bluuebunny/crossref_metadata_2025_split huggingface dataset:
High-intensity focused ultrasound ablation increases lymphocyte infiltration in mouse prostate tumor
Authors: Su Shengchen , Wang Yanping , Lo Eric M , Tamukong Patrick , Kim Hyung L 
Published: August 2023

Abstract

Introduction and Objective
High-Intensity Focused Ultrasound (HIFU) is an FDA-approved procedure for the treatment of localized prostate cancer. HIFU destroys tumor tissue by noninvasively delivering ultrasonic energy, which rapidly heats the targeted tissue. Prostate cancer has few tumor-infiltrating lymphocytes, which are required for antitumor immunity. The objective of this study was to determine if HIFU activates antitumor immunity and increases lymphocyte infiltration into the tumor.

Methods
RM1 tumors were grown on the flanks of syngeneic C57BL/6J mice and one tumor was treated with HIFU. Contralateral tumor growth was monitored, and tumors were collected at the end of the study to assess immune cell infiltration. Tumor infiltrating lymphocytes were assess by flow cytometry.

Results
When one tumor was treated with HIFU, the growth of the contralateral (untreated) tumor decreased when the contralateral tumor was the same tumor type but not when it was a different tumor type. HIFU increased CD4+ and CD8+ lymphocyte infiltration into the contralateral (untreated) tumor. Depletion studies confirmed that the antitumor immune effect of HIFU is CD4+ and CD8+ lymphocyte dependent.

Conclusions
In preclinical models, HIFU generates an antitumor immune response capable of suppressing the growth of mouse prostate tumors. The antitumor immune effect of HIFU is CD4+ and CD8+ lymphocyte dependent. Future studies should investigate mouse models of metastatic prostate cancer, where cytoreductive prostate tumor ablation can be combined with systemic immunotherapies to generate robust antitumor immunity.

Homer and Gloria Harvey Family Chair Fund in urologic oncology in honor of Stuart Friedman, M.D.
Depletion of plasmacytoid dendritic cells and HIF-1α inhibition enhance immunotherapy against hepatocellular carcinoma
Authors: Geng Degui , Wang Yin , Liu Minyue , Ahodantin James , Li Guangming , Su Lishan 
Published: August 2023

Abstract
Hepatocellular Carcinoma (HCC) is one of the deadliest forms of cancer. Recent years have seen significant advances for HCC treatment, but up to 70% of patients remain at high risk for recurrence necessitating the urgent need for more effective therapies. Plasmacytoid dendritic cells (pDCs) heavily infiltrate liver tumor tissues, contribute to an immunosuppressive tumor microenvironment (TEM), and promote HCC growth. Furthermore, hypoxia often occurs in HCC tumor tissues and plays the central role of an immunosuppressive TME. A hypoxic TME enhances pDC recruitment into tumor tissues via the upregulation of hypoxia-inducible factor 1α (HIF-1α). Here we show that the distribution of pDCs does not co-localized with hypoxic areas in HCC. In the immunocompetent HCC mouse model, depletion of pDCs using the antibody or HIF-1α inhibitor echinomycin significantly suppressed tumor growth. The combination of pDC depletion and HIF-1α inhibition suppressed HCC growth more effectively. Both treatments reshaped the TME with changes of the cytokine profile and increased tumor-infiltrating CD8 T cells. Reduced basic helix-loop-helix transcription factor E2-2 expression by treatments abrogated protumor functions of pDCs in HCC resulting in reactivating tumor-reactive CD8 T cells. This study demonstrates that targeting both pDCs and HIF-1α may serve as more effective immunotherapy for HCC.
Avian paramyxovirus 4 has potent antitumor activity that surpasses the oncolytic effect of NDV in colon carcinoma and melanoma in vivo models
Authors: Bykov Yonina , Javaheri Aryana , Mena Ignacio , Garcia-Sastre Adolfo , Cuadrado-Castano Sara 
Published: August 2023

