boyd Jacob T , Deng Yilun , Osta Erica G , Reyes Ryan M , Shen Hailian , Wang Juan , Kari Suresh , Clark Curtis A , Hu Yanfen , Li Rong , Gupta Harshita B , Zhao Weixing , Curiel Tyler J
Published: January 2023

Tumor PD-L1 mediates diverse cell-intrinsic signals that increase cancer pathogenesis including the DNA damage response (DDR). Although immune consequences of tumor-extrinsic PD-L1 blockade are known, novel therapeutic vulnerabilities from inhibiting tumor-intrinsic PD-L1 signals are little studied. To assess tumor-intrinsic PD-L1 DDR control, we depleted PD-L1 by CRISPR/Cas9 or shRNA (PD-L1KO) in mouse B16 (melanoma), 4T1 (breast), ID8agg (ovarian), and human bladder RT4 cancer lines. We identified specific DDR signaling defects in PD-L1KO versus control (con) cells after exposure to distinct DNA damaging agents. 24h gemcitabine (or 2 Gy X-ray) in vitro in PD-L1KO vs. con RT4 cells elevated γH2AX (DNA damage marker) and reduced Chk2 DDR gene expression but not ATM, ATR, Chk1, or BRCA1 (immunoblots), and decreased BRCA1/p-RPA32 nuclear foci formation (confocal) altogether suggesting tumor-intrinsic PD-L1 promotes specific DDR/DNA repair pathways. PD-L1 loss could thus predict synthetic lethality to DDR inhibitors (DDRi) Chk1i or PARPi. PD-L1KORT4, ID8, and 4T1 cells were more sensitive to small molecule Chk1i in vitro (>15 fold vs. con, MTT) and in vivo in NSG mice. Chk1i and PARPi led to γH2AX increase in PD-L1KO B16, RT4, and 4T1 vs. sham treated tumor cells as predicted by DDR defects. Genetic cellular PD-L1 depletion, but not anti-PD-L1 (extrinsic), strongly sensitized B16 and 4T1 tumors to PARPi in vivo. Strikingly, PARPi had no effect on PD-L1KO B16 in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi treatment efficacy. Thus, DDRi plus inhibiting tumor intrinsic PD-L1 signals could improve immunotherapy in immunotherapy-sensitive or resistant tumors.
Targeting GARP-TGFβ axis enhances anti-tumor efficacy of immune checkpoint blockade therapy
Authors: Li Anqi , Wu Bill X. , Riesenberg Brian P. , Li Zihai
Published: January 2023

Although immune checkpoint blockade (ICB) has emerged as a promising cancer therapy, a majority of cancers, such as pancreatic cancer and triple negative breast cancer, fail to respond to ICB. A potential mechanism is the accumulation of transforming growth factor-β1 (TGFβ1) in tumor microenvironment (TME) which drives immune dysfunction by inducing regulatory T cells (Treg), excluding and inhibiting the function of effector CD8+ T cells. Glycoprotein-A repetitions predominant protein (GARP) is a cell surface docking and activating receptor for latent TGFβ1 and highly expressed in breast cancer cells, Tregs and platelets. Previous work from our lab has demonstrated a critical role for GARP in dampening anti-tumor immunity via TGFβ1 pathway. To validate if GARP is a therapeutic target, we generated an anti-human GARP antibody (4D3). We found that 4D3 blocked GARP-latent TGFβ1 interaction and limited active TGFβ1 release. We expressed human GARP in murine 4T1 breast cancer cells. In this orthotopic syngeneic mouse tumor model, combination therapy with 4D3 and anti-PD1 antibody suppressed tumor growth and metastasis more effectively than treatment with either agent alone, resulting in significantly enhanced survival. Mechanistically, the combination therapy reduced p-SMAD3 expression as well as collagen deposition in the TME. In addition, 4D3 treatment reduced tumor-associated macrophages and their PD-L1 expression. Collectively, our preclinical data demonstrated that simultaneous blockade of the PD1 and GARP-TGFβ1 axis elicits potent and superior anti-tumor efficacy relative to monotherapies, suggesting a potentially more effective strategy against cancers that are resistant to current immune checkpoint blockade.
Characterization of the immune microenvironment in melanoma patients resistant to targeted and immunotherapies by a multidisciplinary approach
Authors: Veigas Florencia , Mahmoud Yamil D , Flores Ana , Morales Rosa , Gatto Sabrina , Saez Juan Manuel Perez , Fernandez Elmer , Rabinovich Gabriel A , Girotti María Romina
Published: January 2023

