bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tumor infiltrating T cells: complete workflows allow faster and improved analysis
Authors: Evaristo Cesar , Siemer Ramona , Agorku David , Brauner Janina , Hardt Olaf , Dose Christian , Richter Anne 
Published: January 2023

Abstract
T cells can eliminate tumors, but tumor infiltrating (TI) T cells can become intrinsically anergic or extrinsically suppressed. Identifying the mechanisms of T cell dysfunction is necessary to improve immunotherapy efficacy. Mouse tumor models are useful tools in tumor T cell research, but analysis of TI T cells is technically challenging, time consuming and labor intensive. Their frequency can be very low and small populations may escape analysis as they get lost in the background. Importantly, TI T cells are in an antigen-rich and highly immunomodulatory cell environment, such that cell intrinsic functional analysis is hindered. It is fundamental to use new tools to streamline the workflow and generate reliable data.
We established workflows combining tissue dissociation, T cell isolation and phenotyping. Tumor dissociation was automated using the gentle MACS™ and optimized for epitope preservation. Enzymatic dissociation was essential for analysis of PD1hiTim3+Lag3+CD39+ CD8 T cells in tumors. Using new T cell-specific microbeads for magnetic cell sorting we increased TI T cell purity by up to 500-fold, while maintaining activation status and phenotype. Importantly T cell isolation from the tumor’s highly immunomodulatory cellular environment was essential to assess cell-intrinsic functional properties. Finally, our data show the use of recombinant RE Afinity antibodies eliminated non-specific labeling of cells in the tumor microenvironment.
We conclude that standardized processing of tumor samples and magnetic isolation of tumor infiltrating T cells greatly reduces time and cost of total workflow and downstream analysis while significantly increasing reproducibility and the quality of data obtained from TIL analysis.
Dissection of single-cell gene regulation by simultaneous digital mRNA and protein quantification
Authors: Lam Regina , Shum Eleen , Chang Christina Christina , Lam Gretchen , Bansal Nidhanjali , Shah Hemi , Jensen Devon , Hu Jing , Ghadiali James , Martin Jody , Rosenfeld David , Fan H Christina 
Published: January 2023

Abstract
The immune system consists of complex gene regulatory networks that allow a rapid transition of different cellular states during an immune response. Cell-surface marker analysis using flow cytometry or single cell RNA-seq has allowed characterization of immune subpopulations and a greater understanding of the complexity of immune cells. However, restrictions on protein-only or RNA-only analysis can greatly limit the understanding of how genes are regulated in cells. For example, many cell surface markers – such as CD4 in T cells – have thousands of protein copies per cell, yet is fueled by a small number of mRNA transcripts.
To bridge the understanding of protein and mRNA expression differences, we used BD™ AbSeq on the Rhapsody™ platform to provide digital quantification of both protein and mRNA expression level in single cells. An oligo-conjugated antibody panel against immune-relevant cell-surface markers was created and used for this multi-omic gene expression profiling effort. This approach is coupled with mRNA analysis using the BD Rhapsody Immune Response Panel, a targeted method of RNA-seq that allows a higher sensitivity of immune markers than conventional whole transcriptome assays.
We studied the dynamics of early T cell activation in vitro to understand this response on transcriptional, post-transcriptional, and translational levels. Different time points following anti-CD3 and anti-CD28 treatment were collected and multiplexed on to BD Rhapsody cartridge for single cell capture and analysis. Using AbSeq on BD Rhapsody, we were able to detect the difference in mRNA and protein levels of crucial markers, allowing us to dissect the intricate gene regulatory pathways during an immune response in a single cell level.
Modular, defined assembly of antibody-based constructs using click chemistry
Authors: Langsdorf Chris , Aggeler Robert , Chen Aimei , Dolman Nicholas , Hu Yi-Zhen , Kamen Lynn , Mandavilli Bhaskar , Ordonia Benjamin , Wickett Marcy 
Published: January 2023

