bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dengue virus induces human immune cells to secrete factors capable of tumor cell killing and activation of effector cells against treatment-resistant tumors
Authors: Goldufsky Josef William , Chen Tony , Singh Geeta , Zloza Andrew , Marzo Amanda Lee , Lyday Bruce 
Published: February 2023

Abstract
The use of oncolytic viruses (OVs) as immunotherapies has reinvigorated interest in microbial-based cancer therapies (MBCTs). However, thus far only one OV is FDA-approved and improvement in overall survival is experienced by a small proportion of treated patients. Seeking to take a bold approach, we pursued using Dengue virus (DV) as a novel cancer virotherapy. Thus far, DV has had little to no exploration in cancer, despite being well-studied in the vaccine context, where it demonstrates an outstanding safety profile. In our studies, we identified DV as having the ability to cause tumor cell death. Importantly, co-culture of human peripheral blood mononuclear cells (PBMCs) with DV resulted in cytokine production (including IL-2, IL-6, IL-7, IL-12, TNFα, and IFNγ) rather than cell death. We observed that supernatants from such co-cultures at 72 hours (m.o.i. of 2.0) were able to stimulate T cell- and NK cell-mediated killing of 624.28 melanoma cells, which are resistant to killing due to deletion of the HLA A2.1 gene. Surprisingly, these supernatants alone were also able to cause 624.28 cell death without effector cells or DV present. To determine whether DV could likewise reduce in vivo tumor growth, we utilized our DV plus dendritic cell (DC) platform, in which bone marrow-derived DCs deliver a relevant tumor antigen. C57BL/6 (B6) mice were challenged with B16 cells (5×104) via tail vein injection, resulting in lung melanoma foci. DV treatment (106 pfu, subcutaneous) with gp100-loaded DCs (6×106 via infusion) reduced melanoma foci in the lung by 51% (p&lt;0.05). Future research will focus on mechanisms underlying DV-driven anti-tumor immune responses and tumor cell killing, with the intent of translating these findings to the clinic.
Toward Sézary Syndrome immunotherapy
Authors: Marin Ana V. , Megino Rebeca F. , Viñuela Marcos , Popova Olga , Real-Arevalo Irene , Sanchez-Majano Jose L. , Ortiz-Romero Pablo L. , Castro-Panete Maria J. , Mancebo Esther , Talayero Paloma , Paz-Artal Estela , Paciello Maria L. , Martinez-Lopez Joaquin , Subiza Jose L. , Reche Pedro A. , Lopez-Bigas Nuria , Marcilla Miguel , Paradela Alberto , Moral Manuel Gomez del , Martinez-Naves Eduardo , Serrano Alvaro , Marina-Zarate Ester , Ramiro Almudena R. , Engel Pablo , Dominguez Mercedes , Moreno Inmaculada , Cortegano Isabel , de Andres Belen , Gaspar Maria L. , Garcia-Peydro Marina , Balas Antonio , Moreno Miguel A. , Alenda Raquel , Vicario Jose L. , Luescher Immanuel F. , Toribio Maria L. , Alarcon Balbino , Regueiro Jose R. 
Published: February 2023

