bluuebunny/crossref_metadata_2025_split huggingface dataset:
A pan-allele Class I MHC prediction tool in mice enables tumor immunology studies
Authors: DeVette Christa Innae , Andreatta Massimo , Welm Alana , Nielsen Morten , Hildebrand William H 
Published: January 2023

Abstract
An understanding of tumor-presented Class I MHC peptides is a prerequisite to effective cancer immunotherapy, and more tools are needed to elucidate immune interactions in breast cancer. One tool, the mouse mammary tumor virus (MMTV)-Polyoma Middle T Antigen (PyMT) mouse, models the development of spontaneous metastatic breast cancer in vivo. Surprisingly, little data exists for the MHCI of MMTV-PyMT tumors, which express the H2-q haplotype. To develop the MMTV-PyMT tumor model for tumor immunology, we present an open-access Class I MHC peptide prediction tool for multiple murine haplotypes (including H2-q): NetH2pan (http://www.cbs.dtu.dk/services//NetH2pan/). H2-d/k/b peptides were obtained from the IEDB, while H2-q peptides were eluted from MHC: HeLa and 721.221 cell lines transfected with PyMT and soluble H2-Kq/Dq. Cells secreting these MHC I were expanded, peptide-bearing MHC I affinity purified, and presented peptides analyzed by mass spectrometry. The identified peptide sequence data were used to develop a predictive algorithm to enable in silico candidate peptide searches. Validation of the algorithm using peptides eluted from tumors demonstrated a positive predictive value (34%) improved from other MHC I prediction tools. For a candidate antigen PyMT, the 5 PyMT-peptides eluted from tumors were in the top 25 predicted. Three of 5 peptides were immunogenic as determined by IFNγ ELISpot and peptide:MHCI tetramers in vaccinated mice, and correlated with tumor prevention. These data show that NetH2pan can predict immunogenic epitopes with high-fidelity and that these peptides can be utilized for vaccine development. These studies enable the MMTV-PyMT model for mechanistic studies involving tumor-T cell crosstalk.
Δ42PD-1 inhibitor acts as a PD-1-independent immunotherapy against human hepatocellular carcinoma
Authors: TAN ZHIWU , Chiu Mei Sum , Yan Chi Wing , Yang Xinxiang , Wong YIK CHUN , Man Kwan , Cheng Alfred Sze-Lok , Chen Zhiwei 
Published: January 2023

Abstract
Pathogen infection-induced chronic inflammation is a key contributor to both carcinogenesis and progression of hepatocellular carcinoma (HCC). Persistent toll-like receptor 4 (TLR4) signaling activation can lead to inflammatory responses and chronic inflammation in the tumor microenvironment. Previously, Δ42PD1, a PD1 isoform, has been shown to directly interact with its cognate receptor TLR4 and contribute to innate immune activation and intestinal pathogenesis during acute HIV-1 infection. Here, we aim to determine whether such interaction may also occur in HCC to promote TLR4 signaling activation and in turn induce inflammatory responses, contributing to HCC progression. We studied the relationship between TLR4, Δ42PD1, pro-inflammatory cytokines and tumor development in humanized mouse models and HCC patients. In consistence with others’ findings, TLR4 signal was shown to be associated with chronic inflammation and HCC tumor progression. Furthermore, we demonstrated that Δ42PD1 activated TLR4-mediated inflammatory responses in HCC cells via cJUN MAPK transcriptional activation. Notably, Δ42PD1-TLR4 pathway activation likely formed a feedback loop to induce Δ42PD1-expression CD3+ T cells, a new phenotype of T cell exhaustion, further contributing to tumor development. Importantly, administration of a Δ42PD1-specific monoclonal antibody CH101 interrupted Δ42PD1-TLR4 pathway and displayed therapeutic efficacy in both subcutaneous and orthotopic HCC models. Together, our results demonstrated the importance of Δ42PD1-TLR4-mediated inflammatory responses in promoting HCC and provided conclusive evidence that targeting Δ42PD1-TLR4 pathway reduced inflammatory responses to arrest HCC development.
Cell-intrinsic programmed death ligand-1 (PD-L1) inhibits cytotoxic chemo, promotes DNA damage repair, and enhances ATM/ATR signaling following exposure to DNA damaging agents in bladder, melanoma, and ovarian cancer cells
Authors: Kornepati Anand V , Zhang Deyi , Hambright Heather G. , Kari Suresh C. , Deng Yilun , Clark Curtis A. , Gupta Harshita B. , Chakravarty Rekha , Hu Yanfen , Li Rong , Curiel Tyler J. 
Published: January 2023