Abstract
Immunoglobulin antibodies contribute to a broad range of research assays and also provide the foundation for a diverse variety of biopharmaceutical agents. Here we describe the use of bio-orthogonal click chemistry reactions to synthesize several common antibody-based structures and demonstrate their use in cell-based applications. First, defined antibody-fluorophore conjugates were synthesized, characterized and functionally tested in receptor occupancy and antibody internalization assays. Next, a double conjugation strategy was employed to synthesize antibody drug conjugates which become brightly fluorescent upon endocytosis. ADC uptake and targeted cell killing were demonstrated using cell line and spheroid models. Then bispecific antibodies were constructed using a click chemistry reaction to create tetravalent heterodimeric antibodies. Structure and function of these bispecific constructs are demonstrated in cell-free and flow cytometry assays.
Validation of a Protein A-based ELISA for quantifying Immunoglobulin G in a non-traditional wildlife species, the Steller sea lion (Eumatopias jubatus)
Authors: Kennedy Stephanie N. , Wilhite Brittany , Castellini J. Maggie , Rea Lorrie D. , Kuhn Tom , O’Hara Todd 
Published: January 2023

Abstract
Immunoglobulins are key proteins that function to preserve immune homeostasis and are quantified to infer changes to the acquired humoral immune response in mammals. Measuring immunoglobulins in non-traditional wildlife for immune surveillance often requires ingenuity, and rigorous standardization of methodologies to provide consistent and reliable results especially for species lacking species-specific reagents. We modified and optimized existing ELISA methodology that utilizes the binding properties of Staphylococcus derived Protein A (Prt A) with immunoglobulin G (IgG). We validated the assay for accurately quantifying IgG in Steller sea lion (SSL) serum using critical measures of validation including parallelism, spike and percent recoveries, and internal controls. Of the modifications made, heat inactivation of SSL serum proved pivotal to enhance accuracy and precision of IgG detection by improving linearity and percent recovery in parallelism dilutions and serum spikes. Purified canine IgG standard was not affected by the heat inactivation protocol. These results support that confounding serum proteins likely interfere with the binding of Prt A with IgG, and demonstrate the need for heat inactivation of serum to ensure optimal IgG quantification using the PrtA-ELISA. Further, incorporation of spike and recovery are essential validation measures to ensure proper standardization of this assay. Consequently, the improved and validated PrtA-ELISA guarantees a species-independent IgG detection with validation criteria to enhance accuracy, and precision for addressing future immunological questions concerning SSLs and other non-traditional wildlife in a clinical, ecological, and conservation context.
NCR−ILC3 as the initial responders to liver inflammatory environment promote HCC development through IL-23/IL-17 axis
Authors: Liu Yonghao , Song Yuan , Lei Lei , Huang Yuhui , Liu Haiyan 
Published: January 2023

Abstract
IL-17 is crucial in tumor-promoting inflammation and the development of hepatocellular carcinoma (HCC). However, the role of IL-23, an important IL-17-inducing inflammatory cytokine, as well as the immune cells responsible for initiating IL-17 production in the tumor microenvironment are not clear. In the current study, we assessed the development of HCC and the function of the adaptive anti-tumor immune response, as well as the kinetics of the development of IL-17-producing cells in an IL-23-rich tumor microenvironment. Our results demonstrated that IL-23 promoted HCC tumor growth and inhibited anti-tumor adaptive immune response by enhancing IL-17A production in the tumor microenvironment. Furthermore, our kinetic analysis showed that NCR−ILC3 were the first responders to IL-23, producing IL-17 during the early phase of tumor development. Their proliferation in vivo was facilitated by IL-23, and adoptive transfer of NCR−ILC3 cells promoted HCC development. NCR−ILC3 could suppress IFN-γ production of both CD4+ and CD8+ T cells, and inhibit proliferation and survival of CD8+ T cells in vitro. Moreover, the percentage of IFN-γ-producing ILC1 were significantly reduced by IL-23 in the liver during tumor development, and they were shown to be able to differentiate into ILC3 in the presence of IL-1β and/or IL-23 in vitro. The adoptive transfer experiment further demonstrated that ILC1 could gain RORγt expression in an IL-23-expressing HCC model. Therefore, our findings demonstrated that IL-23 could promote HCC development through enhancing IL-17 production, and NCR−ILC3 as the initial IL-17-producing cells in response to IL-23 in the liver could be the initiator of the IL-17-rich immune-suppressive tumor microenvironment.
Phosphatidylserine targeting antibody in combination with tumor radiation and immune checkpoint blockade promotes anti-tumor activity in mouse B16 melanoma
Authors: Budha Sadnu , Giese Rachel , Gupta Aditi , De Henau Olivier , Zappasodi Roberta , Campesato Luis Felipe , Barker Christopher , Shan Joseph , Wolchok Jedd D , Merghoub Taha 
Published: January 2023