Abstract
Sézary syndrome (SS) is a leukemic form of cutaneous mature T-cell lymphoma characterized by circulating malignant CD4 T lymphocytes (Sezary cells). Patients with SS have a poor prognosis and current treatment options show high rates of relapse, morbidity or mortality. Thus, there is an unmet need for an efficient and safe treatment. Sézary cells have unique clonal potentially targetable epitopes, including their TCR, and TCR- and neoantigen-derived HLA-restricted peptides.
Our general aim is to design a patient-tailored two-pronged strategy against SS. The specific aims are 1) to target SS clonal TCR B cell epitopes using mAb and/or CAR T cells, 2) to target SS HLA-restricted T-cell epitopes using TCR peptide- and/or neoantigen-specific human T cells, and 3) to validate efficacy in vitro and in mouse models.
For the generation of mAb, apheresis-purified SS cells or SS TCR CDR3beta peptides were used for immunizations, and screening was done on SS vs non-SS CD4 cells as defined by flow cytometry using CD26 and/or PD-1. For in vitro expansion of SS peptide-specific T cells, SS patient-derived non-SS PBMC were stimulated in 96-well plates with IL-2 and pooled HLA class I+II SS peptides, 10 μM each, defined by SS WGS, WES and RNAseq-based predictions or peptidome studies. After one week, cells were exposed to autologous DC pre-loaded with peptide pools, and cytokine production was analyzed by flow cytometry.
We have obtained preliminary data on aims 1 and 2 studying two SS patients with monoclonal T cell lymphomas, including potential mouse antibodies against a clonal SS TCR using cell and peptide immunization and T-cell hits that seem to be specific of a SS TCR HLA class-I-restricted CDR3beta sequence.
Evaluation of membrane bound IL-21 expanded natural killer cells for chronic lymphocytic leukemia therapy
Authors: Yano Max , Lance J. Rachel , Zapolnik Kevan , Mo Xiaokui , Lee Dean A , Byrd John , Muthusamy Natarajan 
Published: February 2023

Abstract
Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia and remains incurable despite recent therapeutic advances. Natural killer (NK) cells have potent anti-cancer effects, but efforts to use NK cell therapy in CLL have been limited by poor NK cell growth and anti-leukemia activity. We sought to determine if stimulating NK cells ex vivo with K562-based feeder cells expressing membrane-bound IL-21 (mbIL-21) (Denman et al., 2012) could overcome the limitations of prior techniques to create an effective therapy for CLL.
Our data demonstrate that healthy donor-derived mbIL-21 expanded NK cells have increased antibody-dependent cellular cytotoxicity (ADCC) and direct cytotoxicity (DC; without targeting antibody) against CLL cell lines. This technique can also overcome CLL-induced NK suppression – CLL patient-derived NK cells expanded equally and were as potent as normal donor-derived NKs, including against both allogeneic and autologous CLL tumor cells. CLL tumor cells are not susceptible to DC but are very vulnerable to ADCC with anti-CD20 antibodies (rituximab or obinutuzumab). The lack of direct cytotoxicity of tumor cells was correlated to low NK:CLL quantitative conjugate formation, suggesting a lack of tumor target recognition.
Blocking antibodies to FasL, TNFα, and TRAIL show that only TRAIL contributes modestly to anti-CLL tumor cytotoxicity, while application of folimycin demonstrates that perforin/granzyme release is the major mechanism of cytotoxicity. In vivo, treatment with expanded NK cells plus obinutuzumab significantly prolongs survival in the OSU-CLL human xenograft model. Based on these data, we are planning a clinical trial of mbIL-21 expanded NK cells plus obinutuzumab in CLL patients.
TetTCRII-SeqHD: High-dimensional analysis of antigen-specific ex vivo CD4+ T cells
Authors: Schonnesen Alexandra A , Xia Amanda , Parker Patrik , Jiang Ning 
Published: February 2023

Abstract

Objective:
We present a method for high-throughput and inexpensive generation of DRB1 tetramers to assess CD4+ T cell specificity with special applications in oncoimmunology.

Methods:
Peptides were generated by in vitro transcription and translation (IVTT) of dsDNA. These peptides were then loaded onto DRB1 MHC molecules via chemical exchange and tetramerized using DNA barcoded streptavidin as previously described. As a proof of concept, we generated 250 DNA-tetramers bearing peptide segments from a variety of naïve and experienced antigens. These tetramers were pooled and used to stain primary CD4+ T cells from healthy donors. These cells were then labeled with Abseq antibodies (BD Biosciences), sorted and loaded onto the BD Rhapsody.