Abstract
We have shown that cancer cell-intrinsic PD-L1 signals increase mTORC1 and unbundle actin. To test if tumor cell PD-L1 affects the DNA damage response (DDR) we made PD-L1KO mouse ID8agg (ovarian) and B16 (melanoma), and human bladder RT4 cancer lines. The DNA damaging agent gemcitabine (gem) reduced PD-L1KO ID8agg and RT4 viability in vitro vs. control (con) (ID8agg: 29X vs. 1.3X; RT4: 2X vs 1.1X). 4h gem increased phospho (p)-γH2AX, a marker of DNA double strand breaks: 2X in PD-L1KO vs. con RT4. P-γH2AX stayed abnormally high after gem removal, further showing defective DDR in PD-L1KO. 24h gem also induced more p-γH2AX in PD-L1KO vs. con ID8agg, and 48h gem induced more apoptosis in PD-L1KO vs. con RT4. Cisplatin, a DNA damaging agent distinct from gem, reduced PD-L1KO ID8agg viability 9X vs. 2X in con. No viability difference was seen in cisplatin treated PD-L1KO vs. con RT4. Thus, PD-L1 chemo resistance effects are cell type and/or agent specific. To define DDR mechanism(s), we found that 24h gem decreased p-Chk2 (ATM DDR pathway), due to reduced total Chk2, but not p-Chk1 or total Chk1 (ATR DDR pathway) in PD-L1KO vs. con RT4. 20 Gy X-rays (DNA damage distinct vs. gem), reduced p-Chk1 in PD-L1KO but not con B16 and induced less p-RPA32 in PD-L1KO vs. con B16, suggesting defects specifically in homologous recombination mediated DNA repair. Thus, cell-intrinsic PD-L1 DDR control is insult and/or cell type specific. RT4 DDR effects were independent of mTOR or actin. DDR affects immunotherapy so PD-L1 cell-intrinsic DDR control could be exploited in treatment or response biomarkers. DDR inhibitors, chemo or radiation combos could improve anti-PD-L1 efficacy in distinct cancers, subjects of ongoing work, as is testing tumor cell intrinsic PD-1 effects.
PD1 blockade modulates Ca2+ fluxes in T cells of head and neck cancer patients
Authors: Gawali Vaibhavkumar Shantaram , Chimote Ameet A , Chirra Martina , Janssen Edith , Wise-Draper Trisha , Conforti Laura 
Published: January 2023

Abstract
Immunotherapy offers promising results in head and neck cancer (HNC) albeit it faces the problem of high resistance. PD1 (programmed death receptor-1) and its ligand (PD-L1/PD-L2) interaction reduces T cell receptor (TCR)-stimulated Ca2+ fluxes. Ion channels regulate Ca2+ entry in T cells downstream to the TCR (store-operated Ca2+ entry, SOCE). We hypothesized that anti-PD1 antibody (Pembrolizumab, Pembro) increases SOCE. We studied the effect of Pembro and PD-L1 on SOCE in CD8+ peripheral blood T cells (PBTs) of HNC patients and healthy donors (HD) using Thapsigargin (TG). Additionally, we tested the effect of Pembro on KCa3.1, Kv1.3 and Ca2+ release activated Ca2+ (CRAC) channels in PBTs of HNC patients using patch-clamp electrophysiology. TCR-mediated Ca2+ fluxes were assessed in PD1-expressing NFAT-reporter Jurkat cells. In these cells, Pembro increased the number of cells that responded to TCR stimulation with a sustained increase in Ca2+ (30%, p&lt;0.001). Pembro increased SOCE by 55% (p&lt;0.02) in PBTs of a subset of HNC patients (5/9). Baseline SOCE (before Pembro) was lower in the HNC PBTs that positively responded to Pembro compared to untreated HD PBTs (p&lt;0.006). PD-L1 decreases the Ca2+ response, which was reversed by Pembro in HD PBTs (p&lt;0.05). Pembro did not alter the expression of ion channels, but significantly increased KCa3.1 and Kv1.3 conductance in a subset of patients (5–6/8) while it increased divalent free (DVF) currents through CRAC channels only in 2/5 patients. These data suggest that Pembro enhances SOCE in T cells by modulating ion channels activity. This mechanism may contribute to the improved functionality of T cells in cancer patients undergoing immunotherapy. (DoD CA160714; 2-R01-CA95286)
Single Cell RNA-sequencing Identifies Distinct Fibroblast Subsets in a Human 3D Skin Model of Systemic Sclerosis
Authors: Kosarek Noelle Noreen , Wheeler Tamar , Toledo Diana Maria , Huang Mengqi , Pioli Patricia A , Whitfield Michael 
Published: January 2023