Abstract
Phosphatidylserine (PS) is a phospholipid that is exposed on surface of apoptotic cells, some tumor cells and tumor endothelium. It has been shown to promote immunosuppressive signals in the tumor microenvironment. Radiation therapy (RT) is an effective focal treatment of primary solid tumors, but is less effective in treating metastatic solid tumors as a monotherapy. There is evidence that RT induces immunogenic tumor cell death and enhances tumor-specific T cell infiltration in treated tumors. In this study, we show that irradiation of B16 melanoma causes an increase in PS expression on the surface of viable tumor and immune infiltrates. We subsequently examined the effects of combining RT with an antibody that targets PS (mch1N11) and immune checkpoint blockade (anti-PD-1) in B16 melanoma. We found that treatment with mch1N11 synergizes with RT to improve anti-tumor activity and overall survival in tumor bearing mice. In addition, the triple combination of mch1N11, RT and anti-PD-1 treatment displayed even greater anti-tumor and survival benefit. Analysis of immune responses in the tumors of treated animals revealed a shift towards a pro-inflammatory M1 phenotype after treatment with RT and mch1N11. In addition, analysis of the adaptive immune response revealed an increase in antigen-specific CD8 T cell infiltration in the tumors as well as increased activation, effector function and differentiation in the triple combination therapy. This finding highlights the potential of combining these agents to improve outcome in patients with advanced-stage cancer and may inform the design of future clinical trials.
Blocking colony stimulating factor 1 receptor (CSF-1R) and tropomyosin receptor kinase A (TrkA) improves the antitumor efficacy of immune checkpoint blockade
Authors: Duffy Colm R. , Mok Stephen , Allison James P. 
Published: January 2023

Abstract
Established tumors can escape immune responses by secreting the cytokine colony stimulating factor 1 (CSF-1), stimulating the proliferation and recruitment of immunosuppressive myeloid cells to the tumor microenvironment by binding to colony stimulating factor-1 receptor (CSF-1R). Additionally, the neurotrophin nerve growth factor (NGF) is a ligand for tropomyosin receptor kinase A (TrkA), which is over-expressed on multiple tumor types. Signaling through TrkA via the Akt and MAPK pathways regulates survival, proliferation and invasion of tumor cells. This invasion can occur around nerves, and perineural invasion (PNI) has emerged as a critical pathological feature of multiple malignancies, resulting in poor prognosis.
PLX7486 is a novel orally bioavailable small molecule Trk and CSF-1R dual-inhibitor that is now being studied in Phase I clinical trials. We hypothesized that PLX7486 would synergize with immune checkpoint blockade to result in a greater antitumor effect, by targeting Trk signaling directly on cancer cells and by inhibiting the recruitment of immunosuppressive myeloid cells through CSF-1R, thus enabling an improved antitumor T cell response.
We confirmed the expression of TrkA receptor on multiple murine cancer cell lines in vitro and exposed them to PLX7486, showing a direct cytotoxic effect with an IC50 of 5–8μM on most of the cell lines and an inhibition of AKT pathway signaling in MC38 cells. PLX7486 also had a direct cytotoxic effect on bone marrow-derived macrophages and the murine macrophage cell line RAW264.7 with an IC50 &lt;1μM, while it had no effect on activated T cells in vitro. Combining PLX7486 and anti-CTLA-4 or anti-PD-1 in vivo in MC38 and B16F10 tumor models resulted in increased antitumor effects.
The Curative Effect of Cryptotanshinone and anti-PD-L1 on Mouse Hepatocellular Carcinoma
Authors: Han Zhen , Liu Shuo , Lin Hongsheng , Trivett Anna , Yang De , Oppenheim Joost J. 
Published: January 2023

Abstract
Cryptotanshinone (CT), isolated, purified and crystalized from the dried roots of Salvia militorrhiza, has been reported to have cytotoxic antitumor effects on a human liver cancer cell line in vitro. However, we have shown that CT also has immunoadjuvant effects and augments Th1 antitumor immunity. CT inhibited the proliferation of mouse hepatoma (Hepa1-6) cells by inducing Hepa1-6 cells apoptosis by dose dependent inhibition of the STAT3 signaling pathway. Furthermore, CT could also induce RAW264.7 cells polarize toward a M1 phenotype presumably with cytotoxic anti-tumor effects. CT was effective in the treatment of established Hepa1-6 hepatoma tumors in mice in combination with anti-PD-L1, and can cure Hepa1-6-bearing mice with the induction of subsequent anti-Hepa1-6 specific long-term immunity. Our data indicate that CT and anti-PD-L1 result in an effective immunotherapeutic regimen for the treatment of human hepatocellular carcinoma (HCC) based both on the cytotoxic effects and induction of immunity to the tumor.
Improvement of agonist cancer immunotherapy by CD73 blockade
Authors: Chen Siqi , Fan Jie , zhang minghui , Qin Lei , Dominguez Donye , Long Alan , Zhang Bin 
Published: January 2023

