bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stereotactic radiotherapy increases functionally suppressive regulatory T cells in the tumor microenvironment
Authors: Muroyama Yuki , Nirschl Thomas R. , Kochel Christina M. , Lopez-Bujanda Zolia , Theodros Debebe , Mao Wendy , Carrera-Haro Maria A. , Ghasemzadeh Ali , Marciscano Ariel E. , Velarde Esteban , Tam Ada J. , Thoburn Christopher J. , Uddin Muniza , Meeker Alan K. , Anders Robert A. , Pardoll Drew M. , Drake Charles G. 
Published: January 2023

Abstract
Radiotherapy (RT) enhances innate and adaptive anti-tumor immunity; however, the effects of RT on immune-suppressive regulatory T cells (Tregs), in the tumor microenvironment (TME) have not yet been fully elucidated. Although previous reports suggest a post-RT increase in Tregs, whether these Tregs are functionally suppressive has not been determined. To test the hypothesis that RT enhances the suppressive function of Tregs in the TME, we selectively irradiated implanted tumors using the Small Animal Radiation Research Platform (SARRP), which models stereotactic RT in human patients– followed by flow-cytometric and functional analyses of tumor-infiltrating lymphocytes (TIL). Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Tegs), and that these cells have higher expression of CTLA-4, 4-1BB, and Helios, consistent with activated/suppressive phenotype. This observation held true across several tumor models (B16/F10, RENCA, MC38). Notably, we found that post-RT, TIL-Tregs had equal or improved suppressive capacity compared with non-irradiated tumors. Our data also indicated that Tregs proliferate post-RT, more robustly than the other T cell subsets in the TME. In addition, the post-RT Treg expansion occurred when T cell migration was inhibited by Fingolimod - suggesting that the post-RT Treg increase is likely due to preferential proliferation of intratumoral Tregs. Our data also suggest that post-RT Treg expansion is independent of TGF-β and IL-33. Collectively, these data demonstrate that RT increases the phenotypically and functionally suppressive Tregs in the TME, and provide a rationale for treatment regimens that combine RT with Treg-targeting agents to maximize anti-tumor efficacy.
Guided delivery of the “alarming” cytokine IL-33 to tumor by chimeric antigen receptor T cells
Authors: Chen Yuang , Lu Binfeng 
Published: January 2023

Abstract
Current immune checkpoint-based cancer therapy is limited by low levels of spontaneous antitumor T cells and immune suppressive microenvironment (TME). Interleukin-33 (IL-33) is a member of the IL-1 family of cytokines and is released by damaged tissue cells or activated innate immune cells such as macrophages upon tissue damage or infection. Thus, IL-33 is thought to serve as an endogenous “alarming signal”, which triggers inflammation and cell-mediated immune responses in affected tissues. We have found that IL-33 is expressed in many normal human lining tissue cells but drastically down-regulated in advanced cancer cells indicating the role of IL-33 in inhibiting tumor progression. Thus, IL-33 is a candidate cytokine whose down-regulation is an important mechanism for tumor “immune ignorance”, which is crucial for immune evasion by cancer cells. Consistent with this idea, we have furthered shown that expression of IL-33 in tumor cells inhibit tumor growth. Thus, tumoral delivery of IL-33 can serve as a novel therapy for immune therapy of cancer. In the current study, we “armed” CAR T cells with IL-33 expression cassette, which allows guided IL-33 expression in the tumor site specifically. The antitumor efficacy and toxicity of such strategy is evaluated in a humanized mouse model. The data will show whether and how tumor-site delivery of IL-33 increases efficacy of CAR T cells and immunogenicity of the TME.
Induced MHCII expression on breast cancer cells broadens the responding T cell repertoire, delays tumor-specific T cell exhaustion, and impairs tumor growth
Authors: McCaw Tyler R , Li Mei , Starenki Dmytro , Cooper Sara , Meza-Perez Selene , Buchsbaum Donald , Forero Andres , Randall Troy D. 
Published: January 2023

