bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immune tolerance
Authors: Reticker-Flynn Nathan E , Zhang Weiruo , Belk Julia Ann , Basto Pamela Antonia , Satpathy Ansuman , Plevritis Sylvia Katina , Engleman Edgar G 
Published: January 2023

Abstract
The majority of cancer deaths result from distant organ metastasis. Lymph nodes (LNs) are major sites of anti-tumor lymphocyte education, yet LN metastasis frequently precedes distant metastasis. Here, we find that LN metastasis represents a critical step in tumor progression by inducing tumor-specific immune tolerance, thus enabling further dissemination of tumors to distant organs. Using an in vivo passaging approach, we generated 300 cell lines exhibiting varying degrees of LN metastatic capacity. We show that the LN metastases promote distant organ metastasis in a manner that is tumor specific. Through organism-wide immune profiling by single cell sequencing, we identify multiple cellular mediators of tolerance. In particular, we find that LN metastases have the capacity to both resist NK cell cytotoxicity and induce regulatory T cells (Tregs). Adoptive transfer of Tregs from the LNs of mice bearing LN metastasis to naïve mice facilitates metastasis in a manner that Tregs from mice without LN metastases cannot. Additionally, these Tregs are induced in an antigen-specific manner. Using whole exome sequencing, we show LN metastases do not evolve through the acquisition of driver mutations, loss of neoantigens, loss of MHC class I, or decreases in melanoma antigens. Rather, by RNA-seq and ATAC-seq, we show that a conserved interferon signaling axis is upregulated in LN metastases and is rendered stable through epigenetic regulation of chromatin accessibility. Knockout studies reveal that these pathways are required for LN metastatic seeding, and we validate their significance in additional mouse models and patients. These findings demonstrate a critical role for LN metastasis in promoting tumor-specific immunosuppression.
This work was supported by NIH grants U54 CA209971 and F32 CA189408.
Response to anti-PD-1 and anti-LAG-3 immune checkpoint blockade is associated with induction of pro-inflammatory Tregs
Authors: Rolig Annah S , Sturgill Elizabeth R , Mick Courtney , Rose Daniel , Kaufmann Johanna , Yanamandra Niranjan , Griffin Sue , Smothers James , Redmond William L 
Published: January 2023

Abstract
Only a subset of patients durable clinical responses to aPD-1 and/or aCTLA-4 immunotherapies, thus, developing new therapeutic agents to increase the proportion of responding patients is a priority. Combining aPD-1 with aLAG-3 has shown promising results; however, lack of mechanistic understanding of aPD-1/aLAG-3 synergy remains a barrier for its optimal clinical use. Here, we examined the mechanism of aPD-1/aLAG-3 synergy in multiple mouse models using flow cytometry and single cell RNA sequencing. Combined aPD-1/aLAG-3 immunotherapy significantly improved the survival of CT26 (BALB/c; colon carcinoma) and MCA-205 (C57BL/6; sarcoma) tumor-bearing mice compared to monotherapy. Regulatory T cells (Tregs) suppressed response to this therapy, as in the absence of CD4+ T cells, 100% of mice responded. To understand how responders overcome Treg suppression, we performed an in-depth analysis of tumor-infiltrating lymphocytes (TIL) comparing mice that responded to treatment (decreased tumor size post-treatment) to non-responders (same tumor growth trajectory as control). Responders had reduced Foxp3+ CD4+ Tregs in comparison to non-responders and, in addition, those Tregs had a ‘fragile’ phenotype, including a pro-inflammatory cytokine profile (TNF-a; IFN-g), increased LAG-3, and decreased NRP1 expression. Within responders, CD8+ TIL exhibited increased frequency, effector cytokine production (TNF-a; IFN-g), and LAG-3 expression as compared to non-responders. Together, these data suggest that aPD-1/aLAG-3 can reduce Treg frequency and function leading to expansion of active tumor-specific CD8+ T cells capable of supporting tumor regression and improved survival.
Supported by the Providence Portland Medical Foundation and GlaxoSmithKline
TCF-1 controls Treg functions that regulate inflammation, CD8 T-cell cytotoxicity, and severity of colon cancer
Authors: Khazaie Khashayarsha , Osman Abu , Yan Bingyu , Kazemian Majid , Gounari Fotini 
Published: January 2023

