bluuebunny/crossref_metadata_2025 huggingface dataset:
CD126 negative regulatory cells represents a superior Treg subset in treating autoimmune diseases
Authors: Zheng Song Guo , Chen Ye , Jarjour Wael , Wang Julie
Published: January 2023

Treg cells play an important role in maintaining immunologic homeostasis. Abnormal Treg were found in some autoimmune diseases. Infusion of natural Treg (nTreg) prevents some autoimmune disease but usually fails to treat established diseases. Several studies demonstrated nTreg could be transdifferentiated into Th17 cells and lost its function under inflammatory condition. Given IL-6 is an important inflammatory cytokine and it binds to its receptor (CD126) to exert functional role, whereas nTreg express CD126, we hypothesize that CD126− Foxp3+ cell subsets could be more stable and functional in the inflammatory condition.

CD126+ and CD126− nTreg were sorted from collagen-induced arthritis (CIA) and then were analyzed for transcript difference via RNA-sequence. They were cultured in the presence of IL-6 to determine the stability and function following a colitis model in vivo.

Compared to normal mice, the proportion of CD126+ nTreg in CIA was significantly higher. CD126− nTreg express higher immunosuppressive molecules and present stronger function than CD126+ nTreg in both normal condition and presence of IL-6. Moreover, CD126− nTreg was stable while CD126+ nTreg lost Foxp3 expression and trans-differentiated into Th17 in the presence of IL-6. In addition, CD126− nTreg exhibited reduced Hif-1a and glucose transporter 1(Slc2a1) expression, which are two key regulators of glycolytic metabolism and play an important role in the balance of Th17 and Treg. Finally, CD126− Treg more potent treat colitis compared to control.

We show CD126− nTreg present stronger function and stability under inflammatory conditions Our results suggest that manipulation of CD126− nTreg might be a novel strategy for the treatment of autoimmune diseases.
Antigen-specific suppression of previously activated T cell responses via polymeric GalNAc antigen conjugation
Authors: Tremain Andrew C. , Wilson D. Scott , Hubbell Jeffrey
Published: January 2023

Current treatments for autoimmunity, allergy, and drug hypersensitivity require continuous global immune suppression. Here we demonstrate in vivo therapeutic efficacy of antigen-specific T cell tolerance induction following prior immunization in various models of murine immunity. Our lab has engineered an N-acetylgalactosamine polymer (pGal) that can be conjugated to protein antigen and delivered intravenously to immunosuppressive hepatic antigen presenting cells, thereby inducing lasting immune regulation without off target effects. In this work, we show treatment with pGal-ovalbumin (OVA) conjugates can suppress previously activated T cells in an antigen-specific fashion. We describe the suppressive effects and underlying signatures of tolerogenic T cell reprogramming by comparing treatment with native OVA to pGal-OVA. Therapeutic treatment with pGal-OVA results in reduced T cell expansion and activation after secondary challenge, higher expression levels of suppressive/exhausted markers such as PD1 and TOX, and markedly lower production of cytokines including IFNg, TNFa, and IL-17 following in vitro restimulation with antigen. Further investigation of antigen-specific tolerance induction utilizing pGal-conjugates can lead to potentially curative approaches for controlling aberrant immunity to self or innocuous foreign protein.
Lidocaine improves psoriasis by calming down inflammatory cells
Authors: Wang Honglin , YIN Qianqian
Published: January 2023

Psoriasis is an incurable chronic skin disease. In addition to immune system participation, cutaneous nervous system has long been reported to play a role in psoriasis pathogenesis. However, the clinical application and underlying mechanism of how peripheral sensory nervous system works during psoriasis pathogenesis are largely unknown. Here we report a new and straight-forward clinical strategy using epidural injection of lidocaine to directly manipulate peripheral sensory nervous system, resulting in significant PASI score improvement of psoriasis patients. Imiquimod-induced rat model of psoriasis shows consistent therapeutic effect with epidural injection of lidocaine. Transcriptome analysis of lidocaine-treated rat primary sensory neurons indicates that lidocaine may function through calming down neuronal proinflammatory chemokines (CCL2, CCL7 and CCL21), cytokines (IL-34 and TSLP). Collectively, our results reveal that direct modulation of peripheral sensory nervous system by epidural treatment with lidocaine reveals a novel and promising therapy for psoriasis treatment.
High affinity oligoclonal TCRs define effective adoptive T-cell therapy targeting mutant KRAS-G12D
Authors: Sim Malcolm John Wyness , Lu Jinghua , Spencer Matthew , Hopkins Francis , Tran Eric , Rosenberg Steven A , Long Eric O , Sun Peter Daopin
Published: January 2023

Complete regression of cancer has been observed in a subset of patients following adoptive T-cell transfer (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TIL). However, the low success rate of ACT presents a great challenge to broader clinical application. To provide a rational approach to TIL-based immunotherapy, we developed a three-pronged approach to comprehensively evaluate clinically relevant traits of TIL-TCR. We applied the approach to a successful case of ACT where regression was observed against tumors carrying the hotspot mutation KRAS-G12D. We show that only G12D mutant peptides (a nonamer and a decamer) but not wildtype peptides can be presented by HLA-C*08:02. Structurally, the mutated Asp formed a salt bridge necessary for HLA-C binding. Therapeutic TCRs exhibited high affinity anti-tumor binding both in solution and on reconstituted T-cells. The nonamer G12D is recognized by multiple TCR9 through conserved contacts on their shared CDR2β and CDR3α, allowing CDR3β variation to generate an oligoclonal response. The decamer G12D is recognized by TCR10 through an induced peptide conformation. Interestingly, the antigen binding affinities of these TIL-TCR inversely correlated with their persistence in vivo. Importantly, the successful ACT contained TCRs specific for two non cross-reactive conformationally distinct tumor epitopes. We conclude that successful ACT requires high affinity oligoclonal TCRs with specificity for multiple antigens to optimize tumor clearance and in vivo persistence. This approach has the potential to transform current trial and error aspect of TIL-based immunotherapy to a rational selection of TCRs with optimal therapeutic properties.
Synthetic MyD88 co-stimulation in donor CD8+ T cells enhances T cell activation, function, and tumor control in experimental hematopoietic cell transplant
Authors: Ciavattone Nicholas G , Wu Long , O’Neill Rachel , Davila Eduardo , Cao Xuefang
Published: January 2023