Most melanoma patients who develop resistance to targeted therapies do not respond to subsequent immunotherapy. Therefore, establishing which patients would derive clinical benefit from immunotherapy is a compelling clinical question. We performed a meta-analysis of published transcriptome datasets from melanoma biopsies before and after development of resistance to BRAFi or BRAFi/MEKi. We observed an increase in M2-macrophages infiltrate by CIBERSORT in relapsed biopsies as well as an increase in T-cell exhaustion markers and immunosuppressive cytokines. Our preliminary findings show changes in the galectin/glycan axis in melanoma resistance to BRAFi/MEKi. Particularly, we observed that the expression of Galectin-1 (Gal-1) is increased in human melanoma resistant cells. We analyzed single-cell and bulk RNA-Seq gene expression profiles from melanoma biopsies before and during treatment with anti-PD-1/anti-CTLA-4 and found that macrophages and CD8 T-cells of non-responders upregulate a specific glycosylation-related signature. Macrophages from non-responding patients show an immunosuppressive expression profile accompanied by the appearance of a novel sub-group during treatment associated with resistance. We hypothesize that there are Gal-1/glycan interactions that promote an M2 immunosuppressive microenvironment in resistant tumors preventing response to immunotherapy. Finally, we developed MIXTURE, a novel algorithm to infer the tumor immune infiltrate that overcomes CIBERSORT limitations. MIXTURE analysis revealed a differential immunosuppressive infiltrate in melanoma biopsies with high intratumoral heterogeneity and in biopsies from non-responding patients to anti PD-1 immunotherapy.
Molecular iodine activates cytotoxic immune response in breast cancer tumor microenvironment
Authors: CUENCA OLGA , GONZÁLEZ EVANGELINA DELGADO , VELASCO CARMEN ACEVES
Published: January 2023

Present work analyzes the tumoral immune response of oral molecular iodine (I2) supplementation with the conventional treatment (5-fluorouracil/epirubicin/ cyclophosphamide or taxotere/epirubicin; FEC/TE) in patients with breast cancer from Hospital General of Queretaro, Mexico. Patients diagnosed with stage II were given placebo (vegetable colored water) or I2 from diagnosis to mastectomy (7 to 40 days) while patients with stage III received placebo or I2 with neoadjuvant FEC/TE treatment (40–70 days). Transcriptomic analysis and GESA studies showed that I2 supplementation alone or in presence of FEC/TE up-regulate Th1 and Th17 differentiation pathways, activation by T-cell receptor, cytotoxicity by NK and CD8+, lymphocyte migration and formation of tertiary lymphoid structures. Deconvolution analysis indicate an increase in macrophages/dendritic cells in I2 group while in FEC/TE+I2 group exhibited increased B cells fractions. Analysis of Th1/Th2 response markers showed significant increase expression in Tbx-21 (RT-PCR) and Interferon gamma (immunohistochemistry) in the FEC/TE+I2 group. This anti-tumor immune activation correlated with the longer disease-free period (43%) and 5 years-survival (60%) observed in FEC/TE+I2 patients. Studies to analyze the effect of I2 supplementation in immunotherapies are in course.
Influence of DNA damage repair defects on tumor immunogenicity
Authors: Samstein Robert
Published: January 2023