Abstract
Systemic sclerosis (SSc) is a multisystem autoimmune disease characterized by skin and organ fibrosis, autoantibody formation, and vascular injury. Due to the systemic nature of this disease, 30–40% of patients die within the first 10 years after diagnosis. There are currently no FDA-approved disease-modifying therapies for SSc and available treatments fail to target the origins of skin and organ fibrosis 1. There is a need to identify targetable cell populations and molecular mediators responsible for disease. Here we report single cell RNA-sequencing (scRNA-seq) profiles of 11,140 human fibroblasts, macrophages, and keratinocytes from eight 3D SSc human skin equivalents 2. We isolated 956 macrophages, 606 keratinocytes, and 9578 fibroblasts from this model system. The keratinocyte cluster was identified by high expression of keratin genes such as KRT6A and KRT16, while the macrophage cluster was identified by upregulation of HLA-DR, CD163, and CD206. The fibroblast population was composed of six different subpopulations distinguished by differentially expressed features. Three of the fibroblast subpopulations were driven by genes encoding collagen proteins such as COL1A1 and COL3A1, while the other three subpopulations were driven by genes encoding mitochondrial and ribosomal components, metal and ion binding proteins, and lipoproteins. These distinct fibroblast subpopulations may represent targetable cell types for therapeutic intervention.
Effects of a-lysophosphatidylinositol (LPI) on kinase activation and cytoskeletal re-arrangements leading to migration in mast cells
Authors: Aguilar Lizbeth Magnolia Martinez , Ibarra-Sánchez Alfredo , Macías-Silva Marina , González-Espinosa Claudia 
Published: January 2023

Abstract
Mast Cells (MCs) initiate allergic responses but also participate in innate inflammatory reactions triggered by infection, tissue damage and cancer. MCs are recruited to affected sites where they release preformed and neo-formed mediators of inflammation. Bioactive lipids (such as sphingosine-1-phosphate, S1P) have demonstrated to induce MC chemotaxis. Recently, α-lysophosphatidilinositol (LPI) has awakened scientific interest due to the discovery of its endogenous receptor, the orphan receptor GPCR55. This receptor has been shown to couple to Gα12/13 pathway, controlling actin polimeryzation and cell migration. In this study, we aimed to characterize the role of LPI in the activation and migration of MCs. Using murine bone marrow-derived MCs, we observed that low concentrations of this lipid (10 nM and 100 nM) induces maximal ERK 1/2 phosphorylation at 15 and 10 minutes, respectively. Also, we found that LIMK (an important G12/13 and Rho-dependent kinase) is phosphorylated after 5 and 10 min after 10 nM or 100 nM LPI, respectively. Both ERK 1/2 and LIMK phosphorylation patterns were reproduced with the selective agonist of GPCR55, O-1602, at equi-potent concentrations to those of the LPI. Using Boyden chamber assays, we observed that LPI provocked higher chemotaxis of MCs when compared with S1P. Finally, using confocal microscopy, we observed that LPI triggered depolymerization of cortical actin, as well as the formation of filopodia between 15 and 30 minutes post stimulation. We concluded that GPR55 expressed in MCs induces important changes on MAPK and LIMK activation and its ligand, LPI, is a potent chemotactic factor of MCs.
Complement protein C1q modulates endothelial cell functions in atherosclerosis
Authors: Monsibais Alisha N , Espericueta Victoria , Megliola Leilani , Fraser Deborah A 
Published: January 2023

Abstract
Atherosclerosis is a chronic inflammatory disease characterized by the development of plaque in arteries. Interactions between monocytes, endothelial cells and lipoproteins play a major role in the disease pathogenesis. C1q is traditionally known for activation of the complement cascade, leading to inflammation and clearance of foreign targets. However, we previously showed that C1q bound to modified lipoproteins (e.g. oxLDL) plays a protective role by polarizing monocytes and macrophages towards an anti-inflammatory state during engulfment and increases survival and efferocytic function of macrophage foam cells. Additionally, C1q modulates macrophage chemokine production in in vitro and in in vitro models of atherosclerosis. Here we investigate C1q modulation of both endothelial cell chemokine production, and functions affecting monocyte migration. Human aortic endothelial cells (HAEC) were incubated with oxLDL +/− C1q. RNA and supernatants were collected for chemokine gene regulation and protein analysis, respectively. Monocyte adhesion and transendothelial migration were also measured. The data show that C1q modulates chemokine gene and protein expression, increases monocyte adhesion and transendothelial migration and enhances HAEC monolayer permeability. Thus, C1q interactions with HAEC in hyperlipidemic conditions may result in increased availability of phagocytes for clearance in the early atherosclerotic lesion. However in later stages infiltrating cells may contribute to disease progression through an increased proinflammatory environment and complement activation. Understanding timing and molecular mechanisms in atherosclerosis will be vital for the development of novel treatments.
Inhibition of trans-endothelial migration of HIV-1 infected myeloid cells using virus expression dependent anti-ALCAM CRISPR-Cas9
Authors: Kaminski Rafal , Dabrowski Konrad , Luongo Francesca Paola , Myhlyk Maksym , Ebo Sunday , Rom Slava , Young Won-Bin , Burdo Tricia 
Published: January 2023