Abstract
Avulaviruses make up some of the most widespread avian viral pathogens. Within the genus, APMV-1, also known as Newcastle disease virus (NDV), is the only avulavirus that has been extensively characterized and explored as an oncolytic agent. While an array of clinical and preclinical studies have shown NDV to hold much promise, the oncolytic potential of the other avulaviruses remains largely unexplored. In this study, we evaluated the in vivo anti-tumor potential of prototype strains of APMV serotypes -2, -3, -4, -6, -7, -8 and -9 in syngeneic B16–F10 melanoma and CT26.WT colon carcinoma models. All the selected viruses were able to delay tumor growth, however, treatment with APMV-4 Duck/Hong Kong/D3/1975 imparted the greatest survival benefit as well as a number of complete remissions that were protected against re-challenge in both tumor models. Infection of cancer cell lines with APMV-4 in vitro resulted in potent activation of the IFN response, and in vivo interrogation of the tumor microenvironment revealed high levels of infiltration by T cells as well as various members of the myeloid compartment. Importantly, the development of a reverse genetics approach to rescue recombinant APMV-4 viruses allowed us to successfully express GFP in the virus backbone, highlighting APMV-4 as a versatile tool that can, in the future, include therapeutic transgenes that will optimize its potential benefit in the clinic.
supported in part by a Public Health Service Institutional Research Training Award AI07647
Combination epigenetic therapy activates immune cells in a murine model of ovarian cancer
Authors: Cox Olivia L , Gomez Stephanie , Grundy Erin E , Chiappinelli Katherine B 
Published: August 2023

Abstract
Ovarian cancer (OC) is the fifth leading cause of cancer death in women in the United States. Despite novel therapies and significant advances in our understanding of the disease, the five-year survival rate for OC has not changed in decades. The immunosuppressive microenvironment of OC leads to a low response rate to immunotherapies. We have previously shown that in a murine model of OC epigenetic therapies including DNA methyltransferase inhibitors (DNMTis) and histone deacetylase inhibitors (HDACis) activate type I interferon signaling in tumor cells to drive immune infiltration and sensitization to immunotherapy. This indicates a role for immune control of the tumor, but we did not directly investigate the effects of these epigenetic therapies on the immune cells. We hypothesized that T cells could be activated by epigenetic therapy in this model.
We isolated T cells from the spleens of healthy C57/Bl6 mice and treated the cells with a DNMTi, 5-azacytidine (Aza) at 500 nmol/L, and an HDACi, ITF-2357 (ITF) at 100 nmol/L, singly and in combination. Cell pellets were isolated, RNA extracted, and analyzed via qRT-PCR. Cell supernatants were analyzed for cytokine and chemokine production using a Legendplex assay. The treated bulk T cells, CD8+ T cells, and CD4+ T cells showed anti-tumor cytokines, including CCL5, TNF-α and IFN-γ, were upregulated while the pro-tumor cytokine IL-10 was downregulated. These changes were evident at the transcript and secreted cytokine level, pointing to transcriptional upregulation of the target genes. This study demonstrates direct activation of T cells by DNMTis and HDACis and provides mechanistic insight into the role of epigenetic therapies in the murine model of OC.
Sensitization of Tumor Cells to IFNy-Mediated Cell Death
Authors: Sun Nicholas , Krogman Erica , Chawla Yogesh , Ting Adrian 
Published: August 2023

Abstract
There is increasing evidence that immunotherapies make tumor cells more vulnerable to the host’s own immune system that can lead to long-term and durable remission. To date, only a small percentage of cancer patients have benefited from this approach and advances in this field will likely come from a greater understanding of the interaction between immune cells and their tumor targets. T cells are critical to a successful anti-tumor response due to their ability to directly lyse tumor cells by releasing cytolytic granules, and to produce inflammatory cytokines, such as interferon gamma (IFNγ). IFNγ has several functions in an anti-tumor response. On the immune cell side, IFNγ is a major driver of Th1 differentiation that leads to the activation of T cells and NK cells. On the tumor side, IFNγ can enhance MHC class I presentation but also inhibitory receptors such as PD-1. However, it is not as clear whether IFNγ is directly cytotoxic for tumor cells. If that is the case, it is also likely that due to selection pressure exerted by T cells, tumor cells may have acquired mechanisms to protect themselves against this possibility. Thus, I hypothesized that tumor cells are normally resistant to killing by IFNγ but can be sensitized if the death protection mechanisms are disabled. In preliminary studies using the B16F1 murine melanoma model, we found that while wild-type (WT) melanoma cells were completely resistant, tumor cells with knockouts of two kinases (DKO) became highly sensitized to IFNγ-mediated apoptotic death. Furthermore, the DKO tumors were also more easily killed by T cells in an IFNγ-dependent manner. These findings will provide insight into potential targets for therapeutic intervention to enhance immune-mediated destruction of tumors.
An in vitro T cell Dysfunction/Exhaustion Assay System that is partially reversible by pembrolizumab
Authors: Daftarian Pirouz M , King Jeffrey , Goverse Gera , Kleiman Eden 
Published: August 2023