Allogeneic hematopoietic cell transplantation is a potentially curative treatment for blood cancers. Nevertheless, some patients will relapse and succumb to their cancer. Donor lymphocyte infusion of CD8+ T cells with transplant evidently provides further protection from recurrent malignancies. Further engineering the donor CD8+ T cells to respond to the tumor may provide even further protection in patients. Novel co-stimulation through Toll-like Receptors in CD8+ T cells enhances T cell activation and function through the MyD88 adaptor protein. Previously, we developed a synthetic MyD88 linked to CD8 which drives MyD88 signaling with T cell activation. This tool enables assessment of the role of MyD88 in CD8+ T cell function and helps determine the benefit of this pathway in T cell immunotherapies. Our studies show that donor CD8+ T cells transduced with this construct (CD8-MyD88 T cells) increase the graft-versus-tumor response in experimental mouse transplants with A20 lymphoma. Further studies show engineered CD8-MyD88 T cells proliferate better in the host and elicit greater production of functional cytokines and Granzyme B compared to controls. Increased granzyme B production from CD8-MyD88 T cells did not hinder their activation and these cells displayed enhanced survival. Using in vitro cytotoxicity assays we determined that the tumor control is a direct result of T cell cytotoxicity and that this effect is enhanced with host-Antigen Presenting Cells. Increases in GvHD were indicated in host mice, specifically relating to weight loss, however the symptoms occurred with no mortality. Future experiments will explore the specific mechanism of tumor killing and delineate the cause of GvHD symptoms from donor CD8-MyD88 T cells.
Modulating extracellular TCR-CD3 interaction to identify new immunotherapy targets against cancer
Authors: Krogsgaard Michelle , Natarajan Aswin , Velmurugu Yogambigai , Yuan Zhou , Ge Chenghao , Nadarajah Vidushan , Cardozo Timothy , Bracken William C. , Zhu Cheng
Published: January 2023

T cell recognition of antigen and resulting proximal signaling are key steps in the initiation of the adaptive immune response. Previous studies targeting antigen binding site for enhancing T-cell responses to tumor antigens often lead to off-target effects and toxicity. Recently, we used nuclear magnetic resonance (NMR) spectroscopy, mutational analysis and computational docking to derive a 3D structure of the extracellular TCR-CD3 assembly. Further, biomolecular force probe (BFP) measurements allowed us to determine how 2D affinity and force-modulated TCR-pMHC kinetics depend on TCR-CD3 interaction sites and affect transduction of extracellular pMHC-TCR ligation into T cell function. Based on our TCR-CD3 structural model and binding data, we generated TCR libraries for a melanoma-specific TCR (DMF5) using site-specific mutagenesis in the Cbhelix 3 and helix 4-F strand regions of the TCR to optimize the TCR-CD3 interaction and to select for mutants with enhanced T-cell effector function. One Cb helix 4-F strand mutant, NP202203AA showed increased T cell response to antigen and showed enhanced TCR-pMHC bond lifetime (catch-bonds) in BFP assays leading to prolonged T cell signaling. In the future, DMF5 TCR with reengineered CD3 binding regions will be used in tumor rejection in pre-clinical mouse melanoma models for efficacy and toxicity to develop more effective T cell therapies for human targets.
Bispecific anti-CD22 T-cell engager efficiently promotes cell-mediated cytotoxicity of lymphoma, leukemia and sarcoma targets
Authors: Barisone Gustavo A , Vang Daniel P , Murphy William J , Tuscano Joseph
Published: January 2023

Immunotherapy has revolutionized cancer therapy. Two of the most recently FDA-approved immunotherapies for B-cell malignancies target CD19, in the form of a Bispecific T-Cell Engager (BiTE) antibody construct or CAR-T cells. Blinatumomab, and FDA-approved BiTE, binds to CD19 on B cells and to CD3 on T cells, mediating killer-target cell contact and T cell activation that results in effective elimination of B cells. Although CD19 is expressed by essentially all cases of B cell malignancies at clinical presentation, relapses with loss or reduction of CD19 surface expression are increasingly recognized as a cause of treatment failure.

We have developed a novel BiTE consisting of humanized anti-CD22 and anti-CD3 single chain variable fragments, expressed as a single molecule and purified from tissue culture supernatant. Binding of the anti-CD22 and anti-CD3 moieties to B and T cells, respectively, was confirmed by flow cytometry. CD22-BiTE promoted cell-mediated cytotoxicity in a dose and E:T-dependent fashion, with a maximum NHL and ALL-specific killing activity of 90% in vitro. Besides canonical expression in B cells, CD22 has been reported in non-hematologic malignancies. Our analysis of TCGA data suggests CD22 is expressed in a variety of solid tumors, including sarcoma. We verified expression of CD22 message and protein in solid tumor cell lines. Accordingly, our CD22-BiTE mediated killing of HT1080 sarcoma cells in vitro.