Abstract
The transcription factor TCF-1 is essential for the development and function of T regulatory (Treg) cells, however its function is poorly understood. Here, we show that TCF-1 primarily suppresses transcription of genes that are co-bound by Foxp3. Single-cell RNA-seq analysis identified effector- and central-memory Treg-cells with differential expression of Klf2 and memory and activation markers. TCF-1 deficiency did not change the core Treg transcriptional signature, but promoted alternative signaling pathways whereby Treg-cells became activated and gained gut-homing and TH17 characteristics. TCF-1-deficient Treg-cells strongly suppressed T-cell proliferation and cytotoxicity, but were compromised in controlling CD4+ T-cell polarization and inflammation. In mice with polyposis, Treg cell-specific TCF-1 deficiency promoted tumor growth. Consistently, tumor-infiltrating Treg cells of colorectal cancer patients showed lower TCF-1 expression and increased TH17 expression signatures compared to adjacent normal tissue and circulating T-cells. Thus, Treg cell-specific TCF-1 expression differentially regulates TH17-mediated inflammation and T-cell cytotoxicity, and can determine colorectal cancer outcome.
Supported by NIH R01 AI 108682 (FG & KK), NIH RO1 AI 147652 (FG), NIH R35GM138283 (MK), and Praespero Innovation Award Alberta, Canada (FG & KK)
Tumor-intrinsic factors dictate beneficial effect of microbiota-based therapies
Authors: Lam Khiem C , Huang April , Araya Romina E , Chen Quanyi , Goldszmid Romina S 
Published: January 2023

Abstract
Gut microbiota impacts antitumor immunity and recent studies have shown that manipulating microbiota via fecal transplant can rescue immune checkpoint blockade (ICB) response in refractory melanoma patients. However, not all patients benefited from this approach. We hypothesize that tumor-intrinsic factors (e.g. neoantigen load and immune microenvironment) contribute to the microbiota effect on anticancer response. To test this, we performed preclinical microbiota manipulation studies using dietary intervention. Mice fed high-fiber diet (FD) had improved spontaneous tumor control and response to ICB. Mechanistically, FD-induced changes in microbiota composition skewed the tumor innate immune repertoire towards an antitumor profile marked by increased dendritic cell infiltration. Downstream of the innate compartment, FD reduced the proportion of intratumoral Tregs and exhausted CD8+ T cells. We further assessed the effects of FD across 8 tumor models including EL4 lymphoma, MC38 colon carcinoma, and 6 melanomas (M1–M6) that mimic the variety of human melanoma subtypes. Although FD had a beneficial effect overall, not all models responded to the same extent, and were accompanied by different changes in the tumor immune profile. FD delayed tumor initiation for EL4 and M4, reduced growth rate for M3 and M5, both delayed initiation and reduced growth rate for MC38, M2, and M6, but had no significant effects for M1. Additionally, we found that increasing tumor immunogenicity resulted in a synergistic effect with FD. Our data suggests that tumor-intrinsic factors influence the beneficial effect of microbiota on antitumor immunity. Identifying these factors will help refine the use of microbiota-based therapies in the clinic.
This research was supported by the Intramural Research Program of the NIH (CCR-NCI).
This research was supported by the Intramural Research Program of the NIH (CCR-NCI).
Improving anti-tumor CD8+ T cell function through manipulating glutamine metabolism
Authors: Fisher Emilie L , Madden Matthew Z , Chi Channing , Suguira Ayaka , Rathmell Jeffrey 
Published: January 2023

Abstract
Immunotherapies that bolster the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) have improved outcomes for many, yet most patients fail to achieve complete and durable responses. Investigations into strategies to further enhance the cancer killing capacity of these cells are therefore crucial in the effort to improve immunotherapies. One avenue for manipulation of CTLs is through alterations to cellular metabolic programs. T cells radically alter their metabolism upon activation. Prior studies have shown pan-inhibition of glutaminolysis using the inhibitor 6-Diazo-5-oxo-l-norleucine (DON) enhances the function of CTLs in the tumor microenvironment. However, DON’s toxicity combined with this drug’s lack of specificity demonstrate a need for a more directed glutaminolysis targeting approach. Herein, I utilize a targeted glutaminolysis CRISPR library to investigate the role of individual enzymes targeted by DON. To interrogate the importance of these enzymes specifically in anti-tumor CTLs, I developed a model in which CRISPR-edited antigen-specific CTLs were adoptively transferred into tumor-bearing mice. After 7 days of in vivo selection, next generation sequencing was performed on isolated CTLs to evaluate the survival advantage or disadvantage of these gene knockouts. While multiple DON targets resulted in decreased CTL fitness, deletion of the gene encoding for glutamine synthetase (GS) – the enzyme responsible for catalyzing the conversion of glutamate to glutamine – conferred a fitness advantage to CTLs. In future experiments, inhibition and genetic deletion of GS will be utilized to investigate the role of GS in CTL activation, proliferation, effector cytokine production, and memory formation.
T-cells resist CD5 CAR mediated fratricide by continuously degrading CD5 protein
Authors: Ma Royce , Popat Divya , Chaumette Alex , Carisey Alexandre , Brenner Malcolm K , Mamonkin Maksim 
Published: January 2023