In recent years, immune checkpoint inhibitors (ICI) have dramatically altered the treatment landscape for patients with advanced stage and metastatic cancers. Durable benefit, however, is limited to subsets of histologies and patients, highlighting the importance of identifying predictive biomarkers to select for patients who may benefit. DNA damage repair and response (DDR) deficiency including mismatch repair deficiency and resulting increased tumor neoantigens have been associated with improved response. We hypothesize that a similar concept may extend to other defects in DDR pathways, the most common being homologous recombination (HR). In ongoing work, we have implicated mutations in the DDR pathways including the HR pathway with improved survival after ICI in a large retrospective cohort. However, divergent responses were noted depending on the individual HR gene mutated. Syngeneic murine models were used to study individual DDR genes using CRISPR-Cas9 mutagenesis and their role in altering the tumor microenvironment and response to immune checkpoint blockade in isogenic model systems. We report differential effects of mutations in genes of the homologous recombination (HR) pathway on response after ICI administration in mouse and human tumors, and show that truncating mutations in BRCA2 are associated with superior response to ICI compared to BRCA1-deficient tumors. Single cell RNA-sequencing of CD45+ cells sorted from tumors reveals distinct immune landscapes. These data have significant implications for elucidating the genetic and microenvironmental determinants of response to immunotherapy in the setting of DDR deficiency and ultimately expand the therapeutic window of immunotherapy.
Metastatic colorectal cancer cells harness nonsense-mediated mRNA decay for immune evasion
Authors: Wu Xingxin , Tan Tao , Xu Qiang
Published: January 2023

Immunosuppression microenvironment allows primary tumor growth, while tumor-inherent factors that bring about immune evasion during metastasis remain elusive. Here, we observed a stronger nonsense-mediated mRNA decay (NMD) activity with a higher expression of up-frameshift protein 1 (UPF1) in colorectal cancer (CRC) metastasis than in matched primary cancer cells. In metastatic CRC SW620 cells, the higher UPF1 expression was found to arise from the increased stability of UPF1 by ubiquitin specific peptidase 10 (USP10)-mediated deubiquitination. In contrast, in primary CRC SW480 cells, autophagy promotes proteolysis of ubiquitinated UPF1 and inhibits NMD activity. Interestingly, in SW620 cells NMD selectively controlled a cluster of immune-related genes including ICAM1. Furthermore, NMD inhibition exposed SW620 cells but not SW480 cells to natural killer (NK) cell-mediated cytolysis that depended on ICAM1.

Conclusion

NMD activity can be recommissioned in CRC metastasis cells to escape from NK-dependent immune surveillance, and inhibiting NMD to boost anti-tumor immunity could be a novel strategy to treat CRC metastasis.
Isolation of Tumor-Infiltrating Leukocytes from Mouse Tumors
Authors: Poon Grace FT , Antignano Frann , Boyd Lyz , Ennis Siobhan , Deng Joe , Kokaji Andy I , Woodside Steven M , Eaves Allen C , Louis Sharon A
Published: January 2023

