bluuebunny/crossref_metadata_2025_split huggingface dataset:
E3 ubiquitin ligase NKLAM/RNF19b regulates inflammatory cytokine production in a Sendai virus pneumonia model
Authors: Lawrence Donald W. , Shornick Laurie , Kornbluth Jacki 
Published: January 2023

Abstract
Recent studies have shown that E3 ubiquitin ligases are important mediators of the innate immune response. Studies from our laboratory have defined a role for the ubiquitin ligase natural killer lytic-associated molecule (NKLAM) in mouse and human innate immunity. Here, we present novel data describing a role for NKLAM in regulating the immune response to Sendai virus (SeV)-induced respiratory infection in mice. We found that NKLAM−/− mice demonstrated significantly less weight loss than wild type mice during SeV infection. Immunohistochemical analysis showed that the lungs of NKLAM−/− mice had less perivascular and peribronchial leukocyte accumulation than wild type mice. Using flow cytometry, we found the lungs of NKLAM−/− mice contained a lower percentage of neutrophils, macrophages and NK cells than wild type mice post-infection. The expression of several key pro-inflammatory cytokines/chemokines that are transcriptionally regulated by STAT1 and/or NFκB such as IL-6, IFNγ and MCP-1 was decreased in NKLAM−/− mice. Consistent with these findings, STAT1 and NFκB p65 phosphorylation were significantly lower in NKLAM−/− mice than in wild type mice. Collectively, our results suggest that NKLAM plays a role in orchestrating the innate immune response to SeV by regulating the expression of pro-inflammatory cytokines.
Novel Foxp3 Regulators discovered by a genome-wide CRISPR Screen
Authors: Loo Chin San , Zheng Ye 
Published: January 2023

Abstract
Forkhead box P3 (Foxp3) is an indispensable transcription factor that controls the development of regulatory T cells (Tregs) and programs their suppressive functions. Understanding the mechanisms that regulate Foxp3 expression can potentially lead to the development of therapeutics to manipulate Tregs for treating autoimmune diseases or enhancing cancer immunotherapy. Although a large body of works has been devoted to unraveling the molecular mechanisms controlling Foxp3 expression, a systematic approach is still needed to understand the regulation of Foxp3. In this study, we developed pooled retroviral CRISPR libraries and performed an efficient genome-wide knockout screen in mouse primary T cells to identify the regulators of Foxp3. The outcomes of this screen not only confirmed a number of known Foxp3 regulators but also revealed many novel factors that control Foxp3 expression. Gene ontology analysis showed that positive regulators of Foxp3 are highly enriched with genes involved in chromatin modification, including subunits in the SWI/SNF and SAGA complexes, indicating an underappreciated role for chromatin organization in Treg development and maintenance. Among the negative regulators of Foxp3, we identified a set of genes involved in mRNA processing and methylation, suggesting that transcriptional and post-transcriptional mechanisms actively mediate Foxp3 turnover in Tregs. In summary, our study generated a comprehensive picture of genes and pathways controlling Foxp3 expression and uncovered multiple novel regulators that could be exploited to manipulate Tregs. The genetic tool and strategy used in our study can also be applied to screen for factors determining other traits in mouse primary T cells.
Gata3 plays a supportive role for the development of Tbet-expressing Th17 cells in EAE
Authors: Butcher Matthew J , Gurram Rama K , Zhu Jinfang (Jeff) 
Published: January 2023

Abstract
IFNg+IL-17A+(or Tbet+RORgt+) Th17 cells are known to be key players in the pathology of experimental autoimmune encephalomyelitis (EAE), however the dynamics and overall stability of Tbet+ Th17 cells is less clear. Recent data from our lab (Zhong, C. et.al. 2016) has demonstrated that Gata3 plays a role in deciding the balance between Tbet and RORgt expression in gut NKp46+ ILC3 cells. Here, we hypothesized that Gata3 might be expressed by Th17 cell subsets during EAE, and that Gata3 expression would affect the generation or functionality of Tbet-expressing Th17 cells. Surprisingly, we found that all differentiating polyclonal and 2D2 transgenic Th17 cells upregulate Gata3 and subsequently maintain low levels of Gata3 thereafter, both in vitro and in vivo. Additionally, the re-stimulation or re-polarization of Th17 cells does not further affect Gata3 expression, reaffirming that Gata3 is induced during de novo Th17 polarization. To examine how the loss of Gata3 might affect the generation of Th17 or Tbet+ Th17 cells in EAE, we utilized complimentary Gata3 conditional knockout models. In a tamoxifen-inducible Gata3 knockout system, Tamoxifen treated CreERT2 Gata3Fl/Flmice (d6 P.I.) were resistant to EAE, and failed to mount a robust Tbet+Th17 response in comparison to vehicle controls. Similarly, Tbet-dependent Gata3 knockout Tbx21CreGata3Fl/Flmice failed to mount a robust Tbet+ Th17 response in the dLN or CNS, and were resistant to EAE symptoms in active and passive EAE models. Together, these data suggest that while all Th17 cells induce and maintain Gata3 expression, Tbet+ Th17 cells require Gata3 for their generation and survival in EAE.
This research was supported by the Intramural Research Program of NIAID, NIH.
RAS P21 Protein Activator 3 (RASA3) specifically promotes pathogenic T helper 17 cell generation by repressing T helper 2-biased programs
Authors: Wu Bing , Wan Yisong 
Published: January 2023