Abstract
Developing chimeric antigen receptor (CAR) T cells for patients with T cell malignancies remains challenging as most CARs targeting T-lineage antigens induce T cell fratricide. A notable exception is a CD5-specific CAR, which does not produce extensive fratricide, coinciding with a loss of detectable surface CD5 by an unknown mechanism.
In this study, we used live imaging fluorescent microscopy, Western Blot, and flow cytometry to show CD5 protein is rapidly internalized and degraded in transduced T cells following CD5 CAR expression. Experiments with C-and N-tagged CD5 confirmed complete degradation of the entire protein in CD5 CAR T cells. Physical ligation of CD5 antigen by the CAR in cis is sufficient to drive loss of CD5 protein, independently of either CAR or CD5 signaling, resulting in full protection from CD5-directed fratricide.
In an ongoing Phase I clinical trial (NCT03081910), CD5 CAR T cells expand and produce robust anti-tumor activity in patients with T cell malignancies. However, patients exhibit incomplete aplasia of non-transduced normal T-cells and a population of circulating T cells remain resistant to CD5-directed elimination and persistalongside CAR T cells. These resistant T cells are surface CD5-negative suggesting that in the presence of CD5 CAR T cells there can be removal of the target antigen as a means of evading killing. Indeed, in vitro experiments showed normal T cells degrade CD5 protein upon coculture with CD5 CAR T cells indicating antigen down modulation occurs on target cells not only in cis but also in trans. These data identify a possible mechanism of resistance of normal T cells to CD5-directed CAR T-cell elimination.
Supported by CAGT T32 Training Grant NIH NCI SPORE P2
Glycolipid-loaded nanoparticle immunotherapy cooperates with checkpoint inhibitors to harness iNKT cells for tumor control
Authors: Shute Travis J , Dudley Elizabeth A , Nash Kelly , Leadbetter Elizabeth A 
Published: January 2023

Abstract
Invariant natural killer T cells (iNKT) have a well-documented role in direct and indirect killing of tumor cells through their release of proinflammatory cytokines and cytotoxic compounds. To improve outcomes in poorly immunogenic cancers while avoiding NKT cell anergy, we induced anti-tumor responses by activating NKT cells with polymeric nanoparticles containing a low dose of a glycolipid adjuvant, α-Galactosyl-Ceramide (aGalCer), and a model tumor neoantigen, ovalbumin (OVA). Prophylactic vaccination of mice with aGalCer-OVA nanoP before inoculation with B16F10-OVA melanoma cells reduced tumor growth rate and increased overall survival compared to mice immunized with control nanoparticles. In mice with an established B16 tumor, therapeutic administration of aGalCer-OVA nanoP provided significantly greater anti-tumor effects, reduced tumor growth rate and increased survival, compared to control nanoparticles. Therapeutic treatment with aGalCer-OVA nanoP, but not control nanoPs, also induced significant infiltration of iNKT, NK, and antigen-specific CD8 T cells in the tumor, and enhanced production of granzyme B and perforin by CD8 T cells. Furthermore, immune checkpoint blockade treatment (anti-CTLA-4 + anti-PD-1) enhanced the effectiveness of aGalCer-OVA nanoP, leading to clearance of established tumors nearly 50% of the time. These results provide strong evidence that activation of iNKT cells via nanoparticles containing a glycolipid adjuvant and tumor antigen is a viable anti-cancer strategy that partners well with other clinically used immunotherapies.
Supported by grants from NIH (T32 AI138944) and the CTSA (TL1 TR002467)
A tumor-targeted cytokine/antibody fusion protein to stimulate anti-cancer immunity
Authors: Silver Aliyah Brianne , Spangler Jamie 
Published: January 2023

