bluuebunny/crossref_metadata_2025 huggingface dataset:
It takes a village: STATs control IE-CTL pathogenesis in Celiac Disease
Authors: Borregard Megan Elisabeth , Broekema Roeland , Jonkers Iris , Jabri Bana
Published: August 2023

Celiac Disease (CeD) is an autoimmune-like inflammatory disorder triggered by exposure to dietary gluten in genetically predisposed individuals. Tissue destruction in CeD is driven by tissue-resident, cytotoxic CD8+ intraepithelial lymphocytes (IE-CTLs). Upregulation of tissue alarmins and inflammatory cytokines – including IL-15, IL-21, and type I interferon – is evident in biopsies from CeD patients. It is thought that these cytokines may license IE-CTLs to kill stressed intestinal epithelial cells and contribute to their long-term pathogenic reprogramming. We therefore explored the contribution of these cytokines, interferons, and TCR stimulation in IE-CTL activation and pathogenic conversion. We found that both cytokine and TCR stimulation induced overlapping and distinct transcriptional programs. Pathways associated with activation and effector function were most robustly induced by a combination of cytokines. Further, genes identified from CeD-associated GWAS were also more strongly induced by cytokine stimulation. Cytokine signaling resulted in the activation of signal transducer and activator of transcription (STAT) pathways, which subsequently act as key TFs in IE-CTL reprogramming. To link specific transcriptional responses to particular STATs we performed individual and combined knockdowns of the four STATs associated with these cytokines. Our results showed significant redundancy in the STAT signaling pathways following cytokine stimulation, suggesting that convergence in individual STAT signaling nodes in IE-CTLs is integral to CeD pathogenesis.

Supported by grants from NSF GRFP 1746045 and NIH BIBIB 2 T32 EB 9412.
Cytometric analysis authenticates adjuvanticity of interferon-γ and suggests potential synergy with toll-like receptor agonists
Authors: Bian Yuanzhi , Walter Debra , Zhang Chenming
Published: August 2023

Interferon-γ (IFN-γ) is a cytokine that plays an important role in immune regulation, especially in the activation and differentiation of immune cells. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that sense structural motifs related to pathogens and alert immune cells to the invasion. Both IFN-γ and TLR agonists have been investigated as immunoadjuvants to augment the efficacy of cancer immunotherapies and vaccines against infectious diseases. In this study, we aimed to explore the potentiality of IFN-γ and TLR agonists being applied as dual adjuvant systems. In brief, murine dendritic cells were treated with IFN-γ and/or the TLR agonists, polyinosinic-polycytidylic acid (poly I:C) or resiquimod (R848). Subsequently, the cells were stained for an activation marker, cluster of differentiation 86 (CD86), and the percentage of CD86-positive cells was measured by flow cytometry. IFN-γ efficiently stimulated a considerable number of the dendritic cells, while the TLR agonists by themselves could merely activate a few compared to the control. The combination of IFN-γ with poly I:C or R848 triggered a higher amount of cell activation than IFN-γ alone. For instance, 10 ng/mL IFN-γ with 100 μg/mL poly I:C achieved 59.1% cell activation, which was significantly higher than the 33.4% positive cells obtained by 10 ng/mL IFN-γ (p = 0.0004). These results suggested that IFN-γ and TLR agonists could be applied as a complementary, dual adjuvant system to promote dendritic cell activation and antigen presentation. Additionally, there might be a synergy between the two classes of adjuvants, and further investigation is warranted to ascertain the interaction of their adjuvant activities.

This work was supported by the NIDA/NIH award UG3DA048775.
Reshaping the anti-tumor CD4 T-cell repertoire contributes to oncolytic virus therapeutic efficacy in malignant glioma
Authors: Leavenworth Jianmei Wu , Grimes Jeffrey M , Ghosh Sadashib , Murchison Charles , New J Stewart , Crossman David , Markert James M
Published: August 2023

Malignant gliomas are deadly brain tumors with no cure. We engineered herpes simplex virus (HSV)-1 and developed oncolytic HSV (oHSV) to treat brain tumors, given its potential to selectively kill tumor cells while sparing normal brain cells, and to boost anti-tumor immunity. The therapy shows great promise based on our Phase I trial results. However, the precise mechanisms for its enhanced anti-tumor immune responses remain poorly understood. Using an oHSV secreting murine IL-12 (M002), murine intracranial glioma models and adoptive transfer approaches, we observed that M002 treatment induced unique CD4+ T-cell populations to acquire cytolytic effector phenotypes, promoting anti-glioma responses in an MHCII-dependent manner. Notably, single-cell RNA sequencing and T-cell receptor (TCR) repertoire analysis revealed that M002 treatment shifted the TCR dominance while reducing the clonal diversity. These results suggest that the capacity of oncolytic virus therapy to reshape the anti-tumor CD4+ T-cell repertoire contributes to its improved efficacy. Insights obtained from this study can facilitate the development of better therapeutic strategies for brain tumor patients.