Abstract
Th17 cells that produce the signature cytokine interleukin 17 (IL-17) can be both pathogenic and non-pathogenic. Pathogenic-Th17 (pTh17) cells drive inflammation and immune-pathology, but whether pTh17 cells are a Th17-subset whose generation is under specific molecular control remains unaddressed. We found that Ras p21 protein activator 3 (RASA3), a GTPase activating protein, was highly expressed by IL-1β/IL-6/IL-23 induced-pTh17 cells relative to TGFβ1/IL-6 induced-non-pTh17 cells and was required specifically for pTh17 generation in vitro and in vivo. Mice conditionally deficient for Rasa3 in T cells showed less pathology during experimental autoimmune encephalomyelitis. By RNA-seq analysis, Rasa3-deficient T cells were found to acquire a Th2-biased program that dominantly trans-suppressed pTh17 cell generation via interleukin 4 production. The Th2-bias of Rasa3-deficient T cells was due to aberrantly elevated transcription factor IRF4 expression. Furthermore, RASA3 promoted proteasome-mediated IRF4 protein degradation by facilitating interaction of IRF4 with E3-ubiquitin ligase Cbl-b. Therefore, a RASA3-IRF4-Cbl-b pathway specifically directs pTh17 cell generation by balancing reciprocal Th17-Th2 programs. These findings indicate that a distinct molecular program directs pTh17 generation and reveals targets for treating pTh17-related pathology and diseases.
Akt phosphorylation of hnRNP A1 controls CD4+ T cell fate decisions
Authors: Jin Ye , White Theresa L , Gable Matthew , Morel Penelope 
Published: January 2023

Abstract
The interaction between T-cell receptor (TCR) and a specific peptide-bound major histocompatibility complex (MHC) class II molecule on an antigen-presenting cell stimulates naive CD4+ T cells to differentiate into either effector T (Teff) or regulatory T cells (Treg). Previous studies have shown that CD4+ T cell fate decision is controlled by TCR signal strength. High dose of antigen promotes the differentiation into Teff cells, while low dose of antigen favors the Treg fate. We previously reported that the Akt/mTOR signaling pathway is involved in this process of alternate fate decision. High or low dose of antigen results in differential Akt phosphorylation and subsequent Akt substrate specificity. Upon high dose of antigen, dually phosphorylated Akt (T308 and S473) phosphorylates FoxO1, which excludes it from the nucleus; while under low dose of antigen, phosphorylated Akt (T308) activates RNA processing factors hnRNP L and hnRNP A1. Our lab has shown that both hnRNP L and A1 are necessary for the induction of Tregs in vitro. Other studies reported the identification of S199 as the phosphorylation site of hnRNP A1 by Akt. However, the relationship between Akt phosphorylation on S199 of hnRNP A1 and Treg induction is unclear. In order to investigate this, we are performing RIP-Seq on RNAs bound by hnRNP A1 under low or high stimulatory condition. We have generated a mutant mouse in which the hnRNP A1 S199 site has been mutated to Alanine. We are currently examining the impact of this mutation on the nature of RNAs bound by hnRNP A1 in T cells stimulated by low or high dose of antigen. In conclusion, our goal is to determine the mechanism by which Akt phosphorylation of hnRNP A1 is implicated in the CD4+ T cell differentiation programs.
Substrate rigidity affects human regulatory T cell induction in vitro
Authors: Shi Lingting , Lee Jounghyun Helen , Kam Lance 
Published: January 2023