Abstract
Migration of HIV-1 infected monocytes across the endothelial barrier plays an essential role in establishing and maintenance of viral reservoir in the brain and continues despite antiretroviral therapy (ART). Using the CRISPR-Cas9 gene-editing platform, we created an inducible knockout for activated leukocytes cell adhesion molecule gene (ALCAM/CD166), an HIV-1 host dependency factor shown to play a critical role in CNS entry of infected myeloid cells. First, using lentiviral delivery, we developed several ALCAM knockout clones in monocytic U937, and their latently infected with HIV-1 equivalent: U1 cells. As expected, ALCAM−/− myeloid cells showed markedly reduced adhesion to and transmigration through monolayers of endothelial cells. Next, using AAV6 delivery, we replicated these results in primary human monocytes from three different healthy donors. Finally, to limit CRISPR-Cas9 editing to HIV-1 infected cells, we placed Cas9 expression under the control of minimal HIV-1 Tat responsive LTR promoter (−80/+66). HIV-1BAL-GFP infection of AAV6/adeno-LTR-CRISPR-anti-ALCAM treated CD4+ T cells, monocytes, and monocyte-derived macrophages (MDMs) resulted in the induction of Cas9 expression and CRISPR mediated cleavage of exon 1 of ALCAM gene in Tat expression dependent manner and inhibition of their transendothelial migration. We demonstrated the feasibility of using CRISPR gene editing to disrupt a single CAM gene, i.e, ALCAM to disable adhesion and transmigration ability of HIV-1 infected myeloid cell line cells, primary monocytes, and macrophages. Moreover, we provided data proving the achievability of creating an HIV expression dependent CRISPR platform to restrict Cas9 cleavage activity only to HIV infected cells.
IL-4 regulates resident memory CD8+ T cell formation and persistence
Authors: Mora-Buch Rut , Akbaba Hasan , Chasse Alexandra Y , Sama Andrea M , Mani Vinidhra , Bromley Shannon K 
Published: January 2023

Abstract
Tissue resident memory CD8+ T cells (TRM) persist long-term in lymphoid and non-lymphoid tissues to provide rapid, local defense against pathogens and tumors. Tissue cytokines drive the formation and persistence of CD8+ TRM. However, whether the local microenvironment can inhibit TRM formation is largely unknown. Individuals suffering from conditions associated with T helper 2 (Th2)-type immune responses, such as atopic dermatitis, asthma, and some cancers, present impaired anti-viral or anti-tumor responses. We hypothesize that Th2 cytokines inhibit the formation and/or persistence of CD8+ TRM. We demonstrate that Th2 cell supernatant downregulated TGF-β-induced CD103 expression on in vitro- and in vivo- activated CD8+ T cells in an IL-4- and Stat6-dependent manner. Moreover, IL-4 induced Eomes expression by TGF-β-stimulated CD8+ T cells. In vivo, more CD8+CD103+ TRM populated the skin of Il4ra−/− and Stat6−/− mice compared to wild-type mice at homeostasis. Additionally, CD8+ T cell activation in the presence of IL-4 impaired subsequent CD8+ TRM formation; IL-4-exposed CD8+ T cells expressed decreased CD103 in a mouse model of cutaneous inflammation, and these cells failed to persist within the skin at memory time-points. These data suggest that IL-4 counters TGF-β-mediated signals to inhibit CD8+ TRM formation/persistence and may allow identification of novel treatments to boost anti-tumor immunity and anti-viral responses in patients with type 2 inflammation.
Hot Spot-type Questions offered via ExamSoft to learn Immunology
Authors: Fazal Nadeem , Olk Sandra , Edquiban Marianne , Fadhil Shams 
Published: January 2023