Abstract
The persistence of antigen and chronic stimulation of T cells results in T cells’ functional decay, referred to as T cell dysfunction or exhaustion (TEX). TEX is an essential part of the checks and balances of the immune system chronic T cell stimulation. Assays that model T cell dysfunction/exhaustion with functional readouts are urgently needed for identification and characterization of active drug candidates.
Here, to recapitulate persistent of antigen as it happens in tumor microenvironment, we have optimized an in vitro stimulation set-up using PBMCs from various donors that models key features of T cell exhaustion/dysfunction including main markers and functional features of T cell exhaustion. Various stimulation conditions were assessed, with stimulation lasting up to four rounds, and optimal conditions were selected for anti-CD3/anti-CD28 stimulations of PBMCs that resulted in a) increased expression of the co-inhibitory molecules associated with exhausted T cells, PD-1, TIM3, LAG3, CD38, and CD39, and b) impaired T cell functions as demonstrated by reduced expression of IFN-γ, TNF-α, and reduced cytotoxic killing in an allogeneic setting, using both a 2D cancer cell line and a 3D tumoroid model. This in vitro T cell dysfunction/exhaustion platform is a candidate to test drug candidates for their ability to delay or partially reverse T cell exhaustion.
Crown Bioscience internal R&D
A 2-laser 13-color cFluor®B Cell Monitoring panel for analyzing human B cell subsets
Authors: Miller Heather , Tran Kevin , Edinger Mark , Chen Jingyi , Deng Jun 
Published: August 2023

Abstract
Flow cytometry is valuable for identifying and monitoring B cell subsets in patients with humoral immune disorders and patients treated with immunotherapies targeting B cells. We have designed a 13-color cFluor®B Cell Monitoring panel that identifies plasmablasts/plasma cells (CD19+ CD20− CD27+ CD38++), naïve B cells (CD27− IgD+), IgG class switched memory (CD27+ IgG+) and unswitched memory (CD27+ IgD+ and/or IgM+). Additionally, markers for CD4+ and CD8+ T cells are included for a general status of lymphocytes along with granulocyte (CD15) and monocyte (CD14) markers to allow for cleaner lymphocyte gating.
The panel was tested on healthy PBMCs and peripheral blood samples collected in EDTA, Cyto-Chex BCT, or heparin tubes. The cell populations of interest were well identified in the PBMCs and blood samples collected in the different types of blood tubes. The resolution of each marker in the panel was comparable to the single-color staining. Evaluation of assay reproducibility showed that the coefficient of variance (CV) for the B cell subsets of interest were &lt;15% in triplicate runs of 10 samples. Also, blood sample stability and post staining stability were evaluated over time.
In conclusion, Cytek’s 13-color cFluor®B Cell Monitoring panel was shown to be effective in identifying and enumerating B cell subsets. This may be a useful tool for monitoring B cell subsets of patients with humoral immune disorders or patients treated with immunotherapies targeting B cells in translational research.
Impact of type I IFN signaling on B cell pathogenicity in a mouse model of early Sjögren’s disease
Authors: Allred Merri-Grace , Lieberman Scott M 
Published: August 2023