Abstract
Immunotherapy using regulatory T cells (Tregs) has shown recent successes in the treatment of autoimmune and inflammatory diseases such as type 1 diabetes. While natural Tregs are unstable and dysfunctional in the inflammatory milieu, induced Tregs are more potent and durable. Tregs can be induced from CD4+CD25− T cells in vitro with TGF-β and IL-2 during activation. Chemical pathways in Treg induction have been heavily investigated, but the impact of mechanical cues on Treg induction has not been thoroughly explored. As T cell activation has been shown to be sensitive to the rigidity of the activating substrate, Treg induction may also be modulated by substrate rigidity. To test this hypothesis, Tregs were induced with 10 ng/ml TGF-β and IL-2 on different rigidities of polyacrylamide gels (5 to 110 kPa) coated with anti-CD3 and anti-CD28. The hydrogel stiffness was controlled by adjusting the acrylamide monomer and crosslinker content. The density of activating antibodies was varied by altering the concentration of streptavidin acrylamide that allows the binding of biotinylated antibodies to the gels. High ligand density on the stiffer substrate significantly upregulated the rate of Treg induction, measured by percent Foxp3 high T cells. Decreasing the ligand density shifted the optimal rigidity to softer substrates. This preliminary data has shown that Treg induction is mechanosensitive and dependent on ligand density, which can contribute to the understanding of mechanosensing in Treg induction and the improvement of biomaterial design for generating functional and stable Tregs to advance Treg adoptive therapy.
Harnessing alloreactive responses by patient lymphocytes to achieve anti-cancer responses
Authors: Fast Loren D , Pando Alejandro , Reagan John L. 
Published: January 2023

Abstract
Potent immune responses are induced by encounter with cells from a different (allogeneic) individual. The increased number of responding alloreactive T cells can be explained by the cross-reactivity exhibited by T cell receptors (TCR). This cross-reactivity also allows these TCR to bind to syngeneic major histocompatibility complex molecules expressing peptides derived from pathogens or cancer cells. Initial studies showed that CD3+ lymphocytes isolated from newly diagnosed leukemic patients were able to lyse syngeneic leukemic cells after stimulation with allogeneic cells in about half of the patients. These findings raise the possibility that manipulating the alloreactive responses by patient lymphocytes could result in enhanced anti-cancer responses. Further studies utilized a mouse acute myeloid leukemic cell line, C1498, derived from a C57BL/6 mouse. C57BL/6 splenocytes stimulated with allogeneic stimulator cells were shown to be able to lyse allogeneic target cells as well as the syngeneic C1498 leukemic cells. The level of lysis of the syngeneic leukemic cells was dependent on the strain from which the allogeneic stimulator cells were obtained. Immunization of C57BL/6 mice with C1498 cells or subcellular antigen enhanced the anti-C1498 response when stimulated with allogeneic cells. Ongoing experiments are defining whether naïve or memory effector cells are responsible for the lysis of the syngeneic leukemic cells as well as the nature of the peptide antigens that allow the syngeneic cancer cells to be recognized. The results of these studies will facilitate the use of protocols which harness alloreactive responses by patient lymphocytes to generate anti-cancer responses.
Mandated Cool Housing Temperature and Adrenergic Stress reduce the efficacy of radiation and mask the “Abscopal Effect” in mouse models of cancer
Authors: Chen Minhui , Qiao Guanxi , Hylander Bonnie L , Mohammadpour Hemn , Singh Anurag K , Repasky Elizabeth A 
Published: January 2023

Abstract
Mice used for modeling cancer and anti-tumor immunity experience increased adrenergic stress due to IACUC-mandated cool housing temperatures. We wondered if this source of stress could be impacting the efficacy of ionizing radiation and the frequency of an “abscopal” effect (in which tumors outside the field of irradiation exhibit a response, an effect linked to enhanced systemic anti-tumor immunity). When mice bearing two separate CT26 tumors were housed at a thermoneutral temperature (TT; ~30°C) and 6 Gy of radiation was delivered to one tumor, control of both the irradiated and non-irradiated tumors was much greater than that seen in both tumors of mice housed at standard cool temperature (ST; ~22°C). Moreover, when tumor bearing mice housed at ST were treated with the pan β-blocker, propranolol and radiation, we observed a significantly improved growth control of irradiated and non-irradiated tumors. These effects were lost in SCID mice, and in mice depleted of CD8+T-cells. Experiments using knockout mice specifically identified a role for the β2 adrenergic receptor in mediating these effects. Moreover, we found that intra-tumoral effector T-cells, including IFN-γ+, GzmB+, TNFα+ and T-bet+CD8+T-cells, as well as the ratio of IFNγ+CD8+T-cells to Tregs, increased while numbers of MDSC and Tregs decreased, in mice treated with β-blocker and radiation. Finally, cured mice treated with propranolol and radiation rejected local and distant rechallenges in a tumor-specific manner. In conclusion, these data suggest that adrenergic stress plays a major role in regulating the efficacy of radiation therapy. Blockade of β2 adrenergic signaling could be a useful new strategy in the radiation oncology clinic.
Increasing intratumoral dendritic cells to potentiate anti-tumor effects of Newcastle disease virus-based immunotherapy
Authors: Svensson-Arvelund Judit , Cuadrado-Castano Sara , Hammerich Linda , Yellin Michael , Marsh Henry , Brown Brian D , García-Sastre Adolfo , Brody Joshua 
Published: January 2023