Cell-based immunotherapy is being evaluated in various types of cancer and it is one of the most rapidly growing and promising areas of cancer research. Tumor-infiltrating leukocytes (TILs) consist of highly diverse leukocyte subsets with major roles in cancer immune surveillance. Due to their relatively low frequency, tumor heterogeneity, and the abundance of tissue debris in tumor samples, it is difficult to isolate or analyze TILs with sensitivity and precision. To address this challenge, we have developed a simple method for isolating CD45+ TILs from mouse tumors. Performance was evaluated in three commonly used mouse models, namely the B16 melanoma, CT26 colon carcinoma, and 4T1 mammary tumor models. Solid tumors were induced by subcutaneous implantation of B16, CT26.WT, and 4T1 cancer cell lines into syngeneic recipients. Starting with a single-cell suspension, TILs from tumor samples were labeled with an antibody complex that links CD45+ cells to magnetic particles, then separated using an EasySep™ magnet. Using this method, TILs were enriched from 17.5 +/− 5.8% to 90.1 +/− 6.0% (n = 13) from B16 tumors, 27.9 +/− 9.4% to 74.2 +/− 12.3 % (n = 9) from CT26 tumors, and 39.5 +/− 8.0% to 87.5 +/− 3.7% (n = 6) from 4T1 tumors. The protocol can be easily modified to achieve higher purity or recovery as required by adjusting the addition volume of the antibody complex or particles. Importantly, major immune subsets including T cells, B cells, and myeloid cells are recovered after isolation. The EasySep™ Mouse CD45 TIL Isolation Kit allows researchers to isolate leukocytes from tumors with ease, improving the TIL downstream workflow. Furthering our understanding of TILs will be essential for developing effective immunotherapeutic strategies.
Modulating the cross-talk between the immune and nervous systems to treat rectal cancer
Authors: Uccello Taylor Paige , Mills Bradley N. , Repasky Elizabeth A. , Lord Edith M. , Linehan David C. , Gerber Scott A.
Published: January 2023

Cancer patients with perineural invasion (PNI) have reduced overall survival, enhanced rates of local recurrence, and increased metastasis. Nerves secrete catecholamines (CA) that bind beta 2 adrenergic receptors (B2AR) on various cells, including immune cells. Signaling through this neuronal-immune axis drives immune cells towards a suppressive phenotype. Therefore, the sympathetic peripheral nervous system is considered to be a source of immunosuppression in the tumor microenvironment (TME). The impact that various therapies, such as radiation therapy (RT), have on innervation and CA secretion is unknown. We hypothesize that proinflammatory cytokines induced by RT signal nerves to secrete CAs that propagate tumor growth. We have shown via qPCR and flow cytometry that tumor cell lines exposed to RT in vitro upregulate B2AR transcription and protein level, respectively. Consequently, tumor lines exposed to B2AR agonists are more radioresistant by colony forming assays. To further explore the role of the nervous system in enhancing tumor growth, we have developed an orthotopic model of rectal cancer. Using this model, we visualized PNI by immunohistochemistry. We also detected positive B2AR expression on immune, tumor, and stromal cells excised from the TME. Upon treating mice bearing orthotopic tumors with a B2AR agonist, we observed increased metastatic burden, accompanied by reduced overall survival compared to untreated controls. Moving forward, we will inhibit immunosuppressive neuronal signaling in the TME in combination with RT. Our goal is to elucidate the immunosuppressive role of the nervous system in rectal cancer and use this information to identify a novel RT adjuvant that potentiates a robust antitumor response.
The changes of immunologic properties and their prognostic implications: the paired analysis of initial and recurrent head and neck squamous-cell carcinoma
Authors: So Yoon Kyoung , Cheong Taek Yoon , Byeon Sun-ju , Chung Man Ki
Published: January 2023

Purpose

The immunologic properties of tumors can change during the clinical course. We aimed to compare the expression of PD-L1 and the infiltration of CD8+ tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment between initial and recurrent head and neck squamous-cell carcinomas (HNSCCs). We also evaluated whether the changes of those immunologic properties in recurrent HNSCCs affect the oncologic outcome.

Methods

We included 42 patients who had been treated for both initial and recurrent HNSCCs. Pathologic specimens from initial and salvage surgery were obtained for each patient, and IHC staining of CD3, CD8, PD-1, and PD-L1 were done. Also, neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were collected. The change of each immunologic profile was expressed as the recurrent-to-initial ratio (R/I ratio).

Results

The change of CD3+, CD8+, PD-1, and PD-L1 positive cells varied widely. For more than half the patients, those values decreased in recurrent tumors. The median R/I ratio was 0.5 for CD3+ TILs, 0.6 for CD8+ TILs, 0.5 for PD-1 and 0.4 for PD-L1. In contrast, NLR and PLR increased in recurrent tumors for more than half the patients. The median R/I ratio of NLR and PLR was 1.6 and 1.4, respectively. In multivariate analysis, increased CD8 (R/I ratio >1) was the independent prognostic factor for better OS (hazard ratio 0.228; 95% CI 0.067 – 0.777; p= 0.018).