Abstract
Sjögren’s disease is a chronic autoimmune disease characterized by focal inflammation of the lacrimal and salivary glands. NOD mice spontaneously develop lacrimal gland inflammation by 5–6 weeks. As disease progresses, B cells accumulate in the glands and produce antibodies resulting in glandular dysfunction. Disruption of the type I interferon (IFN) signaling pathway protects NOD mice from developing lacrimal gland disease. While the impact of B cells later in disease has been well-characterized, the role of B cells in early disease development has not been defined. Here, we performed gene expression studies, adoptive transfers, and spectral flow cytometry to define the phenotype of B cell subsets and the potential role of type I IFN signaling in lacrimal gland autoimmunity.
Gene expression studies demonstrated that genes associated with B cell receptor signaling, lymphocyte trafficking and antigen presentation were upregulated at 10 weeks compared to 6 weeks. Flow cytometry of the lacrimal glands showed that most early B cells express IFNAR1. Adoptive transfer studies demonstrated decreased disease when donor cells lacked IFNAR1 expression compared to WT donor cells, but this difference was abrogated when magnetically purified T cells were transferred suggesting a type I IFN dependent pathogenic role on a non-T cell population. In vitro studies show that B cell phenotype can be shaped by IFNα in a dose dependent manner.
Type l IFN signaling drives the development of lacrimal gland autoimmunity in NOD mice. Type l IFN signaling shapes B cell phenotype and function in early lacrimal gland disease. Ongoing in vivo studies will interrogate the impact of IFNAR1 expression on B cell trafficking to the lacrimal glands.
Supported by grants from NIH (R01 EY027731)
Impaired expansion and selection of geminal center B cells in lupus drives autoantibody-producing cells
Authors: Sanchez Gina Marie , Antao Olivia , Mayer Daniel , Hirsch Eden , Lemenze Alexander , Weinstein Jason 
Published: August 2023

Abstract
Lupus is a systemic autoimmune disease characterized by the presence of autoantibodies that form immune complexes in tissues leading to the recruitment of immune cells and subsequent inflammation and organ damage. The origin of autoantibody-secreting B cells is not clear, but the germinal center (GC) is of particular interest as the B cells here undergo somatic hypermutation, which may result in the generation of autoreactive memory or long-lived plasma cells. Within the GC, B cells cycle between two major zones: the dark zone (DZ) where mutation and proliferation occur, and the light zone (LZ) where affinity-based selection occurs. We found in lupus-prone mice that as autoantibodies increased, there was a reduction in proliferation measured by EdU incorporation in conjunction with dysregulation of the GC zones as B cells here became skewed towards a LZ phenotype (CXCR4lo CD86+). As MYC expression in LZ B cells correlates with the strength of selection, we examined MYC levels by flow cytometry and Amnis and found it and its nuclearization decreased as disease progressed. Single cell BCR sequencing of GC B cells throughout the course of disease revealed a loss in BCR repertoire diversity and mutations. Furthermore, we found that despite the accumulation of LZ B cells, there was an increase in the generation of plasma cells and memory-like CD11c+ Tbet+ B cells, suggesting aberrant selection and differentiation into these potentially autoantibody-secreting cells. Thus, our findings suggest that as lupus disease progresses, the ability of GC B cells in the LZ to undergo proper selection is impaired, resulting the permissive selection of autoreactive B cells and differentiation into autoantibody-secreting cells.
Supported by grants from the NIH/NIAMS (R01 AR073912, R01 AR073912 - 03W1)
Rheumatoid arthritis associated with a cytotoxic cell leukemia is characterized by enhanced reactivity to citrullinated antigens
Authors: Ananth Kusuma , Gomez-Banuelos Eduardo , Trejo-Zambrano Isabel , Kirschmann Jessica Marie , Moosic Katharine B , Wang Hong , Feith David J , Robinson William H , Andrade Felipe , Loughran Thomas P , Darrah Erika 
Published: August 2023

Abstract
T cell large granular lymphocyte leukemia (T-LGLL) is characterized by clonal expansion of cytotoxic T cells and neutropenia. Compared to 1% of the general population, about 25% of T-LGLL patients develop rheumatoid arthritis (RA), an autoimmune disease characterized by anti-citrullinated protein antibodies (ACPAs). To investigate the autoimmune response in patients with both T-LGLL and RA (T-LGLL/RA), we analyzed their ACPA positivity and specificity using anti-CCP ELISA (n=30 for T-LGLL/RA and T-LGLL; n=150 for RA) and multiplex array (n=26 per group), respectively. We found that anti-CCP positivity was enriched in patients with T-LGLL/RA (90%) compared to those with RA alone (75%; p=0.09), or T-LGLL alone (33%; p&lt;0.0001). This was supported by multiplex serological profiling of reactivity to 51 individual RA autoantigens, which demonstrated that T-LGLL/RA patients are more responsive to citrullinated (cit)-antigens. T-LGLL/RA patients were overrepresented (61%) in a cluster of highly reactive individuals compared to patients with RA (39%) or T-LGLL alone (0%), and recognized a higher number of cit-antigens than patients with either disease alone: 11.7 vs. 5.8 for RA alone (p=0.2135) and 2.4 for T-LGLL alone (p&lt;0.0001). Among specific antigens, recognition of a cit-clusterin and a cit-fibrinogen alpha peptide were higher in patients with T-LGLL/RA than RA alone (p=0.0253 and 0.0507, respectively). Our results show that T-LGLL/RA patients have more robust anti-CCP responses and recognize a wider diversity of citrullinated RA autoantigens than patients with RA or T-LGLL alone. Investigating immune responses in T-LGLL/ RA, which may consist of an extreme form of RA, may provide insight into RA pathogenesis.
This research was funded by NIH/NIAMS R01-AR079404.
Macrophage extracellular traps require peptidylarginine deiminase 2 and 4 and are a source of citrullinated antigens bound by rheumatoid arthritis autoantibodies
Authors: Bashar S Janna , Holmes Caitlyn , Shelef Miriam 
Published: August 2023