Abstract
Interleukin-2 (IL-2) is a pleiotropic cytokine that has clinical utility in stimulating anti-cancer immunity. However, the clinical efficacy of IL-2 is limited by its short serum half-life, systemic toxicity, and concurrent activation of immunostimulatory natural killer (NK) and effector T (Teff) cells and immunosuppressive regulatory T (Treg) cells. IL-2 can signal down an intermediate-affinity receptor consisting of IL-2 receptor beta (IL-2Rβ) and common gamma chain (γc) or a high-affinity receptor that also includes IL-2Rα. Tregs express high levels of IL-2Rα, making them much more responsive to IL-2 than naïve NK and Teff cells. To extend the serum half-life and selectively bias the immunostimulatory functions of IL-2 in a stable unimolecular format, we created a single-chain fusion protein, denoted F10 immunocytokine (F10 IC), that intramolecularly fuses human IL-2 to an anti-IL-2 antibody that sterically blocks the IL-2/IL-2Rα interaction and allosterically enhances the IL-2/IL-2Rβ interaction. To exploit the abundance and accessibility of collagen in the tumor microenvironment for targeting, we fused collagen binding domains to F10 IC in various topographies. These novel fusion proteins occlude IL-2Rα binding and enhance IL-2Rβ binding, thereby eliminating the Treg advantage conferred by IL-2Rα and preferentially activating immune effector cells. Furthermore, these proteins selectively bind purified collagen and are capable of homing to, and being retained in, the tumor in vivo. Building on these promising results, we are assessing the in vivo therapeutic efficacy of these fusion proteins in various murine tumor models to illustrate the potential of collagen binding F10 IC as a powerful anti-cancer therapeutic.
Supported by a Sanofi Innovation Award and NIH grants (R01 EB029341, T32 AI007417)
Resveratrol, Modafinil and Caffeine’s modulation of B cell’s CD19 activation receptor
Authors: Valenzuela Hector F , Samayoa Giovanni , Martinez Kassandra , Salvador Samantha 
Published: January 2023

Abstract
Resveratrol a natural compound with anti-oxidant properties shown to inhibit cell proliferation and induces cellular apoptosis in Jurkat cell lines. Mechanistically, it was observed that interleukin-2 expression was blocked in T cells, suggesting resveratrol’s anti-inflammatory mechanism may involve downregulation of activation molecules. In this study, we investigated the effects of resveratrol or in combination with Modafinil or caffeine on a B cell line, Nor20. Modafinil is a compound shown to directly act on enzymes in the brain’s free-radical scavenging system and therefore directly reduce free radical levels. Caffeine has been shown to abolish the anti-inflammatory effects of resveratrol in astrocytes. Here we show that Nor20 treated with resveratrol can have up to 47.3% reduction in viability, consistent with previous observations. To determine a mechanism to explain these results, we used flow cytometry to measure the expression of CD19, the dominant receptor for all forms of B cell activation and modulator of B cell activation threshold combined with Modafinil or caffeine. We observed that resveratrol reduced the expression of CD19 up to 51%, Modafinil, inhibited up to 65%, but when resveratrol and Modafinil were combined the effect was not additive remaining at 53% inhibition when compare to the controls. In contrast, caffeine did not affect CD19 expression but when combined with resveratrol, caffeine abolished the downregulation of CD19 by resveratrol returning to the same levels as the untreated controls. Taken together, these results may indicate that resveratrol’s anti-inflammatory effects may initiate at the lymphocyte’s early recognition signaling receptors as a mechanisms to control cell growth.
Supported by RevGenetics and Whittier College’s Faculty Research Grant.
Demonstration of regulatory CD8 T cell prevalence, phenotype, and functions in autoimmune patients treated with a tolerizing peptide vaccine.
Authors: Maurer Meghan , Bowser Justin , Fasnacht Rachael , Gardell Jennifer L , Julien Susan , Templeton Megan , Therriault Jon , Yang Su J , Swiderek Kristine , Crane Courtney 
Published: January 2023

Abstract

INTRODUCTION
Others have described a subset of CD8 T cells with immunosuppressive characteristics in inflammatory disease settings. CD8 Treg activation, through a canonical T cell receptor, results in oligoclonal expansion and perforin dependent elimination of pathogenic CD4 T cells. We have demonstrated the CD8 Treg network in Celiac patients, and that CD8 Tregs can be modulated to enhance their cytolytic elimination of pathogenic CD4 T cells. Here, we evaluate culture conditions to enhance CD8 Treg functions, and describe the impact of a gluten tolerizing peptide vaccine on CD8 Treg in Celiac disease patients.

METHODS
We examined the phenotype and function of Celiac patient-derived peripheral blood CD8 Tregs using flow cytometry, multicolor immunohistochemical staining, supernatant analysis, and TCR sequencing. We then evaluated the impact of a gluten tolerizing peptide vaccine on the CD8 Treg network in Celiac patient derived peripheral blood and tissues.

RESULTS
Using our previously reported phenotypic and functional readouts to define CD8 Treg activity, we describe culture conditions that support oligoclonal CD8 Treg cell expansion, plasticity, and cytolytic function. In patients treated with a gluten tolerizing vaccine, we found evidence for CD8 Treg expansion and preferential recruitment to duodenal tissues; however, pathogenic CD4 T cells were still detectable, suggesting insufficient CD8 Treg functional activation.