Conclusion

The change of immunologic properties along with the recurrence of HNSCC varied widely from patient to patient. Generally, the intratumoral biomarkers decreased, while the systemic inflammatory markers increased. The increased CD8+ TILs in recurrent HNSCCs was the significant prognostic factor for better overall survival.
The role of osteosarcoma cell-derived exosomes in the promotion of lung metastasis
Authors: Mathias Alissa B , Palmer Eric P , Kruh-Garcia Nicole , Regan Daniel P
Published: January 2023

Osteosarcoma (OS) is the most common primary tumor of bone and typically occurs in children and young adults. 30–40% of OS patients develop lung metastases after initial diagnosis, with only 20% of these patients surviving 4 years post-relapse. For over 30 years, there have been no improvements in the treatment of OS, and more importantly, no diagnostic tools that help identify those patients at high risk for lung metastases. Recent data has shown that in other tumor types, tumor-secreted extracellular vesicles (EVs) deliver biological cargo to distant sites, resulting in priming of non-malignant host cells for promotion of metastasis. The aim of this project is to understand how OS EVs influence the response of lung fibroblasts (LFs) and alveolar macrophages (AMs), and if their uptake of EVs results in pre-metastatic priming that can be detected via AM transcriptomic changes.

Hypothesis

LFs are primary targets of OS EVs, which will increase LF IL-6 production and cause paracrine STAT3 activation of AM’s into an M2 tumor-promoting phenotype.

Results

Flow cytometry and confocal microscopy demonstrated in vitro uptake of OS EVs by LF cells. In vitro ‘education’ of LFs with OS EVs induced significant production of IL-6, IL-8, and CCL2, with the greatest mean difference between EV-educated and naïve LFs observed with IL-6 secretion.

Conclusion

This data suggest that OS EVs efficiently prime LFs to induce IL-6 secretion, and this response may be associated with promotion of OS lung metastasis. Further data is being collected to determine the role of IL-6 in OS cell survival and lung metastatic colonization. RNA sequencing of AMs will be used to characterize phenotype which could serve as an effective cellular biomarker to predict lung metastasis.
Characterization of the genomic and immunological diversity of malignant human brain tumors through multi-sector analysis
Authors: Schaettler Max , Richters Megan , Griffith Malachi , Dunn Gavin
Published: January 2023

While the degree of spatial genomic heterogeneity within primary glioblastoma (GBM) has been previously investigated, the extent of this heterogeneity in other malignant brain tumors and its connection to the local immune microenvironment remains unknown. To address this, we performed whole-exome, RNA, and TCR sequencing on multiple spatially distinct sectors from a cohort of malignant brain tumors comprised of both brain metastases and primary and recurrent GBMs. Our results suggest a striking difference in which a majority of mutations and predicted neoantigens are shared between spatially distinct regions of metastatic brain tumors in contrast to the spatial heterogeneity observed within both primary and recurrent GBMs. Additionally, despite substantial differences in immunotherapy responsiveness between brain metastases and GBM, we can detect significantly expanded T-cell clonotypes within both tumor types with some clonal frequencies exceeding 10% of intratumoral T-cells. Similar to the observed distribution of variants and neoantigens, the expanded clonotypes are more shared among spatially distinct sectors in metastatic brain tumors than in primary or recurrent GBMs. Interestingly, the frequencies of most of these enriched T-cell clones are significantly diminished within expanded tumor infiltrating lymphocyte (TIL) cultures, advising caution in the use of expanded TIL for the detection of neoantigen-specific responses. These results provide novel insight into the immunogenomic landscape of malignant brain tumors with implications for our understanding of tumor-immune interactions and the development of immunotherapies.