In conclusion, we report here the development of a new BiTE with killing activity against CD22+ malignancies which could represent a therapeutic option for B-cell malignancies and potentially solid tumors.
Heteroclitic peptide cancer vaccine counters MHC-I skewing in mutant calreticulin-positive myeloproliferative neoplasms
Authors: Gigoux Mathieu , Zappasodi Roberta , Park Joseph J , Pourpe Stephane , Ghosh Arnab , Bozkus Cansu Cimen , Mangarin Levi M.B. , Redmond David , Verma Svena , Schad Sara , Duke William , Leventhal Matthew , Jan Max , Ho Vincent , Hobbs Gabriella , Knudsen Trine Alma , Skov Vibe , Kjær Lasse , Larsen Thomas Stauffer , Hansen Dennis Lund , Lindsley R Coleman , Hasselbalch Hans , Grauslund Jacob H , Andersen Mads H , Holmström Morten O , Chan Timothy , Rampal Raajit , Abdel-Wahab Omar , Bhardwaj Nina , Wolchok Jedd D , Mullally Ann , Merghoub Taha
Published: January 2023

The majority of JAK2WT myeloproliferative neoplasms (MPN) have disease-initiating frameshift mutations in calreticulin (CALR) resulting in a common novel C-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in CALRMUT MPN patients, but the reasons for this phenomenon are unknown. In this study, we examine class-I major histocompatibility complex (MHC-I) allele frequency in CALRMUT MPN patients from two independent cohorts and observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are under-represented in CALRMUT MPN patients. We speculate that this is due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles. As a result of this MHC-I allele restriction, we reasoned that CALRMUT MPN patients would not efficiently respond to cancer vaccines composed of the CALRMUT fragment, but could do so with a properly modified CALRMUT heteroclitic peptide vaccine approach. We found that heteroclitic CALRMUT peptides designed for CALRMUT MPN patient MHC-I alleles elicited a cross-reactive CD8+ T cell response in human PBMC samples otherwise unable to respond to the matched weakly immunogenic CALRMUT native peptides. We also modeled this effect in mice and observed that C57BL/6J mice, which are unable to mount an immune response to CALRMUT, can mount a cross-reactive CD8+ T cell response against a CALRMUT peptide upon heteroclitic peptide immunization and this was further amplified by combining with anti-PD-1. Together, our data underscore the therapeutic potential of heteroclitic peptide-based cancer vaccines in CALRMUT MPN patients.
Tumor-derived lactate inhibits methylation levels of SGK1 in myeloid-derived suppressor cells to promote GLUT1-mediated glucose uptake and metabolic reprogramming
Authors: Ma Jie , Shen Hua , Xu Huaxi , Wang Shengjun
Published: January 2023

Objective

Myeloid-derived suppressor cells (MDSCs) play a critical role in tumor-associated immunosuppressive function, our previous results have confirmed that MDSCs in the tumor microenvironment have stronger glucose uptake capacity and higher glycolysis activity compared with MDSCs in the spleen of mice lung cancer xenograft model. This may be related to metabolic reprogramming, but little is known about the metabolic reprogramming of MDSCs and its regulatory mechanism in the tumor microenvironment.

Methods

We screened changes in gene expression levels before and after lactate treatment in MDSCs by transcriptome sequencing. The methylation levels of SGK1 were confirmed by BSP sequencing.

Results

we found that lactate, a metabolite found in high concentration within the anaerobic tumor environment, increased the glycolysis activity and immunosuppressive function of mouse spleen MDSCs. After the treatment of lactate, the expression of SGK1 was significantly up-regulated, and the methylation level of SGK1 was greatly reduced. Furthermore, methylation of SGK1 increases expression of GLUT1 and transfers to cell membrane, thereby increasing glucose uptake and glycolytic activity of MDSC.

Conclusions

We believe this is a new mechanism through which tumor cells up-regulate the expression of SGK1, thereby promoting the glucose uptake and metabolic reprogramming of MDSCs by regulating the cell localization of GLUT1. Together, our results highlight the ability of tumor cells to alter the metabolic reprogramming and function of tumor infiltrating MDSCs to optimize the tumor growth microenvironment.
Pathways from glutamine to apoptosis: ROS and PP2A in SKW6.4
Authors: Kim YunKyu , Yang Hun Ji , Yeo Myeong Gu , Lee Kyung Jin
Published: January 2023

Altered glutamine metabolism is emerging target of cancer therapy. Glutamine deprivation has been proposed to induce apoptosis in multiple cancers. Dependency on glutamine metabolism of skw 6.4 lymphoma cell was investigated in vitro and in vivo. Glutamine deprivation induces apoptosis of skw 6.4 cell. Intracellular concentration of metabolites in tricarboxylic acid cycle and glutathione concentration were reduced and concentration of reactive oxygen species (ROS) and expression of PP2A were increased in glutamine-deprived skw6.4 cell. Reduction of ROS by antioxidants and knockdown of PP2A inhibited apoptosis induced by glutamine deprivation. Glutamine antagonist reduces skw6.4 tumor size in mouse xenograft model in PP2A-dependent manner. Overall, these results suggest survival of skw6.4 cells depends on the supply of glutamine in PP2A-dependent manner.
Apigenin modulates immune checkpoint molecules in pancreatic cancer enhancing anti-tumor immunity
Authors: Villalobos-Ayala Krystal , Luongo Jessica , Marsh Ari , Areas Javier , Miller Bradley , Williamson Tanika , Husain Kazim , Ghansah Tomar
Published: January 2023

