bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of AMPK restricts Zika virus replication in endothelial cells by potentiating antiviral response and inhibiting viral-induced glycolysis
Authors: Kumar Ashok , Singh Sneha , Singh Pawan , Giri Shailendra
Published: January 2023

Viruses are known to perturb host cellular metabolism to enable their replication and spread. However, studies examining Zika virus (ZIKV) infection and metabolism are limited. Here, using primary human retinal vascular endothelial (HRvEC) cells and HUVEC cell line, we investigated the role of AMPK, a master regulator of energy metabolism, in response to ZIKV challenge. ZIKV infection caused a time-dependent reduction in the phosphorylation of AMPK and its downstream target ACC. Pharmacological activation of AMPK using AICAR or metformin attenuated ZIKV replication, and similar findings were observed using GSK621, a specific activator of AMPKα. This phenomenon was reversed by incubation of cells with an AMPK inhibitor, compound C. Lentivirus-mediated knockdown of AMPK and the use of AMPK−/− MEFs further supporting the notion that AMPK restricts ZIKV replication. Interestingly, AMPK activation potentiated the expression of genes with antiviral properties (e.g., IFNs, OAS2, ISG15, MX1) and inhibited inflammatory mediators (e.g., TNFα, CCL5). Bio-energetic analysis revealed that ZIKV infection evoked a glycolytic response, as evidenced by elevated ECAR (extracellular acidification rate) levels and increased expression of key glycolytic genes (GLUT1, HK1, TPI, MCT4); AICAR treatment reduced this response. Consistent with this, 2-deoxyglucose (2DG), an inhibitor of the glycolysis, augmented AMPK activity, and attenuated ZIKV replication. Collectively, our study demonstrates that AMPK activation restricts ZIKV replication by promoting an antiviral responses and inhibiting ZIKV-induced glycolysis in endothelial cells. AMPK signaling thus represents a new potential antiviral target for ZIKV infections.
Listeria hijacks host cell mitophagy to evade killing through a novel mitophagy receptor
Authors: Qian Youcun
Published: January 2023

Mitophagy, a selective form of autophagy, plays a critical role in eliminating damaged or excessive mitochondria during cell homeostasis or challenge to maintain the organelle quality and function. Listeria monocytogenes (L.m) is an intracellular pathogen of humans and animals, and an important and widely used model organism to study bacterial pathogenesis and host defense. It is still unknown if bacterial pathogens can induce host cell mitophagy. We made use of an L.m model system to show that intracellular bacterial pathogens induced host mitophagy in macrophages. Through a mitophagy inhibitor and a mitophagy promoter, we demonstrated that L.m-induced mitophagy provided a survival advantage of the bacterial pathogen. We further identified an LC3-interacting region (LIR)-containing protein as a novel mitophagy receptor for L.m-induced mitophagy. Consistent with the results of the mitophagy inhibitor, deficiency of the mitophagy receptor resulted in much reduced L.m titers both in macrophages in vitro and in mice in vivo. Thus our study discovers a previously unappreciated strategy by pathogens to hijack a host cellular homeostasis system for their survival. Mitophagy inhibitor or targeting mitophagy machinery may be useful for developing efficient therapeutic strategies against infections.
Inhibitory effect of short-chain fatty acids on the induction of nitric oxide by Staphylococcus aureus lipoproteins in macrophages
Authors: Han Seung Hyun , Park Jeong Woo , Kim Hyun Young , Kim Min Geun , Jeong Soyoung , Yun Cheol-Heui
Published: January 2023