Results:
We analyzed ~5000 CD4+ T cells from healthy peripheral blood and assessed their phenotype, specificity and TCR sequence. We demonstrated that this technique robustly identifies and characterizes antigen-specific CD4+ T cells. It replicates previous findings in low-throughput systems in terms of clonality and phenotype distribution across memory and naïve T cell phenotypes. Given these results, we then analyzed tumor infiltrating lymphocytes and identified foreign specific cells within this compartment. We also found explicit differences in the phenotypes of antigen specific cells within the tumor and a paired healthy tissue sample.

Conclusions:
In conclusion, we validate the use of a high-throughput technology for assessing the specificity, phenotype, TCR, and gene expression patterns in antigen-specific CD4+ T cells in peripheral blood and tumor infiltrating lymphocytes.
Accelerating ADCP screening using the iQue® Advanced Flow Cytometry Platform
Authors: McBain Kirsty A , Weldon Caroline , Szybut Clare , Dale Tim , Carter Mark 
Published: February 2023

Abstract
Antibody-dependent cellular phagocytosis (ADCP) is an immune process stimulated by monoclonal antibodies (mAbs) that promotes specific recruitment of phagocytes to eliminate targeted cancer cells. In vitro functional assays are used extensively in drug development to characterize the effects of mAbs on a range of mechanisms of action, including ADCP.
We describe a high throughput assay for ADCP quantification via co-localization analysis of labeled target cells with CD14 positive effector cells. Target cells were incubated in 96 or 384 well plates with clinically relevant mAbs prior to addition of PBMCs or isolated immune cells (e.g. monocytes or macrophages). Cells were analyzed using the iQue®3 Advanced Flow Cytometer and inbuilt software. Various effector:target (E:T) cell ratios were used to validate the assay.
Maximal ADCP of non-adherent Ramos target cells by PBMCs in the presence of Truxima was 43 ± 2% with an E:T ratio of 20:1, reducing to 19 ± 6% with a 5:1 ratio. Further pharmacological data was obtained for adherent AU565 cells, comparing the response between anti-HER2 mAb isotypes. The potency of response to anti-HER2-IgG1 mAb (Trastuzumab) was five times greater than to another native human isotype, anti-HER2-IgA2, with EC50 values of 6.6 ng/mL and 35 ng/mL, respectively. These data are in line with previous characterizations of isotype effector functions which showed increased ADCP functionality with IgG1 compared to IgA2 mAbs.
The iQue® platform provides a solution for rapid profiling of ADCP effector function in both adherent and suspension target cell models. This accelerates antibody discovery by highlighting potential efficacy and toxicity of antibody drug candidates, leading to greater productivity and insight.
Characterizing tumor-infiltrated immune cells with spatial context using an integrated RNAscope-immunohistochemistry workflow in FFPE tissues
Authors: Dikshit Anushka , Phatak Jyoti , Hernandez Lydia , Doolittle Emerald , Murlidhar Vasudha , Zhang Bingqing , Ma Xiao-Jun 
Published: February 2023

Abstract
Characterizing heterogenous populations of tumor-infiltrating immune cells requires a multi-omics approach. Here we demonstrate a newly developed integrated in situ hybridization (ISH) and immunohistochemistry (IHC/IF) workflow that can substantially improve RNA-protein co-detection, enabling the visualization and characterization of tumor immune infiltrates at single-cell and spatial resolution.
To characterize tumor-infiltrating immune cells in a tumor TMA (tumor microarray), we utilized the RNAscope Multiplex Fluorescence assay in combination with the RNA-Protein Co-detection Kit to detect multiple immune cell populations. Immune cells such as macrophages, T cells and NK cells were detected using antibodies against CD68, CD8, CD4 and CD56 in combination with probes targeting CCL5, IFNG, GNZB, IL-12, NCR1 etc.
We identified CD4+ regulatory T cells and CD8+ cytotoxic T lymphocytes. Additionally, we determine the activation states of CD8+ T cells by visualizing IFNG and GZMB expression. Furthermore, infiltrating macrophages were detected by CD68 protein expression while the M1 and M2 subsets were differentiated by using the M2-specific marker, CD163. NK cells were identified by detecting CD56 protein in combination with CCL5 and NCR1 RNA expression. The degree of immune cell infiltration varied based on the tumor type.
In conclusion, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies, including many previously incompatible antibodies with RNAscope . This new workflow provides a powerful approach to identifying and characterizing tumor infiltrating immune cells.
Immunogenomic determinants of tumor microenvironment correlate with survival in high-risk neuroblastoma
Authors: Bao Riyue , Spranger Stefani , Hernandez Kyle M. , Zha Yuanyuan , Pytel Peter , Luke Jason J , Gajewski Thomas , Volchenboum Samuel L , Cohn Susan L , Desai Ami V 
Published: February 2023