Pancreatic cancer (PC) remains as a very aggressive malignancy with an extremely poor prognosis and an overall 5-year survival rate of only 8%. Immunotherapies are promising against solid tumors with exception of PC. This poor prognosis is partly due to PC’s inflammatory tumor microenvironment, resulting in an increased expression of both Programmed Death-Ligand (PD-L1) in tumors and Programmed cell death protein 1 (PD-1) on effector CD8+ T cells. Upon PD-L1/PD-1 interaction, CD8+ T cells exhibit anergy, allowing PC evasion and tumor progression. We hypothesize that reducing PC-induced inflammation will increase anti-tumor immune responses and promote tumor regression. Therefore, we examined the expression of checkpoint molecules (PD-L1, PD-L2, PD-1, Major Histocompatibility Complex-I, etc.) in different murine PC cell lines (i.e. Panc02, UN-KPC-960, UN-KC-6141) and modulation by an anti-cancer natural bioflavonoid called apigenin (API). To do this, we treated the PC cell lines, in vitro, with and without API along with interferon-γ (potent inducer of PD-L1) to detect the expression profiles of these checkpoint molecules using flow cytometry. Subsequently, we tested T cell proliferation in response to untreated/treated PC cells. Our results show that API down-regulated PD-L1 expression on PC cell lines, which coincides with increased T cell responses. Our results suggest that API may reduce PC-induced immunosuppression allowing increased anti-tumor immunity. Therefore, clinical application of API as an adjuvant may potentiate the efficacy of current PC immunotherapies.
Humanized Rag2 mice as patient-derived orthotopic mouse models for immune checkpoint blockade and conventional chemotherapeutic drug treatment for human solid tumors
Authors: Li Li , Zhang Xin , Maresh Grace , Mathew Neeha , Bhattarai Amita , Hellmers Linh , Yip Henry , Moret Ravan , Baste Lara McKean , Green Heather , Latsis Maria , Bardot Stephen , Margolin David
Published: January 2023

To evaluate therapeutic efficacy of immune checkpoint blockade (ICB) and conventional chemotherapy in pre-clinical models, we developed humanized patient-derived orthotopic xenograft (PDOX) models for colorectal cancer (CRC) and renal cell carcinoma (RCC).

Rag2 mice were humanized by donor peripheral blood mononuclear cells (PBMC) transfer. Luciferase-tagged CRC and RCC tumor cells were injected intra-rectally and intra renal subcapsular, respectively. Groups of mice (n=5–10) received ICB, anti-PD-1 and anti-PD-L1 antibodies (Nivolumab and Atezolizumab, 200 μg each/mouse) for 4 weeks, and conventional therapy of 5FU (200 mg/kg, for CRC) alone or in combination. Tumor growth was measured by weekly bioluminescent image (BLI) and tumor weight at necropsy. The presence of human immune cells and tumor infiltrating lymphocytes were confirmed by flow cytometry and immunohistochemistry staining.

Humanization was evidenced by >45% HLA-ABC+CD45+ circulating human cells in both CRC and RCC models. CRC tumor weight showed significant reduction in group treated with combination of ICB and 5FU compared with untreated controls or 5FU monotherapy group (p<0.05) using patient tumor cell CoCa302. The lung/liver metastasis detected by ex vivo BLI were reduced from 50% of mice in control group to 33% and 11%, respectively. For RCC, tumor size was reduced by >30% from controls in ICB treated group using patient tumor cell KiCa118 and cell line SN12K1. The later had 20% reduction of liver metastasis.

The combination of ICB and conventional chemotherapies for CRC and RCC can be tested in our humanized PDOX mouse models. The effect of the combination therapy using MHC matched donors or patient autologous PBMC will be further investigated.
Radiation-Induced Immunity in Kidney Cancer Patients: Seq-ing Commonality Between Peripheral and Intratumoral T cell Repertoires
Authors: Chow Jacky Louis , Hoffend Nicholas , Abrams Scott I , Schwaab Thomas , Singh Anurag , Muhitch Jason Bryan
Published: January 2023

Radiation has long been part of the standard of care for cancer treatment. Clinical studies combining immunotherapy with radiation have shown promising response rates implicating radiotherapy as an inducer of tumor immunity. Due to the scarcity of human samples treated in situ with radiation, exploration of this phenomenon has been largely limited to murine models. While these data draw connections between intratumoral immune responses and wider effects in peripheral blood, advances in patient care are restricted by inherent differences between murine models and human disease. Therefore, to identify commonality between local intratumoral and systemic responses to radiation, we analyzed clinical trial samples from renal cell carcinoma (RCC) patients treated with stereotactic body radiation therapy (SBRT) at the primary tumor. With access to patient tumors and peripheral blood, we hypothesized that SBRT initiates local and systemic anti-tumor immunity. Pathway analysis from tumor RNAseq data showed SBRT-specific immune processes: TCR activation, increased costimulation, and chemokine signaling. Deeper examination through single cell RNA sequencing identified the sources of these signals. In parallel, analysis by T cell receptor sequencing (TCRseq) showed increased tumor T cell clonality with SBRT treatment. TCRseq of serial blood samples revealed tumor-enriched T cell clonotypes reaching peak abundance 2 weeks post-SBRT. This expansion was not sustained and contracted towards baseline levels 4 weeks after SBRT. Collectively, these results indicate a critical interval following radiation, wherein tumor-enriched T cell clones are expanded in circulation, that could be utilized to bolster responses to immunotherapy.
Harnessing the power of trained immunity using yeast-derived β-glucan to prevent pancreatic cancer
Authors: Geller Anne E , Ding Chuanlin , Guo Haixun , Yan Jun
Published: January 2023