Staphylococcus aureus is a Gram-positive bacterial pathogen that can cause severe inflammation often resulting in life-threatening diseases. Lipoproteins of S. aureus are a major virulence factor to induce excessive inflammatory mediators such as nitric oxide (NO) in macrophages. Short-chain fatty acids (SCFAs) such as butyrate, propionate, and acetate are commensal bacteria-derived metabolites in the gut that are known to have anti-inflammatory effects. In this study, we investigated the effects of SCFAs on S. aureus lipoprotein (Sa.LPP)-induced NO production in mouse macrophages. Butyrate and propionate, but not acetate, inhibited Sa.LPP-induced production of NO in RAW 264.7 cells and mouse bone marrow-derived macrophages. Butyrate and propionate inhibited Sa.LPP-induced expression of inducible NO synthase (iNOS). However, acetate did not show such effect under the same conditions. Furthermore, butyrate and propionate, but not acetate, inhibited Sa.LPPinduced activation of NF-κB, expression of IFN-β, and phosphorylation of STAT1, which are essential for inducing transcription of iNOS in macrophages. In addition, butyrate and propionate induced histone acetylation at lysine residues in the presence of Sa.LPP in RAW 264.7 cells. Moreover, Sa.LPP-induced NO production was decreased by histone deacetylase (HDAC) inhibitors. Collectively, these results suggest that the SCFAs butyrate and propionate attenuate the inflammatory responses caused by S. aureus lipoproteins through the inhibition of NF-κB, IFN-β/STAT1, and HDAC, resulting in attenuated NO production in macrophages.
Leishmania major degrades CXCL1 to subvert host immune responses
Authors: Gurung Prajwal , Yorek Matthew , Poudel Barun , Mazgaeen Lalita
Published: January 2023

Leishmaniasis is a global health problem with an estimated report of 2 million new cases every year. Innate immune system plays a central role in controlling L. major infection by initiating a signaling cascade that results in production of pro-inflammatory cytokines and recruitment of both innate and adaptive immune cells. Upon infection with L. major, CXCL1 is produced locally and plays an important role in the recruitment of neutrophils and macrophages to the site of infection. Herein, we report that L. major specifically targets murine CXCL1 for degradation and this allows L. major to establish infection in the host. The degradation of CXCL1 is not dependent on host factors because L. major can directly degrade recombinant CXCL1 in a cell-free system. Using mass spectrometry, we have identified that the L. major protease cleaves at the C-terminal end of murine CXCL1. Finally, our data suggest that L. major metalloproteases are involved in the direct degradation of CXCL1. Currently, we are testing the hypothesis that the peptide spanning the cleavage site can be used to inhibit L. major protease activity and pathogenesis.
Functional elucidation of miR-494 in modulation of HCV infection
Authors: Lin Tsun-Mei , Eng Hock-Liew
Published: January 2023

Hepatitis C virus (HCV) infection is considered one of the most important causes of chronic liver diseases due to major alteration in the cross-talk between hepatic cells and immune cells. Many reports have shown that the proteins of HCV might interact with the genes to regulate cellular pathways and miRNA expression contribute to the pathogenesis of liver diseases. In this study, the profiling of miRNAs in hepatocytes transfected with HCV structure proteins was compared by microarray analysis. MiR-494 was significantly decreased in E1 transfected HepG2 cells, but increase in core-transfected or E2-transfected HepG2 cells. However, the expression and activation of TLR7/8 and inflammatory cytokines were dramatically enhanced by E1 protein. Interferon treatment was demonstrated to enhance miR-494 expression but reduced TLR7 and TLR8 signaling in E1 protein transfected HepG2. MiR-494 was proposed to regulate TLR7 and TLR8 expression as confirmed by directly targeting 3′-UTR of TLR7 and TLR8. However, the molecular functions of the involvement of miR-494 in HCV infected hepatocytes remained to be elucidated. The expression of miR-494 in serum samples of HCV infected patients was increased as compared with corresponding controls (mean ± SEM: 23,100 ± 9,700 vs.32.5± 14.2, p< 0.001). Determined through analysis using XTT and transwell assays, the HCV core-overexpression and miR-494 restoration were demonstrated to significantly enhance HepG2 proliferation and migration, In conclusion, our results have shown that HCV structure proteins might play a role in the pathogenesis of chronic hepatic diseases via interaction with miRNA-494 to modulate TLR7/8 expression.
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