Abstract

Background:
Tumor-infiltrating CD8+ T cells and neoantigens are predictors of a favorable prognosis and response to immunotherapy in adult cancer, but little is known about their role in pediatric malignancies. Here, we analyzed the prognostic strength of T cell-inflamed (Tinfl) expression and neoantigen load (neo) in high-risk neuroblastoma (NBL). We investigated transcriptional programs associated with the non-T cell-inflamed (non-Tinfl) phenotype for mechanisms of immune exclusion.

Methods:
Using a defined Tinfl gene expression signature, we categorized high-risk NBL from the TARGET (Therapeutically Applicable Research to Generate Effective Treatments, n=123) and the GMKF (Gabriella Miller Kids First, n=48) programs into non-Tinfl, Tinfl, and intermediate groups. Cox proportional hazard models were used to test the association between survival and tumor group, neo, MYCN amplification. Pathways activated in non-Tinfl vs. Tinfl tumors were identified.

Results:
Patients with high-risk Tinfl tumors showed improved overall survival (OS) compared to those with non-Tinfl tumors (P&lt;0.05), independent of MYCN amplification, in both TARGET and GMKF. Higher neo was associated with better event-free survival and OS (P&lt;0.005) and was independent of Tinfl. Activation of MYCN, ASCL1, SOX11, and KMT2A was inversely correlated with the Tinfl signature in both cohorts.

Conclusions:
Our results indicate that tumors from children with high-risk NBL harboring a strong Tinfl signature have a favorable clinical outcome, and neo is a prognosis predictor, independent of Tinfl. Strategies to target SOX11 and other signaling pathways associated with non-Tinfl tumors should be pursued as potential immune-potentiating interventions.
Ag-dependent Engagement of CD8+ T Cells with H-2Kb on Brain Endothelium Required for CD8+ T Cell entry and Cerebral Manifestation of Malaria
Authors: Fain Cori E. , Zheng Jiaying , Jin Fang , Ayasoufi Kathy , Tritz Zachariah Peter , Khadka Roman , Hansen Michael , Johnson Aaron J 
Published: February 2023

Abstract
Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection with high mortality. The immune system is a key participant as the cerebral manifestation is heavily reliant on CD8+ T cell infiltration and neuroinflammation. In this disease etiology, elevated blood cytokine levels lead to increased adhesion and antigen (Ag)-presenting molecules on brain endothelium prior to CD8+ T cell entry and onset of neurological symptoms. Using the animal model of experimental cerebral malaria (ECM) we sought to further define these interactions in vivo at the blood-brain barrier. While direct interaction with MHC class I on cerebrovascular endothelial cells (CECs) has been put forward as a putative mechanism for ECM, it has not been directly confirmed in vivo. We therefore hypothesized that Ag presentation by discrete MHC class I molecules on CECs would be required for CD8+ T cells to enter the brain and establish ECM. To test this, we crossed CDH5-Cre mice with novel H-2Kb LoxP and H-2Db LoxP mice to create endothelium-specific, individual class I cKO mice. In CDH5-Cre H-2Kb mice, we show that with T cell development fully intact, CD8+ T cell infiltration into brain is decreased in frequency and number at baseline and during the infection. T cells that do enter are increasingly naive with effector responses attenuated. Importantly, mice with loss of H-2Kb on CECs show extended survival and protection from cerebral complications; as assessed by 2-photon imaging, small-animal MRI and immunofluorescence. Together, these data demonstrate that parasite-specific CD8+ T cells must undergo Ag-dependent engagement with H-2Kb class I molecule on CECs in order to fully activate CD8+ T cells to enter brain parenchyma and mediate ECM development.
Immunopeptidomics analysis of Lyme arthritis: insights into infection and autoimmunity
Authors: Danner Rebecca , Pereckas Michael , Rouse Joseph , Wahhab Amanda , Lochhead Robert 
Published: February 2023