Despite the remarkable success of immunotherapy, pancreatic cancer has yet to benefit. This creates a desperate need for therapies that target pancreatic cancer and utilize novel approaches to prevent and treat this lethal disease. Innate immune cells are vital in antitumor immunosurveillance. Recent studies have shown that innate immune cells possess a form of memory termed Trained Immunity (TI), where cells exposed to an initial stimulus undergo epigenetic and metabolic reprograming that causes hyper-responsiveness when exposed to a second signal. It is yet unknown whether inducing TI could protect against tumors, and specifically pancreatic tumors. Here we show that yeast-derived particulate β-glucan, a known TI inducer, results in 90% of an intraperitoneal dose trafficking to the pancreas. This leads to a robust, dose-dependent influx of innate and adaptive immune cells into the pancreas that have enhanced Th1 function. Despite the cellular influx, signs of pancreatitis are absent. In vivo and in vitro studies of β-glucan trained pancreatic macrophages show increased TNFα and IL-6 production when exposed to LPS, pancreatic tumor cells and their supernatant. This indicates that pancreatic macrophages can be trained to protect against pancreatic tumor cells. Depletion of various immune populations suggest that pancreatic macrophages are directly trained by β-glucan, which then stimulate NK and T-cells. In an orthotopic model of pancreatic cancer, β-glucan trained mice show reduced tumor burden and augmented antitumor functions of innate and adaptive cells. Together, this suggests that β-glucan can train pancreatic innate immune cells to have antitumor memory, which can serve as a novel approach to treating pancreatic cancer.
Dietary administration of alpha-tocopheryloxyacetic acid induces CD4+ T cell activation and prolongs survival in murine rhabdomyosarcoma
Authors: Capitini Christian M. , Szewc Fernanda , Kabakov Sabrina A , Rinella Sean P , Akporiaye Emmanuel , Redmond William L
Published: January 2023

Relapsed sarcomas remain essentially incurable for most children with cancer. Alpha-Tocopheryloxyacetic acid (αTEA) is a scalable semi-synthetic derivative of alpha-tocopherol that has shown promising activity preclinically and clinically in adult cancer, but has not yet been studied in pediatric sarcoma. We hypothesized that αTEA would mediate anti-tumor effects in rhabdomyosarcoma (RMS) by modulating the immunosuppressive milieu. Treatment of murine M3-9-M RMS in vitro with αTEA (2.5–100uM) resulted in increased apoptosis by flow cytometry (Annexin V+/7AAD+) and Incucyte analysis (Annexin V+) within 24 hours (p < 0.05). Using an established orthotopic luciferase+ M3-9-M in vivo RMS model, αTEA-supplemented chow administered for 21 days significantly reduced tumor volume (p < 0.001) and bioluminescence (p < 0.05) by 28 days, resulting in a significant prolongation of survival compared to matched control chow (p < 0.05), up to 4 weeks after the feed was stopped. Analysis of spleens from RMS mice fed αTEA-supplemented diets showed an increased percentage of Ki-67+ and IFNγ+CD4+ T cells (p < 0.01) at the end of treatment as compared to recipients of the control diet. In addition, a decrease in the percentage of CD11b+Arg-1+ (p < 0.01) and PD-L1+ (p < 0.05) cells within the spleen were observed in αTEA-treated RMS tumor-bearing mice. RNA-Seq analysis of RMS tumors is underway to explore changes in αTEA vs. control-treated mice. These results suggest that αTEA causes direct induction of apoptosis of RMS in vitro and in vivo, leading to prolonged survival through enhanced CD4+ T cell stimulation and reduced myeloid cell-mediated immunosuppression in RMS-bearing mice. Further investigation of αTEA as an immunomodulating agent is warranted.
The tumor associated antigen LEDGFp75 interacts with oncogenic transcription factors in docetaxel resistant prostate cancer cells
Authors: Ortiz-Hernandez Greisha L , Sanchez-Hernandez Evelyn S , Casiano Carlos A.
Published: January 2023

Our group and others established that the stress oncoprotein LEDGF/p75 is a tumor-associated antigen (TAA) targeted by serum autoantibodies in certain patients with prostate cancer (PCa). LEDGF/p75 is also upregulated in clinical prostate tumors and contributes to docetaxel (DTX)-resistance in advanced PCa cells. The C-terminus of LEDGF/p75 contains a domain called the Integrase Binding Domain (IBD), which in T cells is responsible for tethering the HIV-integrase complex to transcriptionally active chromatin. In cancer cells, the IBD interacts with oncogenic transcription complexes to promote cell survival. However, the relevance of these LEDGF/p75 protein-protein interactions (PPIs) in PCa and chemoresistance has never been explored. This study aims to test if the interactions between LEDGF/p75, JPO2, Menin, and MLL in DTX-resistant PCa cells contribute to chemoresistance. Also, we want to target these interactions with repurposed HIV-based small molecule inhibitors (SMIs) of LEDGF/p75, which target the IBD, to abrogate this resistance. We demonstrated a significant co-upregulation of these proteins in DTX-resistant PC3-DR and DU145-DR cell lines, compared to the parental cells. Also, we observed nuclear co-localization by immunofluorescence microscopy. Using human anti-LEDGFp75 autoantibodies, we confirmed that endogenous proteins co-immunoprecipitate in the DTX-resistant PCa cell lines. Besides, we are evaluating repurposed HIV-based SMIs for their efficacy in sensitizing resistant cells to DTX. Our goals are to use anti-LEDGFp75 autoantibodies to establish its endogenous-interactome, contribution to transactivation of DTX-resistant genes, and target this interactome to overcome PCa chemoresistance.
Inflammation-induced tumor antigen expression on epithelial cells reveals the mechanism of their generation and provides a system for identification of new tumor targets
Authors: Jacqueline Camille , Lee Amanda , Frey Nolan , Minden Jonathan S , Finn Olivera J
Published: January 2023