Abstract
We recently reported that aberrant expression of Major Histocompatibility Class II (MHCII) on human triple negative breast cancer cells correlates with increased tumor infiltrating lymphocytes and prolonged progression free survival. This observation led us to hypothesize that expression of MHCII enhanced the intratumoral CD4+ T cell response, thereby bolstering the tumor-specific CD8+ T cell response, leading to impaired tumor growth. To test this, we transfected the murine breast cancer line TS/A with human class II transcriptional activator (hCIITA) or empty vector, creating MHCII-expressing and MHCII-negative cell lines, respectively. We found that MHCII-expressing tumors grew slower than controls in immunocompetent recipients, but that this difference was abrogated in CD4-depleted and nullified in SCID mice. CD4+ T cells within hCIITA-transfected tumors produced more IFNγ for longer times than those from control tumors. Similarly, CD8+ T cells in MHCII-expressing tumors displayed a more activated phenotype and produced more IFNγ and granzyme B for longer times. Nevertheless, both CD4+ and CD8+ T cells eventually became exhausted in both groups. In addition to boosted effector function, TCR repertoire analysis demonstrated an increase in both breadth and amplitude of T cell response to MHCII-expressing tumors. We next investigated the possibility of inducing MHCII on non-expressing tumors using the histone deacetylase inhibitor Entinostat. Indeed, Entinostat turned on MHCII expression, which correlated with significantly reduced tumor burden. Thus, epigenetic modifiers capable of inducing MHCII expression on tumor cells could avail this augmented T cell response to all patients for enhanced tumor control.
Combining impedence-based viability measurements and flow cytometric analyte quantitation to evaluate effector cell killing of T lymphocytes
Authors: Guenther Garret , Tang Wei , Ye Peifang , Le Ming , Cerignoli Fabio , Xi Biao , Zhou Leyna , Abassi Yama 
Published: January 2023

Abstract
Cancer immunotherapy is increasingly being evaluated in the lab, as well as the clinic as an approach for cancer treatment by harnessing and enhancing the immune system to attack cancer cells. Moving this research from the bench to the clinic is critically important, but we need reliable tools to be able to translate the in-vitro protocols and results for use in-vivo. Here, we have used a combination of impedence-based technology and flow cytometry to evaluate both the target and effector cells in an immune-cell based killing assay. The impedence based Real Time Cell Analysis technology, provides us with a continuous readout of target cell viability and flow cytometry allows us to measure responses using specific labels, such as effector cell viability and cytokine production in the supernatant of these assays. Here, we demonstrate cell killing assays measuring the cytotoxic effect of T-lymphocytes on a B-cancer cell line in conjunction with cytokine concentration measurements of IL-2, IL-4, IL-5, IL-10, TNF-a, IFN-g, IL-8, IL-1b, IL-6, IL-10, and IL-12p70 to simultaneously understand the T-lymphocyte response in these assays. Using both of these methods allows us to thoroughly evaluate immune cell killing efficacy and kinetics. The potency assays described in these studies have great potential for use in-vivo models and clinic.
Immunization with Human Papillomavirus type-18 E1 protein induce CD8+ T cells with cytotoxic activity against cells expressing E1 from HPV-11, 33 and 53.
Authors: Amador Alfredo , Romero Damaris , Lizano Marcela 
Published: January 2023

Abstract
CD8+ mediated immune response plays a major role in the clearance of Human Papillomavirus (HPV) infected cells. Although HPV vaccines are effective in preventing HPV infections through humoral response, they fail to protect against some HPV types and established infections. In the natural history of an HPV infection, the expression of E1 helicase during viral replication makes it a potential immunological target. The early expressed E1 protein is one of the most conserved proteins among papillomaviruses. We demonstrate in previous work, that immunization of HPV type 18 E1 protein along with α-GalactosylCeramide (α-GalCer) adjuvant elicited CD8 T cell-immune response, able to eliminate tumor cells expressing HPV18 E1 protein. For this study, we compared the amino acids sequence of E1carboxi-terminal (CT) domain from HPV18, 6, 11, 16, 30, 31, 35, 45 and 53. The sequences have 60% of identity and preserve two motifs previously described for being common between many Helicase types. We cloned E1-CT domain of HPV11, HPV33 and HPV53, and its expression in B16-F0 cells was determine.
We show that CD8 T cells from mice immunized with HPV18 E1 protein plus α-GalCer, are actively cytotoxic in co-culture with B16-F0 cells expressing E1-CT of HPV11, HPV33 and HPV53 in comparison with unimmunized mice. Cytotoxicity activity was evaluated by detection of CD107a and CD107b on the surface of CD8 T cells as part of cytotoxic granule exocytosis. Ongoing experiments will examine if these CD8 T cells from mice immunized with HPV18 E1 protein plus α-GalCer are also actively cytotoxic in presence of other E1 HPV types.
The characterization of new immunological targets against HPV, could help to develop new strategies to clearance HPV established infections.
CD8+ T cell-independent mechanisms of PD-1-mediated suppression of anti-tumor immunity in mice
Authors: Pauken Kristen E , Juneja Vikram , McGuire Kathleen , LaFleur Martin , Kuchroo Juhi , Sage Peter , Chevrier Nicolas , Freeman Gordon J , Sharpe Arlene H 
Published: January 2023