Abstract
Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis, but the sources of citrullinated antigens remain incompletely defined. Neutrophil extracellular traps (NETs) appear to be a source of ACPA targets. However, while the citrullinating enzyme peptidylarginine deiminase (PAD)4 is required for producing citrullinated NETs, it is not required for citrullination in murine arthritis. Conversely, PAD2 has a limited role in producing NETs but is a major contributor to citrullinated proteins. Thus, additional sources of citrullinated antigens likely exist. We investigated if macrophage extracellular traps (METs) could be a source of citrullinated proteins in a PAD2 or PAD4 dependent manner. Thioglycolate induced macrophages from wild-type, PAD2−/− and PAD4−/− mice were activated with a variety of stimulants, then fixed and stained with DAPI and either anti-citrullinated histone H4 (H4C3) antibody or sera from ACPA+ or ACPA− rheumatoid arthritis subjects. METs were visualized by immunofluorescence, confirmed to be extracellular using DNase, counted in a blinded manner, and compared by paired-T test. We found that ionomycin and monosodium urate (MSU) crystals induce citrullinated METs (H4C3 stained). In the absence of PAD2 or PAD4, citrullinated METs were reduced in response to ionomycin and MSU when compared to wildtype, with a complete absence of citrullinated METs in PAD4−/− macrophages. Also, IgG from ACPA+ sera, but not ACPA− sera, bound to METs. Finally, in the absence of PAD2 or PAD4, ACPA bound METs were reduced in response to ionomycin and MSU. Thus, METs appear to contribute to the generation of citrullinated antigens targeted by ACPAs in rheumatoid arthritis in a PAD2− and PAD4-dependent manner.
Funding for this work was provided by the U.S. Army MedicalResearch Acquisition Activity through the Peer Reviewed MedicalResearch Program (W81XWH-18-1-0717) to M.A.S.
Dissecting the functional role of SENP2 in B cells and systemic lupus erythematosus
Authors: Huang Chien-Hsin , Lin I-Ying , Lin Kuo-I 
Published: August 2023

Abstract
SUMOylation is a post-translational modification that can dynamically regulate protein activity. SUMOylation involves covalent binding to a lysine residue of the target proteins, and the SUMO-specific peptidase (SENP) catalyzes deconjugation. In our laboratory, we analyzed the changes in the expression of genes involved in SUMOylation and found a significant reduction of SENP2 in peripheral navie B cells isolated from patients with SLE as compared to that from healthy donors. We thus stimulated peripheral human B cells and transfected them with siRNAs against SENP2. Our results showed a significant increase in IgG antibodies after knocking down SENP2. We also stimulated naive splenic mouse B cells with T cell-independent (TI) or T cell-dependent (TD) stimulation to examine the expression level of SENP2 after B cell activation. The results showed downregulation of SENP2 protein after TD stimulation, but not after TI stimulation. Taken together, because the increase in IgG antibodies is often due to an enhanced germinal center (GC) response, we next investigated the role of SENP2 in B-cell differentiation and SLE pathogenesis. Hence, we developed a mouse model in which Senp2 is deleted in GC B cells. However, after immunization with TD stimulation, we found a reduction in the frequency of GC-B cells, plasma cells, IgG1-expressing cells, and the amount of antibody production in GC-specific Senp2 knockout mice, which can be recapitulated by using an in vitro induced germinal center culture (iGB) system. We thus hypothesized that the role of SENP2 for limiting IgG production may act in early-phase before GC B cells. The exact timing and mode of action of SENP2’s activity to restrain the production of IgG will be investigated.