Abstract
Oncolytic viruses (OV) represent a novel, mechanistically distinct therapy with significant anti-tumor potential and minimal toxicity. Crucial to OV efficacy is their induction of immunogenic cell death resulting in tumor associated antigen (TAA) uptake by dendritic cells (DCs) and cross-presentation to TAA-reactive T cells. Therefore, we hypothesized that OV efficacy would be increased by increasing intratumoral DCs. Because DCs are potently activated by type I IFNs and Newcastle disease virus (NDV) is a potent type I IFN inducer, we tested our hypothesis by treating A20 lymphoma-bearing mice with intratumoral Flt3L and NDV. In vitro, NDV significantly enhanced the susceptibility of A20 cells to CD8 T cell killing, in a TAA-specific manner. In vivo, the growth of A20 tumors was significantly inhibited by NDV; however NDV alone failed to eradicate some tumors. The addition of Flt3L potentiated the effect of NDV and led to sustained complete remissions in the majority of mice. Ex vivo characterization showed that Flt3L induced a potent accumulation of immature DCs in the tumor, spleen and lymph nodes, and NDV induced the activation of these DCs, as well as activation of NK and T cells. Further, Flt3L enhanced the ability of APCs to cross-prime TAA-specific T cells upon NDV-induced tumor cell death, as shown by increased proliferation and production of IFN-γ and TNF. The therapeutic effect of the Flt3L and NDV combination was completely lost in Batf3−/− mice (which lack cross-presenting DCs), demonstrating the importance of cross-priming in vivo. These findings support the use of NDV and Flt3L as a combined OV-immunotherapy with the potential to activate a powerful anti-tumor immune response and promote long-term immunological memory.
Fatty acid depletion is a reversible cause of kynurenine induced T cell apoptosis
Authors: Beier Ulf H , Cully Michelle D , Siska Peter J , Singer Katrin , Jiao Jing , TeSlaa Tara , Quinn William J , Hancock Wayne W , Baur Joseph A , Rabinowitz Joshua D , Kreutz Marina 
Published: January 2023

Abstract
Metabolic conditions in the tumor microenvironment (TME) are a barrier for anti-tumor immunotherapy. The TME metabolite kynurenine binds aryl hydrocarbon receptor which has been linked to immunosuppressive effects. We questioned if kynurenines could be utilized for therapeutic immunosuppression, and examined the effect of kynurenines on human and murine effector T cell (Teff) metabolism and function. We co-stimulated C57BL/6 Teff for three days and measured bioenergetic function with Seahorse. Injection of 1 mM L- or D-kynurenine reduced extracellular acidification by 4.2 ±0.2 and 7.1 ±0.4% over 1 hr, respectively (p&lt;0.05). Next, we labeled activated Teff with 60 mg/dL [13C] glucose for 3 hr. We observed a 44.2% and 49.3% reduction of [13C]-glucose derived M+3 pyruvate production with D- and L-kynurenine, respectively (p&lt;0.05, 3/group). We also noted a reduction in lipid pools, with a 56.5/33.8%, 40.1/43.9%, 52.5/43.6%, and 70.7/43.2% decline in the palmitate, oleate, lineolate, and myristate with L-/D-kynurenine, respectively. These data pointed to an important role for lipid catabolism in kynurenine rich environments. We observed, that 3-day co-stimulated Teffs ±1 mM L- or D-kynurenine had 98.9 ±0.55 or 97.3 ±0.55% cell death (vs. 69.7 ±18.84% in untreated Teff, 4/grp). Addition of 0.2–0.6 mM oleate/palmitate rescued the apoptosis phenotype, restoring Teff viability and proliferation. Our results show lipid consumption as a reversible cause of kynurenine-induced Teff apoptosis, indicating Teff apoptosis as a potential in vitro artifact of low lipid content in traditional cell culture media, the immune modulatory potential of TME fatty acids, or the possible use of kynurenines to induce lipid catabolism therapeutically.
CD8+ T cells regulate tumor ferroptosis by targeting the system xc− during cancer immunotherapy
Authors: Wang Weimin , Green Michael , Choi Jae Eun , Gijón Miguel , Kennedy Paul , Liao Peng , Lang Xueting , Kryczek Ilona , Sell Amanda , Johnson Jeffrey , Cieslik Marcin , Vatan Linda , Xia Houjun , Zhou Jiajia , Li Jing , Li Gaopeng , Wei Shuang , Zhang Hongjuan , Gu Wei , Liu Rebecca , Lawrence Theodore , Stone Everett , Georgiou George , Chan Timothy , Chinnaiyan Arul , Zou Weiping 
Published: January 2023