Abstract
Over expression of the inhibitory receptor PD-1 commonly occurs in cancer, and PD-1 signaling suppresses protective anti-tumor immune responses. While early clinical trials with PD-1 inhibitors in cancer have been promising, the majority of patients have not shown durable clinical benefit, and the mechanisms underlying both successes and failures remain poorly defined. CD8+ T cells can directly kill tumor cells, and CD8+T cell infiltration of human tumors positively correlates with responses to PD-1 blockade. CD8+ T cells in tumors (TILs) often show high expression of PD-1; however, PD-1 is also expressed by other cell types, including CD4+ T cells, regulatory T cells (Treg), B cells, and some myeloid cells. Consequently, we hypothesized that loss of PD-1 only on CD8+ T cells may not be sufficient to control tumor burden. To address this question, we compared tumor responses in mice completely deficient for PD-1 (referred to as cKO) or selectively deficient for PD-1 on CD8+ T cells (E8i-cre PD-1f/f mice, referred to as CD8ΔPD-1). Following subcutaneous implantation of MC38 adenocarcinoma tumor cells, 100% of PD-1 cKO mice cleared the tumor. In contrast, only 70% of CD8ΔPD-1 mice controlled the tumor. Moreover, in CD8ΔPD-1 mice that controlled tumor, the average time to clearance was delayed compared to PD-1 cKO mice. In both PD-1 cKO and CD8ΔPD-1 mice, CD8+ TILs showed elevated granzyme B levels and an increased CD8 to Treg ratio, suggesting that CD8+ T cell-independent mechanisms contribute to immunity following loss of PD-1 in MC38 tumors. On-going work examining both tumor cells and infiltrating immune cells is focused on identifying these CD8+T cell-independent mechanisms of PD-1-mediated suppression.
SETD1B compensates loss of IRF8 expression to activate iNOS expression in myeloid-derived suppressor cells through an epigenetic mechanism
Authors: Redd Priscilla S , Ibrahim Mohammed L , Klement John D , Sharman Sarah K , Paschall Amy V , Yang Dafeng , Nayak-Kapoor Asha , Liu Kebin 
Published: January 2023

Abstract
Inducible nitric oxide synthase (iNOS) generates nitric oxide (NO) in myeloid cells that acts as a defense mechanism to suppress invading microorganisms or neoplastic cells. In tumor-bearing mice, elevated iNOS expression is a hallmark of myeloid-derived suppressor cells (MDSCs). MDSCs use NO to nitrate the T cell receptor and STAT1 to inhibit T cell activation and the anti-tumor immune response. The molecular mechanism for iNOS expression regulation in tumor-induced MDSCs is unknown. We show that IRF8 deficiency results in diminished iNOS expression in mature CD11b+Gr1− and immature CD11b+Gr1+ myeloid cells in vivo, indicating that IRF8 is an essential transcriptional activator for iNOS expression in myeloid cells under physiological conditions. Though IRF8 is silenced in tumor-induced MDSCs, iNOS expression is dramatically elevated in tumor-induced MDSCs suggesting that the expression of iNOS is regulated by an IRF8-independent mechanism under pathological conditions. Tumor-induced MDSCs exhibit diminished STAT1 and NF-κB Rel protein level, the essential inducers of iNOS in myeloid cells. Instead, tumor-induced MDSCs show increased SETD1B expression compared to their equivalent in tumor-free mice. Chromatin immunoprecipitation revealed that H3K4me3, a product of SETD1B enzymatic activity, is enriched at the nos2 promoter in tumor-induced MDSCs. Inhibition/silencing of SETD1B diminished iNOS expression in tumor-induced MDSCs. Our data determine that IRF8 is an essential transcriptional activator of iNOS in myeloid cells under physiological conditions. Tumor cells use the SETD1B-H3K4me3 epigenetic axis to compensate for loss of IRF8 expression to activate iNOS expression in MDSCs under pathological conditions.
Delineating the epigenetic regulation of myeloid derived suppressor cell generation in hepatocellular carcinoma associated with cirrhosis
Authors: MAN LIU , Zhou Jingying , Wong John , Chan Anthony W.H. , Chen Zhiwei , Cheng Alfred S.L. 
Published: January 2023