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

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

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

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

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

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

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

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

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

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<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<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

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

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.
Tumor cell IDO1 increases intratumoral immunosuppressive Tregs independent of enzyme activity
Authors: Zhai Lijie , Bell April , Qian Jun , Ladomersky Erik , Lauing Kristen L , Wainwright Derek A
Published: January 2023

Objective

Indoleamine 2, 3 dioxygenase 1 (IDO1) is a rate limiting enzyme that converts tryptophan (Trp) into kynurenine (Kyn). IDO1 knockdown in murine glioblastoma (GBM) cells suppresses intratumoral regulatory T cell (Treg) recruitment and increases survival, but unexpectedly, has no effect on intratumoral Trp levels. Moreover, while GBM cell IDO1 overexpression increases intratumoral Treg levels, treatment with a pharmacological IDO1 enzyme inhibitor fails to reverse this effect. These novel data led us to question whether GBM cell IDO1 recruits Tregs independent of enzyme activity, in vivo.

Methods

Ido1−/− mouse GBM cells reconstituted with wild-type or enzyme-null (H350A) murine Ido1 cDNA, and IDO1−/− human GBM cells were created. HPLC for Trp/Kyn levels, cell proliferation, cell viability, and splenic macrophage-GBM cell co-cultures, were studied in vitro. Syngeneic or humanized mice intracranially-engrafted with modified mouse or human GBM cells, respectively, were studied for the effect(s) on Treg accumulation and/or survival.

Results

As compared with IDO1 WT GBM cells, IDO1 H350A GBM cells show a significant decrease of Trp metabolism without affecting cell proliferation and viability. As compared with vector control GBM cells, co-culture for both IDO1 WT and H350A GBM cells induces greater numbers of mature macrophages. Independent of enzyme activity, GBM cell IDO1 expression increases intratumoral Tregs and decreases animal survival.

Conclusions

We created novel GBM models for elucidating the mechanism of metabolism-independent, IDO1-mediated intratumoral Treg accumulation. Given the majority of cancers with IDO1 expression, we suspect that our work may be generalizable to many types of malignancies.
Blockade of IDO/TDO downstream effectors restricts cancer immune suppression
Authors: Campesato Luis Felipe , Budhu Sadna , Tchaicha Jeremy , Jaiswal Abhinav , Gigoux Mathieu , Pourpe Stephane , Liu Cailian , Zamarin Dmitriy , Manfredi Mark G , McGovern Karen , Wolchok Jedd D , Merghoub Taha
Published: January 2023

Immune checkpoint blockade (ICB) results in clinical benefit for a subset of cancer patients, yet multiple mechanisms of resistance can impair optimal response. The catabolism of tryptophan into metabolites known as kynurenines (Kyn) by the expression of enzymes such as IDO or TDO is a frequent phenomenon that plays a suppressive role in tumor immunity. Recently it was shown that Kyn acts as agonist of the aryl hydrocarbon receptor (AHR). Here we sought to characterize the mechanisms of immune suppression associated with the AHR pathway and to evaluate its potential as therapeutic target. RNAseq analysis of human cancers revealed a correlation between the expressions of AHR-related genes with markers associated with immunotherapy resistance (PD-1, FOXP3, CD206). By using IDO or TDO-overexpressing variants of a melanoma cell model (B16-F10), we found that myeloid cells, such as tumor-associated macrophages (TAMs) and dendritic cells (DCs), present up-regulation of the AHR. IDO-expressing tumors (B16-IDO) show higher myeloid cell infiltration, which present a tolerogenic phenotype. Tumor-antigen specific CD8T cells present reduced expression of activation markers and proliferation rate when primed by Kyn-treated BMDCs. Treatment of B16-IDO-bearing mice with an AHR-specific antagonist (CH-223191) leads to an increase of MHC II in TAMs, of activation markers in CD8 T cells and reduced frequency of T-regs. AHR inhibition delays progression of tumors with an active IDO/TDO/Kyn pathway (B16-IDO and B16-TDO), and efficacy is further improved when ICB is used in combination. In summary, our findings demonstrate that targeting the Kyn pathway through AHR inhibition could overcome key suppressive mechanisms and sensitize tumors to ICB.
B Cell Subset Changes During Ovarian Cancer
Authors: Riggs James Edward , Maslanka Jeff , Londregan Jennifer , Goldman Naomi , DePierri Kelley , Somerville John
Published: January 2023