Abstract
Agonist antibodies (Ab) can directly target costimulatory molecule on the surface of antigen-primed T-lymphocytes. Among those, tumor necrosis factor receptor superfamily (TNFRSF) agonists are attractive and some of them are currently in various stages of pre-clinical and clinical trials, including anti-4-1BB, anti-GITR and anti-OX40, albeit with limited therapeutic benefit as single agents. Here, we examined the inhibitory role of ecto-enzyme CD73 (generating adenosine) on the antitumor efficacy of agonistic anti-4-1BB/CD137 Ab therapy, as anti-4-1BB treatment preferentially drove CD73− effector T cell response within TGF-β-low tumor microenvironment. The anti-CD73/anti-4-1BB combination therapy induced tumor regression associated with enhanced antitumor CD8+ T cell responses and reduced CD4+Foxp3+ regulatory T cell (Treg) accumulation. However, the TGF-β-rich tumor milieu confered resistance to anti-4-1BB therapy with sustained CD73 expression on both infiltrating CD4+ and CD8+ T cells across several tumor models. Our study establishes a novel mechanistic link between the CD73 axis and engagement of 4-1BB and possibly other costimulatory molecules. Thus, CD73 blockade has great potential for more effective clinical targeting of TNFRSF agonists.
Immunological Insights Into Liver Metastasis Associated Resistance To Checkpoint Blockade Cancer Immunotherapy
Authors: Lee James , Mehdizadeh Sadaf , Tsai Katy , Algazi Alain , Rosenblum Michael , Daud Adil , Bluestone Jeffrey A. 
Published: January 2023

Abstract
Despite advancements in cancer immunotherapy, we have previously reported that stage IV melanoma and lung cancer patients with liver metastasis have decreased response rate to PD-1 blockade and shorter survival. A uniquely tolerogenic organ, the liver is the most common site of metastases of the majority of cancers, and cancer patients with liver metastasis have much worse survival overall. Thus, we hypothesized that cancer cells that metastasize to the liver may utilize liver-mediated tolerance mechanisms to decrease systemic anti-tumor immunity and render immunotherapies less effective.
Metastatic melanoma patients with liver metastasis or other organ metastasis were biopsied pre and post PD-1 treatment and their distant primary site’s tumor immune infiltrates (TILs) were analyzed by FACS. Patients with liver metastasis had significantly lower CD8/Foxp3+ Treg ratio and decreased percentage of activated PD-1+/CTLA-4+ CD8 cells from their biopsy samples, suggesting a systemic influence on tumor immunity. To investigate the mechanism, C57BL/6 mice were implanted with “primary” subcutaneous (SQ) tumors and experimental “metastatic” tumors in the liver or control organ (lungs) and given systemic PD-1 blockade. Mice with liver metastasis had shortened survival and lower response rate to PD-1 blockade at the SQ site. TILs analysis within the SQ tumor of animals with liver metastasis shows lower CD8/Foxp3+ Treg ratio, lower expression of post-activation makers PD-1, CTLA-4, ICOS, and Ki67, and decreased tumor antigen tetramer-positive CD8 cells in a Treg dependent manner. These results indicate liver metastasis may induce systemic immune suppression to the detriment of stage IV cancer patients undergoing PD-1 blockade therapy.
Commensal dysbiosis modulates the tumor microenvironment in breast cancer and diminishes efficacy of PD-L1 inhibition
Authors: Rutkowski Melanie R. , Rosean Claire M. , Bostic Raegan , Feng Tzu-Yu 
Published: January 2023