Abstract
Hepatocellular carcinoma (HCC) is the most common liver tumor with dismal prognosis. Since most liver tumors arise in a context of chronic inflammation associated with fibrosis, tumor microenvironment plays a critical role in liver carcinogenesis. Myeloid derived suppressor cell (MDSC) is a population of immature myeloid cells that undermine immune surveillance and facilitate tumor immune escape. As a key driver of liver fibrosis, activated hepatic stellate cell (HSC) has been recently shown to induce MDSC generation through cell-cell contact and/or secreted soluble factors. Given the crucial role of epigenetics in cell lineage specification, we aim to elucidate the chromatin regulation of MDSC in the immunosuppressive liver tumor microenvironment. Using human PBMCs from healthy donors, we have successfully recapitulated the immunomodulatory effect of HSCs on monocytic MDSC generation. Comprehensive epigenetic drug screening and expression analysis indicated that accumulation of histone H3 lysine 27 acetylation (H3K27ac), an active enhancer histone mark, is involved in the process of HSC-induced MDSC generation. We further demonstrated that inhibitors of BET family, which disrupts BRD4 association with H3K27ac-enriched active enhancers, abrogates MDSC generation. These results highlight the importance of epigenetic regulation in MDSC accumulation in the fibrotic microenvironment. Ongoing work is directed towards the identification of HSC-activated enhancers that orchestrate MDSC identity and functions. As we showed high level of monocytic MDSC accumulation in patients with cirrhotic HCC, our study may provide novel therapeutic targets to restore effective anti-tumor immune response in this dreadful disease.
TIPE2 is a novel direct target of STAT3 in MDSC and inhibition of its expression on MDSC enhanced T cell activation in tumor
Authors: Yan De hong , Wang Jing hui , Wan Xiao chun 
Published: January 2023

Abstract
MDSCs represent an immature population of myeloid cells that inhibit both innate and adaptive immunity in most cancer patients and mice bearing various tumors through several inflammatory mediators. TIPE2, a member of tumor necrosis factor (TNF)-alpha-induced protein 8 (TNFAIP8 or TIPE) family, is a negative regulator of innate and adaptive immunity. In this study, we investigated the influence of tumor-derived factors on TIPE2 expression in MDSCs. We demonstrate that tumor-derived IL-6 induced TIPE2 up-regulation expression of MDSCs. This effect was dependent on phosphorylation signal transducer of activator of transcription 3 (STAT3) and blockade of STAT3 signaling also reversed the effect of tumor-derived IL-6 on TIPE2 expression in MDSCs. Chromatin immunoprecipitation and luciferase reporter assay revealed direct binding of STAT3 to a transcriptionally active STAT3-response element (SRE) in the TIPE2 proximal promoter. Inhibition of TIPE2 expression on MDSCs by shRNA technology enhanced T cell activation and abrogated the suppressive activity of MDSCs. More importantly, downregulation of TIPE2 level on MDSCs by stattic, a STAT3 inhibitor, significantly delayed tumor growth in tumor-bearing mice. Targeting of TIPE2 therapy may represent a novel approach for cancer immunotherapy.
In vitro model of Rich foci: mycobacterium-induced granuloma formation in the central nervous system
Authors: Walter Fruzsina R , Gilpin Trey E , Mergaert Aisha , Sandor Matyas , Fabry Zsuzsanna 
Published: January 2023