Abstract

Background:
Lyme arthritis (LA) is caused by infection with the Lyme disease spirochete Borrelia burgdorferi (Bb) and in humans is often accompanied by autoimmune T and B cell responses. In this study, we used an immunopeptidomics approach to gain further insight into mechanisms of infection-induced autoimmunity.

Methods:
C57BL/6 (B6) mice, which develop mild, self-limiting LA, and B6 Il10−/− mice, which develop chronic, autoimmune-like LA, were inoculated with 2×104 Bb. Inguinal and popliteal lymph nodes (LN) were harvested from infected mice at 4 weeks and 16 weeks post-inoculation. MHC class II molecules were isolated by immunoaffinity capture and MHC-bound peptides were identified by LC/MS/MS.

Results:
Nearly 10,000 MHCII-bound peptides were identified. At 4 weeks post-inoculation, representing the peak of LA, proteins involved in leukocyte trans-endothelial migration, tissue repair, and immune activation, including a known Lyme autoantigen ApoB-100, were over-represented in both B6 and Il10−/− mice. Peptides from these proteins returned to near baseline levels in LN from B6 mice at 16 weeks post-inoculation, when arthritis resolves, but were further enriched in Il10−/− mice at 16 weeks, during the chronic, autoimmune-like phase. Surprisingly, only 27 peptides derived from Bb proteins were identified, all but one of which were from proteins found in the inner membrane, periplasm, or cytosol.

Conclusions:
This study identified immune-relevant proteins presented by APCs in draining LN that are associated with LA development, which included ApoB-100, a known Lyme autoantigen in humans. Further studies are underway to assess T cell responses to identified Bb and self-peptides, including ApoB-100, during Bb infection.
INF-λ enhances expression of MHC class I molecules on thymic epithelial
Authors: Benhammadi Mohamed , Dumont-Lagacé Maude , Mathé Justine , Kobayashi Koichi S. , Gaboury Louis , Brochu Sylvie , Perreault Claude 
Published: February 2023

Abstract
Introduction: The major histocompatibility complex class I (MHC-I) molecules is a cell surface glycoprotein involved in the presentation of endogenously derived peptides, to the TCR of CD8 T cells. Moreover, several vital processes specifically depend on the interaction between TCR and the MHC-I presented by the epithelial cells (ECs). However, the regulation of the MHC-I expression has been studied almost exclusively in hematolymphoid cells and very little is known about this process in ECs. In the present work, we performed a deep analysis of MHC-I expression in primary ECs freshly harvested from the thymus, skin, gut, and lung.
Methods and Results: Our flow cytometry analysis showed that the abundance of cell surface MHC-I was overall 10- to 100-fold greater in thymic ECs (TECs) than in extrathymic EC types. Our transcriptomic analysis revealed a dominant role of IFN signalling in the differential MHC-I expression found in TECs vs. extrathymic ECs. In fact, superior MHC I expression in TECs is unaffected by deletion of Ifnar1 or Ifngr1, but is lessened by deletion of Aire, Ifnlr1, Stat1 or Nlrc5, and is driven mainly by type IFN-λ produced by TECs. Moreover, Ifnlr1−/− mice showed impaired negative selection of CD8 thymocytes, and at 9 months of age, present autoimmune manifestations.
Conclusion and Relevance: In conclusion, our study opens the door to a new level of understanding of MHC-I regulation in ECs and will pave the way for a more detailed exploration of the impact of IFN-λ signaling in thymic functions.
TSLP rather than IL-33 signaling drives intestinal mucosal mast accumulation and allergen-induced diarrhea in mice following epicutaneous sensitization to food allergen.
Authors: Brandt Eric B , Bolcas Paige E , Shik Dana , Ruff Brandy P , Hershey Gurjit K Khurana 
Published: February 2023