Tumor-associated antigens (TAAs) are self-molecules abnormally expressed on tumor cells that elicit humoral and cellular immunity and are targets of effective cancer immunosurveillance. Surprisingly, immunity to TAAs is found in many healthy individuals with no history of cancer. We previously showed that other events in addition to cancer, such as viral and bacterial infections, can cause the expression of some TAAs and correlate with effective tumor immunosurveillance. We are now testing in mice the ability of two LCMV strains, one that causes acute (Armstrong) and one that causes chronic (Cl-13) infection, to elicit expression of and immune memory to TAAs and protect from a later tumor challenge. We observed that infection with the acute strain elicits immunity to specific TAAs expressed by two tumor cell lines that were then used in tumor challenge experiments. So far, we were able to show a protective effect of a previous Armstrong infection against our model of lymphoid tumor (EL4). We also developed a highly reproducible in vitro model of acute inflammation of primary epithelial cells and confirmed that short-term exposure to pro-inflammatory cytokines IL-1β, IL-6 and TNF-α leads to abnormal expression of three well known TAAs, MUC1, CEA and HER2/neu. These cells were found to activate macrophages, which should promote antigen uptake and presentation of these and other TAAs. We have used this system and 2D Difference Gel Electrophoresis (2D-DIGE) combined with mass spectrometry to identified so far seven new TAAs. Because these TAAs are generated first as disease-associated antigens (DAAs), they represent highly predictable shared antigens for safe and effective preventative cancer vaccines.
Single-cell systems neuroimmunology reveals a highly immunosuppressive microenvironment in human glioblastomas contacting the ventricular stem cell niche
Authors: Bartkowiak Todd , Barone Sierra , Mistry Akshitkumar M , Greenplate Allison R , Sinnaeve Justine , Leelatian Nalin , Hayes Madeline , Roe Caroline E , Mobley Bret C , Chambless Lola B , Thompson Reid C , Weaver Kyle D , Ihrie Rebecca A , Irish Jonathan M
Published: January 2023

Glioblastomas make up more than 60% of adult primary brain tumors and carry a 15 month overall survival despite aggressive standard-of-care therapy. Recent advances in immunotherapy offer an appealing alternative that may improve outcomes for patients with glioblastoma; however, clinical trials have proven unsuccessful due in part to a lack of predictive features that may inform responsiveness to immunotherapy. We have recently shown a strong correlation between 1) immune infiltration, 2) tumor cell phenotype, and 3) patient outcome. Further, patients whose tumors demonstrate radiographic contact with the ventricular-subventricular zone (V-SVZ) have reduced survival compared to patients whose tumors do not contact the V-SVZ. We therefore hypothesized that the V-SVZ acts as a previously unappreciated immunosuppressive microenvironment within the brain that promotes tumor growth by suppressing anti-tumor immunity. Primary human glioblastomas were disaggregated into single-cell suspensions and mass cytometry (CyTOF) measured >30 parameters in thirteen immune populations infiltrating human glioblastomas. Cutting-edge machine-learning tools identified key differences in the abundance and phenotypes of T cells, B cells, NK cells, microglia, and peripheral macrophages infiltrating ventricle-contacting gliomas. Further, enriched expression of immune checkpoint receptors (PD-1, TIGIT, LAG-3, TIM3) correlated with ventricular contact and outcome. These results provide key insights into the immune microenvironment of glioblastomas and elucidate several clinically actionable immunotherapeutic targets that may be used to optimize treatment strategies for glioblastoma patients based on V-SVZ contact status.
The impact of the tumor microenvironment on granzyme production by CD8 T cells
Authors: Hay Zachary LeeZer , Kim Dale , Silva Alexa Lynn , Slansky Jill E
Published: January 2023

Granzymes are a class of proteases produced by CD8 T cells to mediate their cytotoxic activity against target cells. The objective of this project is to determine the mechanism through which CD8 T cells are executing their granzyme-mediated cytotoxic function and how the tumor microenvironment (TME) impacts the specific granzymes produced. By micro-array and qPCR we determined that CD8 T cells in the TME have altered expression of specific granzymes. Granzyme F had one of the most altered expressions of all genes examined in antigen-specific CD8 T cells taken from a spleen relative to those taken from a tumor. We further examined the effects of the TME on granzyme expression by sorting tumor infiltrating CD8 T cells on exhaustion markers and found a relationship between exhaustion and granzyme expression, characterized by a decrease in granzyme A and an increase in granzyme B mRNA expression. We will determine the influence different ex vivo culture conditions have on differential granzyme expression and function of CD8 T cells. The expression of different granzymes may have further implications for modulation of the TME through the form of cancer cell death induced, especially the increase in granzyme F which has been suggested to induce a more immunogenic form of cell death. Additionally, the impact of exhaustion on the cytotoxic activity of CD8 T cells from the TME may represent a means for exhausted CD8 T cells to regulate their cytotoxic activity by altering the granzymes they express.
Molecular cytometry identifies a wide range of translationally relevant markers in tumor and peripheral immune cells of lung cancer patients
Authors: Chattopadhyay Pratip K , Gao Guo-Jian , Taylor Ian , Guidry Kayla , Labbe Kristin , Almonte Christina , Nakamoto Margaret , Kin-Wong Kwok
Published: January 2023

Many immunotherapy drugs, used singly or in combination, are emerging. To realize precision oncology, and better design clinical trials, in-depth immune profiling is key. Many new technologies are available; the most promising simultaneously analyzes >102 proteins and 400 mRNA cell-by-cell (molecular cytometry). Using this technology, we deeply profiled TIL, from freshly resected lung tissue, and PBMC collected at surgery (n=10).