Abstract
Central nervous system (CNS) involvement is the most devastating manifestation of tuberculosis (TB) infection. 5–10 % of the extrapulmonary cases affect immunocompromised individuals and children. Although CNS TB is a global phenomenon with extremely high mortality, understanding how bacteria disseminate into the brain parenchyma is not well understood. In the 1930s Rich and McCordock proposed that bacteria travel via the blood and form a tuberculoma (Rich foci). These foci may rupture resulting in diffuse meningitis. Previously our laboratory showed that bone marrow derived dendritic cells (DCs) migrate across the blood-brain barrier (BBB) into the CNS during inflammation in vitro and in vivo. We also described Mycobacterium bovis (BCG) and M. tuberculosis (Mtb) dissemination from pulmonary granulomas by DCs. We hypothesize that DCs play a main role in the dissemination of mycobacteria and formation of Rich foci. Our goal was to understand the mechanisms and cellular interactions between brain endothelial cells and leukocytes in the formation of the foci in the CNS. Here we show that using an in vitro, primary mouse cell based BBB co-culture system, cellular aggregates resembling granulomas form on the endothelial cell layer, which after four days consisted of infected cells including DCs that were surrounded by leukocytes. More importantly aggregates migrated together to the other side of the monolayer as a migrating island of cells showing that cellular migration of mycobacterium infected cells across the BBB involves relocalization of cellular aggregates. The potential interference through the formation of inflammatory foci on the surface of the brain endothelial layer may provide a way to decrease Mtb dissemination.
Early disruption of the intercelullar junction proteins leads to alveolar-capillary barrier dysfunction and pulmonary edema in a mouse ricin intoxication model of Acute Respiratory Distress Syndrome.
Authors: Sapoznikov Anita , Ehrlich Sharon , Gal Yoav , Falach Reut , Kronman Chanoch , Sabo Tamar 
Published: January 2023

Abstract
Acute respiratory distress syndrome (ARDS), charactarized by an intense pulmonary inflammation involving neutrophil recruitment, is accompanied by a severe disruption of the alveolar-capillary barrier leading to life-threatening pulmonary edema. Ricin, a plant-toxin derived from the seeds of Ricinus communis, irreversibly inactivates ribosomes by site-specific depurination, thereby arresting cell protein synthesis, while its clinical manifestation following pulmonary exposure is that of rapidly developing severe ARDS. We evaluated the role of the members of tight, adherens and gap junction proteins in pulmonary edema formation in a clinically relevant mouse model of ARDS induced by ricin intoxication, since the respiratory endothelium and epithelium barrier function depends on the integrity of intercellular junctions. We found a significant reduction in Claudin5, Claudin18, VE-Cadherin and Connexin43 in mice lungs 3 hours after ricin intoxication. Neutrophil depletion markedly attenuated pulmonary edema and in combination with a matrix metalloproteinase (MMP) inhibitor restored the expression of VE-Cadherin and Connexin43, providing an evidence for MMP-dependent proteolysis of these proteins. Unlike the aforementioned proteins, disruption of Claudin18 following ricin intoxication was found to be neutrophil-independent and involved tyrosine phosphorylation. These results provide an in vivo mechanism for early impairment of the alveolar-capillary barrier by intercellular junction protein disruption, which at later time points, is enhanced by the specific loss of type II alveolar epithelial cells that no longer provide resistance to edema formation and altogether lead to respiratory failure and death.
A potential target for methotrexate in macrophages : AMP-activated protein kinase.
Authors: Cudrici Cornelia D , Pelletier Martin , Siegel Richard 
Published: January 2023

Abstract

Background
Methotrexate (MTX) remains a cornerstone of treatment in multiple forms of inflammatory arthritis. AMPK is a highly conserved protein kinase present in all eukaryotic cells and is activated by an increasing intracellular ADP/ATP levels. Once activated, AMPK will promote ATP production by switching on catabolic and turning off anabolic pathways. We hypothesize that AMPK activation mediates a major portion of the anti-inflammatory effects of MTX.

Methods
We investigated the role of the anti-inflammatory effect of MTX via AMPK in human monocytes-derived macrophages (MDM) and mouse bone marrow-derived macrophages (BMDM) along with AICAR and A769662 (AMPK activators) and compound C (a selective AMPK inhibitor). AMPK phosphorylation and total AMPK were measured by Western blotting. Cells were then stimulated with LPS or TNF-α, and production of pro-inflammatory cytokines were measured in the supernatant.

Results
MTX induced AMPK activation with effects comparable to the AMPK activators (A769662, AICAR ) in hMDM and BMDM. MTX-induced AMPK activation was associated with a reduction in the production of IL-6, IL-1 β, and TNF-α in response to LPS and TNF stimulation. Compound C is able to partially reverse the effects of MTX.

Conclusions
MTX is able to induce AMPK activation in both MDM and BMDM, and suppress pro-inflammatory cytokines in a manner dependent on AMPK activity. These results have been confirmed genetically in macrophages deficient in AMPK subunits and in serum transfer arthritis. Our findings raise the possibility that some anti-inflammatory effects of MTX are mediated by AMPK, suggest that AMPK may be a target for the anti-inflammatory agents and a target for the development of new anti-inflammatory drugs.