Abstract
For patients with HR+ breast cancer, non-responsiveness to PD-1/PD-L1 blockade cannot be attributed to poor immunogenicity, as immune-stimulatory gene signatures are associated with greater long-term survival and enhanced responsiveness to therapy. Targeting myeloid-driven immune suppression has also been shown to enhance efficacy of PD-L1 blockade in non-responsive tumors. However, we find that in HR+ breast cancer, synergy with PD-L1 blockade is negatively influenced by a pre-established loss of commensal homeostasis, or commensal dysbiosis. Using a clinically relevant non-responsive HR+ breast tumor model, establishing commensal dysbiosis prior to breast tumor initiation drives significant and irreversible T cell dysfunction within primary tumors. Additionally, commensal dysbiosis alters the cellular composition within the tumor microenvironment, culminating in the recruitment and polarization of M2-like macrophages with distinct tumor-suppressive attributes. Systemically, tumor-bearing animals with established commensal dysbiosis have significantly higher levels of prostaglandin E2 with the serum, which is independent of tumor volume. However, blockade of COX-2 only synergizes with PD-L1 blockade in animals without commensal dysbiosis. Unexpectedly, we found that commensal dysbiosis enhances COX-1/PGE2 signaling within the mammary epithelium during early tumor progression. Inhibition of COX-1-mediated PGE2 synthesis during early stages of tumor progression resulted in delayed tumor progression only in dysbiotic tumor-bearing mice, suggesting that dysbiosis-induced COX-1 mediated tissue inflammation is sufficient to drive irreversible immune suppression during advanced breast cancer.
IL-17C inhibits the development of hepatocellular carcinoma through the modulation of gut microbiota
Authors: Gong Huanle , Ma Shoubao , Dong Chen , Liu Haiyan 
Published: January 2023

Abstract
IL-17C has been recently reported to play an important role in infections and inflammatory diseases. However, its function in anti-tumor immune response is not clear. Here, we investigated the role of IL-17C in murine hepatocellular carcinoma (HCC) models and found that genetic depletion of IL-17C significantly promoted HCC growth, whilst overexpression of IL-17C inhibited HCC development. Flow cytometry analysis further demonstrated that the percentage of CD8+ T cells was reduced in IL-17C−/− mice. Moreover, the activation of T cells was also significantly reduced when IL-17C was deficient. Interestingly, depletion of gut microbiota with antibiotics treatment diminished the difference in tumor burdens between WT and IL-17C−/− groups, indicating microbiota might be involved in the process of IL-17C regulating HCC development. In addition, 16sRNA sequencing results revealed that IL-17C deficiency significantly reduced the Lactobacillus acidophilus colonization. Collectively, we found that IL-17C suppressed HCC growth in a gut microbiota-dependent manner. Further studies are needed to confirm the contribution of L.acidophilus to HCC development and investigate the detailed mechanisms of IL-17C regulating anti-tumor immune response.
Tim-3 and TIGIT mark Natural Killer cells susceptible to effector dysfunction in human bladder cancer
Authors: Farkas Adam M. , Audenet Francois , Anastos Harry , Galsky Matthew , Sfakianos John , Bhardwaj Nina 
Published: January 2023

Abstract
Natural Killer cell (NK) effector functions are diminished during cancer but the phenotype and mediators of this process are unclear. In contrast, dysfunctional T cells express markers of “exhaustion” (e.g. CTLA-4 and PD-1), and inhibition of signaling through these molecules has improved clinical outcome in multiple tumors. Our data from 100+ patients with bladder cancer (BC) suggests that NK undergo an analogous process of exhaustion marked by up-regulation of Tim-3 and TIGIT in both the periphery and tumor. Tim-3 and TIGIT are induced on both immature and mature NK subsets suggesting implications for normal NK maturation. The magnitude of Tim-3 expression in both tissues is tuned to the invasiveness of the primary tumor, while TIGIT is not, making Tim-3 function as a rheostat for primary tumor invasiveness. Both molecules are expressed at equivalent frequencies in both tissues independent of the magnitude of overall expression but define NK with different effector functions. Peripheral NK from BC patients are functionally comparable to NK from healthy donors in their ability to produce IFNγ and degranulate in response to target cells, while tumor NK are refractory to both stimuli. NK from tumors are not terminally exhausted as they make IFNγ, TNFa, and degranulate after “resting” ex vivo prior to stimulation. Blockade of Tim-3, but not TIGIT, enhances effector function in peripheral NK from BC patients, but is ineffective for NK in tumor tissue implicating tumor-specific factors in mediating NK dysfunction. Tim-3 blockade was most efficient in NK from BC patients after activation with IL-15 suggesting specific inflammatory cues affect responsiveness to checkpoint blockade. Ongoing work will develop strategies to reverse NK exhaustion.