Early biomarkers of ovarian cancer (OvCa) are needed for timely disease detection. Monitoring alterations in the immune system might be informative in this regard. To study humoral immunity during OvCa, we transplant murine epithelial carcinoma cells (ID8) into the peritoneal cavity (PerC). As the PerC is enriched for B1 cells, we assessed B cell subset composition via flow cytometry. As OvCa developed PerC B1 cells were lost while B2 cells persisted in the PerC and spleen (SP). This difference might reflect that self-reactive B1 cells have a role in housekeeping and lack progenitors in adult mice. We tested the latter hypothesis by monitoring transitional (T) B cells, and found the T1, T2, and T3 subsets were markedly reduced in late stage disease, suggesting depletion of B1 B cells, as opposed to expansion of B2 B cells. Intriguingly, marginal zone B cells (MZB) expanded early then contracted as OvCa progressed. We speculate that housekeeping burden grows with OvCa progression and the cells most associated with this vital homeostatic process, notably B1 and MZB cells, are most at risk. These findings highlight the disruption of B cell biology in mice challenged with OvCa, and may help to inform strategies to develop biomarkers for the disease.
Potent anti-tumor effector functions in tumor-infiltrating MAIT and γδ T cells isolated from colon cancer patients
Authors: Rodin William Carl Ivar , Sundström Patrik , Ahlmanner Filip , Lindskog Elinor Bexe , Järbrink Marianne Quiding
Published: January 2023

In colon cancer, tumor progression and patient outcome is affected by the cytokine balance in the tumors. IFNγ, TNFα and Granzyme B expression are associated with favorable patient outcome, while high IL-17 expression is associated with accelerated tumor progression. However, knowledge of the regulation and activation of unconventional T cell subsets in colon tumors is limited. The aim of this study was to characterize unconventional T cells in colon tumors and unaffected tissue, determine their capacity to produce cytokines affecting tumor progression as well as the In vitro cytotoxic capabilities of MAIT and γδ T cells. Using flow cytometry, we show that MAIT cells accumulate in colon tumors and that the frequencies of γδ T cells are reduced in the tumor epithelium. Using polyclonal stimulation, we show that IFNγ production by tumour infiltrating MAIT cells is impaired whilst tumour infiltrating γδ T have an increased expression of IFNγ, TNFα and Granzyme B. IL-17 expression was also elevated in tumour infiltrating γδ T cells, but at lower levels than the TH1 - associated cytokines. Tumor infiltrating MAIT cells had an exhausted (PD-1highTim-3+) phenotype compared to MAIT cells from unaffected tissue. Analyzing cytokine expression, we show that while no single molecule is lost the polyfunctional capacity of tumour infiltrating MAIT cells is decreased compared to MAIT cells from unaffected tissue. Altogether, this study shows that γδ T cells and MAIT cells contribute to the cytokine balance in colon tumors with a TH1 – dominated profile and that they have potent cytotoxic capacity, which may reduce tumor progression and improve patient outcome.
Regulatory T cells suppress the cytotoxic phenotype of T cells in intestinal tumors of APCMin/+ mice
Authors: Szeponik Louis , Akeus Paulina , Järbrink Marianne Quiding , Rodin William Carl Ivar
Published: January 2023

The presence of activated T cells in colorectal cancer (CRC) tissues is a strong predictor of patient survival. Our previous research have shown that regulatory T cells (Treg) are able to reduce T-cell transendothelial migration in vitro and accumulation of effector T cells in intestinal tumors in vivo in a murine model for intestinal adenomas (APCMin/+).