Abstract

Background:
A major route of sensitization to food allergen is through an impaired skin barrier. IL33 and TSLP have both been implicated in epicutaneous sensitization and development of food allergy.

Method:
We assessed the respective contributions of TSLP and IL33 to the development of food allergy in TSLP and IL33 receptor deficient mice following epicutaneous food allergen sensitization, where mice were exposed to thrice weekly skin patches of either saline, OVA or a combination of OVA and Aspergillus fumigatus extract (ASP).

Results:
ASP+OVA-patched but not OVA-patched mice developed an atopic dermatitis (AD)-like skin phenotype, while sensitization to OVA occurred in both OVA and OVA+ASP patched mice. OVA-specific IgE levels were significantly lower in OVA(±ASP)-patched TSLPR−/− mice compared to wild type mice and ST2−/− mice. Repeated intragastric challenges with 50mg of OVA, induced intestinal accumulation of mast cells but not ILC2s in OVA(±ASP)-patched WT and ST2−/− mice, and to a much lesser degree in TSLPR−/− mice. While OVA-induced mast cell degranulation assessed by measuring MCPT1 blood levels and the development of food allergy (diarrhea occurrences) was observed in almost all WT mice and two thirds of OVA+ASP patched ST2−/− mice, all TSLPR−/− mice were protected from developing food allergy and had significantly lower MCPT1 levels.

Conclusion:
Epicutaneous sensitization to food allergen and subsequent development of food allergy does not require atopic dermatitis skin lesions and is dependent on TSLP, suggesting that prophylactic targeting of TSLP may be useful in mitigating the development of food allergy early in life.
Analysis of transdermal sensitization and oral anaphylaxis using salt-soluble protein extracts from diploid wheats in an adjuvant-free mouse model
Authors: Gao Haoran , Jorgensen Rick , Raghunath Rajsri , Chandra Shivam , Wiersma Andrew , Nagisetty Siddharth , Olson Eric , Ng Perry KW , Gangur Venugopal 
Published: February 2023

Abstract
Wheat allergies are growing at an alarming rate for reasons that are not well understood. The commonly consumed wheats belong to the AABB and AABBDD genotypes. The AA wheat is less commonly consumed, and the DD wheat is not commercially available. It is unknown at present whether the diploid wheats elicit allergic reactions in mice. Here we tested the hypothesis that the salt-soluble protein extracts (SSPEs) from AA and DD wheats will induce food allergy in Balb/cJ male mice in an adjuvant-free model. We grew the DD wheat at our university and used it in this study. The AA wheat was obtained from www.einkorn.com. Balb/cJ male mice were bred and maintained on a plant protein-free diet. Mice were exposed to SSPEs or saline via skin once a week for nine weeks. SSPE-specific IgE (sIgE) was measured using an ELISA. After sensitization, mice were orally challenged with SSPEs or saline to elicit a hypothermia shock response (HSR). We found that both AA- and DD-SSPEs elicited significant and comparable levels of sIgE responses upon skin exposures. Oral challenge with DD-SSPE but not saline in DD-SSPE sensitized elicited a mild HSR lasting up to only 15 minutes. In contrast, oral challenge with AA-SSPE but not saline in AA-SSPE sensitized mice elicited a severe HSR lasting for 30 minutes. These data together suggest that the diploid wheats induce comparable sensitization, but DD wheat is less potent in inducing oral anaphylaxis compared to the AA wheat.