Abstract
Cytotoxic T cells recognize specific antigens expressed on tumor cells and mediate tumor cell apoptosis mainly through perforin–granzyme-mediated and FAS-mediated pathways. Ferroptosis is a recently discovered form of cell death that differs from apoptosis and results from iron-dependent lipid peroxide accumulation. The potential contribution of CD8+ T cell-mediated cytotoxic activity and immunotherapy to tumor ferroptosis remains unknown. Here, we find that immunotherapy-activated CD8+ T cells sensitize tumor cell ferroptosis. Mechanistically, IFNγ released from CD8+ T cells downregulates expression of SLC3A2 and SLC7A11, two subunits of glutamate-cystine antiporter system xc−, restrains tumor cell cystine uptake, and as a consequence, promotes tumor cell lipid peroxidation and ferroptosis. In preclinical models, depletion of cyst(e)ine by cyst(e)inase in combination with checkpoint blockade synergistically enhances T cell-mediated anti-tumor immunity and induces tumor cell ferroptosis. Expression of glutamate-cystine antiporter system xc− is negatively associated with CD8+ T cell signature, IFNγ expression, and cancer patient outcome. Transcriptome analyses before and during nivolumab therapy reveal that clinical benefits correlate with reduced expression of SLC3A2 and increased IFNγ and CD8. Thus, T cell-promoted tumor ferroptosis is a novel anti-tumor mechanism. Targeting tumor ferroptosis pathway constitutes a therapeutic approach in combination with checkpoint blockade.
Mature neutrophils acquire a T cell suppressor phenotype in the ovarian cancer microenvironment that requires complement and NADPH oxidase activation
Authors: Emmons Tiffany Rose , Singel Kelly L. , Khan ANM Nazmul , Alqassim Emad , Blom Anna Marie , Ram Sanjay , Holland Steven , Odunsi Kunle , Zsiros Emese , Segal Brahm H. 
Published: January 2023

Abstract
Metastatic epithelial ovarian cancer (EOC) is associated with ascites and accumulation of immunosuppressive myeloid cells. Granulocytic myeloid-derived suppressor cells are an immature population that impairs anti-tumor immunity. We previously found that granulocytes from the ascites of patients with newly diagnosed EOC were morphologically mature and suppressed ex vivo anti-CD3/CD28-stimulated T cell proliferation. Circulating neutrophils (PMN) from EOC patients were not suppressive, but acquired a suppressor phenotype (defined as ≥ 1 log10 reduction of T cell proliferation) after ascites supernatant exposure. Healthy donor PMN exposed to ascites supernatants acquired the same suppressor phenotype and also inhibited T cell activation and cytokine production. Our goal was to delineate the mechanisms for this PMN suppressor phenotype. Treatment of ascites supernatants with compstatin, an inhibitor of C3 activation, fully abrogated the PMN suppressor phenotype. Inhibition of C5 activation by anti-C5 or OmCI partially abrogated T cell suppression. Neutralizing Ab against C7, a required component of the membrane attack complex had no effect. Malignant effusions from patients with various metastatic cancers also induced the C3-dependent PMN suppressor phenotype, showing generalizability of the phenotype. NADPH oxidase-deficient PMN from patients with X-linked chronic granulomatous disease did not acquire a suppressor phenotype following ascites supernatant exposure, indicating that the suppressor phenotype is NADPH oxidase-dependent. Our results point to PMN impairing T cell expansion and activation in the tumor microenvironment and the potential for targeting C3 and NADPH oxidase as therapeutic approaches.