In this study we investigated the effect of Treg depletion on the cytotoxic potential of conventional αβ T cells and γδ T cells in intestinal adenomas. We used the APCMin/+ \DEREG mouse model, which harbours a high affinity diphtheria toxin receptor under the control of the FOXP3 promoter, to deplete Treg in tumor bearing mice. We found that the number of CD8αβ T cells per mg tissue in the lamina propria was significantly increased in the Treg depleted intestinal adenomas in comparison to the non-depleted tumors. We could confirm this finding with IHC staining in tissue sections. The number of CD8αα T cells and γδ T cells remained unchanged. Furthermore, ex vivo frequencies in the lamina propria of Granzyme B+ CD8αβ T cells were increased in the Treg depleted intestinal adenomas. Following in vitro stimulation of lymphocytes from lamina propria with PMA/ionomycin, there was a trend towards a higher frequency of IFNγ+ CD8αβ T cells in Treg depleted adenomas, but frequencies of CD107a+ CD8αβ T cells were decreased. The frequency of TNF+CD8αβ T cells was elevated in the tumor in comparison to the unaffected tissue regardless of Treg depletion. We are investigating the combined effect of Treg depletion and PD-1 immunotherapy on the cytotoxic phenotype of T cells in the intestinal adenomas.

The results indicate that the modulation of Treg in colorectal cancer could have apositive effect on T cell cytotoxicity.
Tumor infiltrating B cells co-localize with CD4 T effector cells within organized tertiary lymphoid structures to present antigen and educate the anti-tumor immune response in human primary tumors
Authors: Bruno Tullia Carmela , Ruffin Ayana , Cillo Anthony , Liu Dongyuan , Kunning Sheryl , Ferris Robert L , Vignali Dario A.A. , Bruno Tullia Carmela
Published: January 2023

Only 20% of cancer patients produce a durable response to current immunotherapies. Thus, a need exists to develop additional therapeutic strategies to treat these patients, which includes evaluation of other tumor infiltrating immune cells that could further augment the T cell response. Tumor infiltrating B cells (TIL-B) represent a possible target for immunotherapy due to their crucial role in the immune response. In fact, current data demonstrate that detection of TIL-Bs within tertiary lymphoid structures (TLS) correlate with increased survival in patients with solid tumors and enhanced response to anti-PD1 immunotherapy. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer by presenting tumor antigens to CD4 TILs within TLS.

We observed increased numbers of activated TIL-Bs in the primary tumors of head and neck squamous cell carcinoma and non-small cell lung cancer patients. We further assessed the transcriptional signature and location of TIL-Bs within the TME and demonstrated that they are adjacent to Th1 and Tfh CD4+ tumor infiltrating lymphocytes (TILs) within organized, germinal center containing TLS. Further, we generated an antigen presentation assay in vitro, and we observed increased CD4+ TIL responses when TIL-Bs presented autologous tumor antigens. These data suggest that TIL-Bs influence the function of CD4+ TILs in patient tumors within TLS. Ultimately, results from this study will increase effective targeting of TIL-Bs within patient primary tumors.
Altered phenotypes of tumor-infiltrating B cells in response to exhausted CD8+ T cells
Authors: Avalos Christian A , Egelston Colt , Simons Diana , Lim Min Hui , Huang Yinghui , Lee Peter P.
Published: January 2023