Abstract
There are many ways to promote meaningful student learning of Immunology. Hot Spot question format is a new type of active learning tool. It indicates that having planned repetition over an extended period of time leads to better retention by including images with text during instruction. This leads to long-term retention and improved student outcomes. In Hot Spot, users indicate the answer by clicking a specific area of an image. First year pharmacy students participated in a 3-hour long Immunology workshop. The students reviewed a video and handout explaining Hot Spot before coming to the workshop. Hot Spot questions containing basic Immunology were prepared for the workshop. A class of sixty two students were given ExamSoft online platform to take a pre-workshop quiz. The students were split into small learning groups and were given half an hour to discuss the pre-workshop content of basic Immunology. After half an hour, the students were randomly asked to pick up a partner to take ExamSoft quiz assessment again in pairs. The difference between pre- and post-workshop was that the students were paired in post-workshop quiz and were allowed to discuss concepts of Immunology with their partners. This pairing exercise and use of Hot Spot type assessment enhanced learning of basic concepts of Immunology and was evident from post-workshop quiz grades. The students not only did better on assessment grades but the post-workshop student feed back surveys also greatly commended this type of learning method. Therefore, we recommend learning basic Immunology through workshops, pairing of students and use of Hot Spot assessments. We also advocate teaching all basic pharmaceutical sciences via interactive educational tools for enhancing student learning.
Optimization of a T cells education protocol to treat hematological cancers
Authors: MATHIEU Cedric , MINGUY Annabelle , TROTTIER Jessica , SANCHEZ-DARDON Jaime , DAVE Vibhuti , ROY Denis-Claude 
Published: January 2023

Abstract
Allogeneic hematopoietic stem cell transplantation is the sole curative treatment for patients with high-risk hematological cancers. Most of its activity relies on the ability of engrafted T cells to recognize minor histocompatibility antigens (MiHAs) expressed on malignant cells resulting in their elimination. However, donor lymphocytes can also recognize MiHAs expressed on non-hematopoietic cells leading to graft-versus-host disease. Hence, the expansion of T lymphocytes displaying potent cytotoxic activity toward MiHA(s) preferentially expressed on leukemic cells would be greatly beneficial for engrafted patients. Using a proteo-genomic approach, novel MiHAs that are predominantly expressed on hematopoietic cells have been identified. We previously showed that these novel MiHAs can be exploited for ex vivo expansion of the antigen specific T cells. However, the frequencies of these MiHA specific T cells are extremely low. Thus it is challenging to generate an optimal ex vivo expansion of antigen specific T cells to a single MiHA. In this context, we aim to improve this complex manufacturing process by using simultaneously multiple MiHAs to enhance the probability of obtaining response to at least one MiHA. This approach allowed us to expand T cells populations with distinct MiHAs specificities. Further, upon restimulation, MiHA specific T cells express high levels of inflammatory cytokines Interferon-γ and Tumor Necrosis Factor-α thus suggesting their functionality. In conclusion, multipeptide T cell stimulation allows for simultaneous expansion of functional T cells specific for several MiHA. This could be translated in improved therapeutical approach reducing the probability of immune escape of malignant cells.
QuickSwitch™: a validated platform for both screening MHC binding SARS-CoV-2 peptides and making SARS-CoV-2 specific MHC class I and II tetramers.
Authors: Poluektov Yuri O , Delcommenne Marc , Daftarian Pirouz M , George Marybeth 
Published: January 2023

Abstract
The success of SARS-CoV-2 vaccines is measured by their ability to mount long-lasting memory responses. To this end, it is important to identify viral protein fragments, or peptides, that generate the best immune response and lead to the development of memory T cells. For this purpose, researchers need a system that would allow them to rapidly test the ability of an antigenic peptide to be presented by the immune system and to identify T cells that can be stimulated by such a peptide in the context of the right MHC molecule. The QuickSwitch™ platform contains two modules for performing both functions. First a large number of SARS-CoV-2 peptides, including mutant peptides were incubated with QuickSwitch™ monomers or tetramers and exchanged against their exiting peptides in the first module. Peptide binding affinities to various human MHC Class I and Class II alleles, which are correlated with their exchange rates, were determined by the capture binding assay, the second QuickSwitch™ module. MHC tetramers built with SARS-CoV-2 high affinity MHC binding peptides were then used for T cell staining. Cell staining indicated that tetramers generated with the QuickSwitch™ platform were able to stain and identify T cells specific for the tested peptides. Further, peptides that bound to multiple MHC alleles, including potentially dominant peptides, were also detected. In conclusion, since QuickSwitch™ allows the screening of MHC binding SARS-CoV-2 peptides and, at the same time, the generation of corresponding tetramers, this platform is a useful tool for developing SARS-CoV-2 vaccines, monitoring T cell populations from vaccinated subjects and assessing the durability of mounted responses.