Antibody staining was robust, with all canonical cell populations at expected frequency. We identified markers uniquely upregulated in TIL, including CXCR6, CD39, CD26, CD69, CD103, and RGS. We also asked what markers were uniquely enriched in PD1-TIL, in order to find other drug targets for patients who fail Nivolumab. We found many molecules upregulated in PD1-TIL, including CD326, CD98, LGALS3, TIM3, CD54, CD235ab, CXCL8, CD141, and CD117. We further identified precise combinations of these markers that inform design of combination immunotherapy.

We also characterized immune landscapes in metastatic disease vs. localized adenocarcinoma, finding drug targets and tumor-immune interactions are different across these settings. For example, T-cell activation markers are downregulated in metastatic tissue, e.g., HLADR (p = 1.5E-16), CD69 (p=2.9e-10), and CD38 (p=3.2e-16). CD103 was also highly downregulated (p=1.9E-14). Notably, in metastatic disease various myeloid proteins are elevated, including CD206 (p=0.002), CD32 (p=0.02), and CD61 (p=0.006). We also characterized the degree of immune exhaustion, using ratios of ZBED2:LGALS in cells. In summary, we demonstrate the utility of molecular cytometry for providing unique (and translationally-relevant) insight into lung cancer.
Inflammation synergistically promotes cigarette smoke carcinogen-induced lung tumorigenesis and determines immunotherapy efficacy
Authors: Di Yuanpu Peter , Liu Chia-Hsin , Chen Zhong , Chen Kong , Liao Fu-Tien , Keohavong Phouthone , Leikauf George
Published: January 2023

Chronic obstructive pulmonary disease (COPD) is a chronic lung inflammatory disease with an increased risk of lung cancer. Pseudomonas aeruginosa (PA) are opportunistic bacteria frequently found in patients with COPD, and associated with increased lung inflammation and acute exacerbations. The impacts of COPD-related inflammation, especially those induced by the PA-produced lipopolysaccharides (LPS), on lung tumorigenesis and the efficacy of immune checkpoint blockades remain poorly understood. In this study, we established a novel murine lung cancer model by treating mice with cigarette smoke carcinogen nitrosamine (NNK) combined with recurrent LPS exposure. LPS-mediated chronic inflammation in NNK-treated mice generates an immunosuppressive microenvironment characterized by the accumulation of myeloid-derived suppressive cells (MDSCs) and regulatory T cells, induced T-cell exhaustion, and enhanced PD-1/PD-L1 activity that resulted in synergistic increases of lung tumorigenesis than NNK treatment alone (>8-fold increase in tumor numbers, P<0.0001). Immunotherapy using anti-PD-1 antibody or anti-Ly6G antibody (to deplete MDSCs) alone reduced tumor growth, and in combination further enhanced the anti-tumor activity. We also identified immune gene signatures from the RNA transcript expression data of our murine cancer model and the published datasets of PD-1 blockade, which effectively predicted treatment responses and survival outcome in cohorts with immunotherapy and conventional treatments. Our results indicate that LPS-mediated chronic inflammation creates a favorable immunosuppressive microenvironment for tumor progression and correlates with the efficacy of anti-PD-1 treatment.
PD-L1 lncRNA splice promotes lung adenocarcinoma proliferation and metastasis via enhancing c-Myc activity
Authors: Liang Hongwei , Qu Shuang , Zen Ke
Published: January 2023

Although blockade of programmed death-ligand 1 (PD-L1) to suppress T cell immune responses shows great promise in tumor immunotherapy, the efficacy of such immune-checkpoint inhibition strategy is low for patients with solid tumors. The mechanism underlying the limited efficacy of PD-L1 inhibitors remains unclear. Here, we show that, via alternative splicing, human PD-L1 protein positive or negative lung adenocarcinoma all produce a long non-coding RNA isoform of PD-L1 (PD-L1-lnc), which promotes lung adenocarcinoma proliferation and metastasis. Analyses of various lung adenocarcinoma cells show that PD-L1-lnc is significantly upregulated by IFNg in a manner similar to PD-L1 mRNA. Both in vitro and in vivo studies demonstrate that PD-L1-lnc increases proliferation and metastasis, but decreases apoptosis of lung adenocarcinoma cells. Mechanistically, PD-L1-lnc directly binds to c-Myc and enhances c-Myc signaling downstream in lung adenocarcinoma cells. Therefore, targeting PD-L1-lnc/c-Myc axis provides a novel strategy for lung cancer therapy.
Low immunogenicity of intracranial tumors limits immune response to brain malignancies
Authors: Stasiak Katarzyna , Lindsay Robin , Engelhard Victor H.
Published: January 2023

The presence of CD8 T cells in tumors, including melanomas, is correlated with longer survival. Cutaneous melanoma is a highly immunogenic malignancy with high rate of dissemination into central nervous system but the brain is considered immunologically privileged due to the blood-brain barrier, which limits T cell entry. To determine if brain tumors are also poorly infiltrated by T cells we evaluated intracranial (IC) and subcutaneous (SC) B16 melanomas expressing different model antigens.

CD8 T cells were present in IC tumors 7 days post tumor implantation in B16 melanomas expressing chicken ovalbumin (B16OVA) or influenza M1 protein (B16M1), but infiltrates were only 10% of those observed in SC tumors. We also observed that expression of homing receptor ligands (HRLs) known to mediate T cell infiltration was lower on IC than SC tumor-associated vasculature. Despite the fact that HRL expression decreased over time in tumors grown in both locations, T cell infiltrates increased, suggesting that HRL are not the only factor limiting T cells entry into IC tumors.