During chronic infections, such as cancer, tumor antigen presentation and inflammation mobilizes a heterogeneous T cell response, consisting of cells in unique states of differentiation and functional capacity. We assayed tumor-infiltrating lymphocytes from human breast, colorectal and melanoma tumors. By flow cytometry, we observed T cells undergoing exhaustion as characterized by a loss of effector functions (IFNy, TNFa, and IL-2) and expressing surface markers PD-1, TIM-3 and CD39. Although this cell intrinsic signature of T cell exhaustion has been well studied, its association with B lymphocytes is still obscure. CXCL13 expression was one of the most significantly upregulated genes in tumor exhausted T cells, as observed by single-cell sequencing. We identified infiltration of CD138+ CD38− plasma cells and CD138−CD38−CD19+ B cells in human breast, colorectal and melanoma tumors. In addition, we observed, by immunohistochemistry, large tertiary lymphoid structures (TLS) in human breast cancer tumors composed of CD20+ B cells and T cells in close proximity. Finally, we evaluated the differential expression of immune modulatory molecules GARP, CD39 and CD73 on B cell subsets from tumors. Further work will investigate the inhibitory or stimulatory role of B cells on tumor-infiltrating T cells.
The death receptor Fas mediates local bystander killing of antigen-negative variants by antigen-specific CD8 T cells in a heterogeneous tumor
Authors: Upadhyay Ranjan , Wroblewska Aleksandra , Baccarini Alessia , Brown Brian D , Brody Joshua D
Published: January 2023

Antigen-specificity is one of the hallmarks of the adaptive immune system, and CD8 T cells are particularly crucial for eliminating malignant cells with aberrant expression of tumor-associated self-antigens or neoantigens. We established a high-throughput pooled CRISPR/Cas9 screen for functionally assessing cancer cell genes involved in modulating the cytotoxic activity of antigen-specific CD8 T cells. As validation of our approach, we identified many genes known to be altered in cancers refractory to immunotherapy, including MHC component B2m and the checkpoint molecule Cd274. Interestingly, the cell surface death receptor Fas was identified as a strong hit in mediating the killing of antigen-positive lymphoma and was further verified with breast cancer, despite the availability of other cytotoxic effectors such as perforin/granzymes. Using a 2-antigen system, we surprisingly observed that Fas was also mediating the killing of neighboring GFP-negative cancer cells by T cells highly specific for a GFP epitope. This phenomenon was not dependent on MHC Class I expression but was potentiated by interferon gamma, which is known to upregulate Fas expression. Even for T cells that were previously primed, this bystander cytotoxicity required the presence of neighboring antigen-positive cells, suggesting a very hyperlocal and temporally regulated off-target effect that was not dependent on natural killer cells in vivo. We believe this phenomenon to be crucial to the durable remissions seen with highly antigen-specific approaches such as CAR-T cell therapy. Modulating this pathway may be a viable approach for preventing immune escape of rare antigen-loss variants or treating heterogeneous tumors with no clear single target antigen.
Characterizing unconventional T cells in the tumor microenvironment of metastatic osteosarcoma
Authors: Appel Nicole , Christie John , Blattman Joseph
Published: January 2023

Osteosarcoma is the most common bone cancer and typically affects patients in the second decade of life. Current treatment methods have not proven effective for treating reoccurring or metastatic osteosarcoma (mOS) given the 5-year survival rate of 15–30%. Previous work has shown anti-PD-L1 combined with anti-CTLA-4 improves survival of mOS in mice from 0% in no treatment mice to 60% in treated mice. To further improve survival, we analyzed tumor infiltrating lymphocytes (TILs) in mice over time and discovered CD4hiCD8hiPD-1hi T cells consistently appeared in the tumor microenvironment (TME) after approximately two months. To determine whether these CD4hiCD8hi, or double positive (DP) cells, were candidates for immune therapy, TILs from mice with long-term cancer were stained for CD3 to confirm T cell lineage, CD44 to verify activation, and Qa-2, which is first expressed during the stage IV single positive phase of T cell maturation and is therefore a marker to distinguish mature from immature T cells. The DP TILs were found to be activated, mature T cells that are distinct from DP immature thymocytes. The DP TILs were further compared to splenocytes and were determined to be concentrated in the TME rather than resulting from a systemic change in the immune system. Finally, the presence of DP T cells in the lungs was found to be unique to long-term lung metastases when lung tissues from age-matched healthy mice were obtained and processed alongside experimental lung metastases. Overall, mature and activated DP T cells concentrate in the TME of long-term lung metastases of mOS.