CONCLUSION
We describe parameters that support cytolytic CD8 Treg function, as well as the impact of a gluten tolerizing vaccine on the mobilization of CD8 Treg. Collectively, our data support the potential for CD8 Treg directed therapies to eliminate pathogenic CD4 T cells in autoimmune diseases.
Development of no-wash, rapid Fcγ Receptor Binding Immunoassays using NanoBiT® Technology
Authors: Swanson Brad , Nath Nidhi , Godat Becky , Flemming Rod , Urh Marjeta 
Published: January 2023

Abstract
Antibody driven therapies including monoclonal antibodies (mAbs), intravenous IgG (IVIG), subcutaneous IgG (SCIG), and vaccines have made a significant impact in the treatment of many diseases including multiple cancers and autoimmune, metabolic, viral, and infectious diseases. A key mechanism of action (MOA) that makes antibody-based therapies so effective is the ability of the Fc domain of the antibody to recruit immune effector cells through their interaction with a variety of Fc receptors (FcγR). However, assessing the combined impact of interaction of the Fc domain of various antibody isotypes with diverse FcγRs (Fc/FcγR interaction) on the efficacy of the drug is challenging, primarily due to the lack of simple to use and reproducible Fc/FcγR binding assays. Furthermore, existing technologies like SPR can introduce artifacts based on assay format, sensor chip characteristics, and immobilization methods.
To meet the need for reliable, simple to use assays, we have developed a suite of bioluminescent biochemical assays for the following receptors: Lumit™ FcγRI Binding Immunoassay Lumit™ FcγRIIA (H131) Binding Immunoassay Lumit™ FcγRIIA (R131) Binding Immunoassay Lumit™ FcγRIIIA (V158) Binding Immunoassay Lumit™ FcγRIIIA (F158) Binding Immunoassay
These assays are homogeneous, meaning no immobilization or washing steps are required. In addition, they are rapid, easy to use in a 96-well format, and require a simple luminometer. These assays can enable better understanding of antibody MOA and hence better therapies.
Plant derived Nano-11 particle adsorbed with stimulator of interferon genes adjuvant and split influenza virus antigens elicits the cross-protective immunity in pigs
Authors: Gourapura Renukaradhya J , Patil Veerupaxagouda , Hernandez-Franco Juan F , Yadaigiri Ganesh , Bugybayeva Dina , Dolatyabi Sara , Feliciano-Ruiz Ninoshkaly , Schrock Jennifer , Hanson Juliette , HogenEsch Harm 
Published: January 2023

Abstract
We evaluated the performance of split swine influenza A virus (SwIAV) H1N2 antigens with a plant-derived nanoparticle adjuvant (Nano-11) or its combination with a synthetic STING agonist ADU-S100 (NanoS100). Specific pathogen free pigs were vaccinated twice via intramuscular (IM) and intradermal (ID) routes and challenged with a virulent heterologous H1N1. Animals vaccinated IM or ID with H1N2-ADU-S100-Nano-11 had significantly increased cross-reactive IgG and IgA titers in serum, nasal secretion and bronchoalveolar lavage fluid. There was a significant increase of IFNγ+ effector memory T-helper/memory cells and late effector cytotoxic T cells (CTLs), IL-17A+ central and effector memory T-helper/memory cells, IL-17A+ early effector CTLs, and CXCL10+ plasmacytoid dendritic cells in the PBMCs of the ID injected H1N2-ADU-S100-Nano-11 compared to H1N2-Nano-11 group. The frequency of IFNγ+ late effector CTLs and effector memory T-helper/memory cells and IL-17A+ late effectorCTLs, as well as in lungs CXCL10+ plasmacytoid dendritic cells was increased in PBMCs of IM injected H1N2-ADUS100-Nano-11 pigs. Increased expression of IL-4 and IL-6 mRNA was observed in tracheobronchial lymph nodes of IM Nano-11 based vaccinates following challenge. Furthermore, the challenge virus load in the lungs and nasal passage was undetectable in IM H1N2+ADU-S100-Nano-11 vaccinates by day 6 post infection along with reduced macroscopic lung lesions. In conclusion, despite vast genetic difference (77% HA gene identity) between the H1N2 and H1N1 SwIAV, the NanoS100 adjuvanted vaccine elicited cross protective immune responses, suggesting the potential role of this combination adjuvant in inducing cross-protective immunity in pigs.
Supported by USDA National Institute of Food and Agriculture, AFRI project 2019-67015-29814