To determine whether the IC tumor infiltrate size was limited by inefficient stimulation of effectors cells, we vaccinated mice to generate fully activated effector T cells able to recognize tumor associated antigen. Vaccination normalized the infiltrate sizes in IC and SC tumors. Interestingly, this was also dependent on expression of Ag by the tumor cells. This suggests that IC tumors are not able to efficiently elicit a robust immune response, possibly due to limitations on drainage to lymph nodes, while IC tumor vasculature does not impose inherently greater limitations on the ability of T cells to enter.
Evidence of ongoing immune surveillance to cancer-associated phosphopeptides in healthy individuals
Authors: Lulu Amanda M , Cummings Kara L. , Engelhard Victor H.
Published: January 2023

Dysregulated protein phosphorylation plays a major role in cancer cell malignancy. We showed that phosphopeptides are presented by MHC-I molecules, representing a new class of cancer neoantigens. We found that healthy donors with no evident prior cancer had robust CD8+ T cell responses to a cohort of HLA-B7+ leukemia-associated phosphopeptides. Here we report that CD8+CD45RO+ T cells purified from blood of different healthy donors showed responses to 2–25% of HLA-A2+ and 0–23% of HLA-B7+ cancer-associated phosphopeptides, confirming that individuals with no evident cancer history have pre-existing phosphopeptide specific immune memory. Six of 59 HLA-A2+ phosphopeptides were “immunodominant,” with memory responses evident in the majority of analyzed donors. This suggests that these phosphopeptides result from processes common in cellular transformation or viral infection. In contrast, no immunodominant HLA-B7+ phosphopeptides were identified among the 168 analyzed, suggesting that mechanisms driving their expression in healthy donors are less common. Robust day 14 responses were observed against a small cohort of HLA-B7+ phosphopeptides in 2 donors. Responses to some of these phosphopeptides could be detected ex vivo and were evident over 3 years, suggesting they reflected chronic antigen exposures. In contrast, responses to 2 phosphopeptides evident ex vivo diminished or disappeared over 14 days in culture, suggesting a recent acute immune response composed of terminal effectors unable to persist long term. These results provide evidence of ongoing immune surveillance in healthy individuals to phosphopeptides known to be expressed on cancers.
Tumor microenvironment dictates function of tumor-expressed PD-L1
Authors: Klement John David , Redd Priscilla S , Lu Chunwan , Yang Dafeng , Liu Kebin
Published: January 2023

To evade immune clearance by cytotoxic T lymphocytes (CTLs), tumors express immunosuppressive checkpoints such as PD-L1. Tumor-expressed PD-L1 is thought to suppress CTL effector functions by binding CTL-expressed PD-1. To investigate the molecular consequences of CTL engagement with tumor PD-L1, we generated PD-L1 knockouts in murine sarcoma, breast and colorectal cancer cell lines by Crispr/Cas9. Surprisingly, PD-L1 expression conferred no protection from CTL-mediated cytotoxicity in an in vitro co-culture model. Similarly, loss of PD-L1 had no effect on in vivo growth following subcutaneous or orthotopic challenge in immunocompetent mice. However, tumor PD-L1 facilitated metastasis to the lung in both spontaneous and experimental metastasis models. Bulk RNA-seq of tumor-bearing lungs revealed global enrichment in interferon-stimulated gene expression following loss of tumor PD-L1. Flow cytometric analysis and single-cell RNA-sequencing localized this signature to an inflammatory macrophage population that expressed PD-1 and selectively expanded following injection of PD-L1 deficient cell lines. In vitro and in vivo experimentation showed that engagement of macrophage PD-1 by tumor PD-L1 reduced the expression of interferon-stimulated genes and antigen processing machinery. We propose a model by which tumor-expressed PD-L1 regulates CTL function indirectly in the lung microenvironment by suppressing the recruitment and activation of CTLs through myeloid cells.
Single cell sequencing identifies novel metastasis-associated macrophage subsets in the lung
Authors: Huggins Danielle , LaRue Rebecca , Knutson Todd P , Schwertfeger Kathryn L
Published: January 2023

Macrophages are among the most abundant immune cell type found within primary mammary tumors and their metastatic lesions. Within the lung, a common site of metastasis, two major subsets of macrophages have been characterized under homeostatic conditions. However, how the complexity and heterogeneity of macrophage subsets changes with the development and progression of metastatic lesions remains unknown. We aim to identify macrophage populations that can be targeted to either suppress pro-tumorigenic function or enhance anti-tumorigenic potential in order to improve patient outcomes. In the present study, macrophages were isolated from the lungs of mice bearing 4T1-Luc mammary gland tumors in which metastatic lesions were evident. Cells were sequenced using the 10X Genomics single cell sequencing platform and at least seven distinct macrophage clusters were identified displaying unique gene expression signatures. These include clusters exhibiting upregulation of genes related to antigen presentation (H2-Aa, Cd74) and interferon signaling (Isg15, Ifitm3). Interestingly, one cluster demonstrates an expression profile highly concordant to lipid-associated macrophages found within atherosclerotic plaques (Lgals3, Trem2). Gene set enrichment analysis of this cluster reveals enrichment for genes implicated in hallmark pathways involved in anti-tumorigenic immune responses as well as for pathways related to cholesterol and metabolism. Understanding the unique localization and regulation of these cells will aid in the development of novel approaches for selectively targeting them to promote polarization toward a more anti-tumorigenic phenotype.