Supported by DoD W81XH-18-1-0315 and UAB Faculty Start-up Funds (to JWL)
RNA-based Personalized Cancer Vaccine for AML immunotherapy
Authors: Liu Haiyan , Tham Guo Hong , Ren Xi , Yang Henry
Published: August 2023

Acute Myeloid Leukemia (AML) is a rapidly progressing malignancy relying primarily on chemotherapy as the frontline treatment option. Success of personalized neoantigen vaccines in melanoma patients have garnered great attention in the field of immunotherapy. We hypothesize that personalized cancer vaccine, in the form of mRNA, can be a viable treatment strategy for AML patients. Next generation sequencing was performed on both C57BL6Jinv mouse’s and C1498 cell line’s nucleic acid materials. Three different neoepitope prediction pipelines were utilized to identify potential immunogenic neoepitopes. Candidate neoepitopes within intron polyadenlyation sites (iPAS), retained intron (RI) sites and somatic mutation (SM) sites were found. A total of 285 SM-derived neoepitopes were identified. Top 38 highly expressed genes were selected for further analysis. Using Sanger sequencing, we confirmed 37 out of the 38 mutations. A total of 5 iPAS-derived neoepitopes were identified. A total of 26 RI-derived neoepitopes were identified in our first RNAseq analysis and a separate 476 RI-derived neoepitopes were identified in our second RNAseq analysis. With these neoepitope candidates, we narrowed down our selection and did a preliminary screen for immunogenic epitopes using a total of 71 synthetic peptides in an ELISpot assay. 10 neoepitope candidates were short-listed for further in-vivo evaluation. mRNA vaccine was synthesized and evaluated in a murine AML model. T cell responses against the 10 neoepitopes were analyzed to provide pre-clinical evidence for patient studies. Neoepitope screening platform with human PBMCs has also been established to validate the prediction pipelines in AML patients.

Supported by grant from NMRC (MOH-CIRG21jun-0008)
Can Engineering IL-2Rα Expression Improve NK Cell Immunotherapy?
Authors: Lee Seung-Hwan , Marr Bryan , Jo Donghyeon , McComb Scott
Published: August 2023

Natural killer (NK) cells are innate lymphocytes central to anti-viral and anti-tumour responses. Activated NK cells upregulate IL-2Rα, promoting the formation of a high-affinity heterotrimeric IL-2Rαβγ that mediates NK cell expansion and enhances cytotoxicity. Several applications of NK cancer immunotherapy, including using chimeric antigen receptors (CAR), have shown early success in clinical studies. Allogeneic NK cells can illicit anti-tumour activity without toxicities, making them attractive for a biomanufactured “off-the-shelf” immunotherapy. Following adoptive transfer, low-dose IL-2 injections are commonly administered to enhance NK cell activity. However, expansion, persistence, and tumour clearance remain largely inadequate. Moreover, regulatory T cells (Tregs) can use their IL-2Rα expression to sequester injected IL-2, leading to Treg expansion and NK cell suppression. We hypothesize that CAR NK cell therapy can be improved by engineering high expression of IL-2Rα alongside the CAR. We predict the resulting CAR-IL2Rα NK cells will be more sensitive to low-dose IL-2 injections. We used lentiviral transduction of a multicistronic transgene to force high IL-2Rα expression alongside a CAR. I found CAR-IL2Rα NK cells were more responsive to low-dose IL-2 in-vitro than conventional CAR NK cells. This novel strategy may improve clinical success by enhancing CAR NK cell expansion, persistence and anti-tumour activity, and limiting Treg suppression in the tumour microenvironment.

NRC Disruptive Technology Solutions Cell and Gene Therapy Challenge Program and the Canadian Institutes of Health Research
Synthetically mannosylated antigens impair the antigen-specific humoral immune response and prolong the therapeutic efficacy of immunogenic protein drugs
Authors: Wallace Rachel P , Tremain Andrew C , Wilson D Scott , Hubbell Jeffrey
Published: August 2023

Immunogenic protein drugs trigger an anti-drug antibody (ADA) response in patients which reduces drug efficacy and increases adverse reactions. Our lab has previously shown that targeting antigen to the liver microenvironment can reduce antigen-specific T cell responses; herein, we present a novel strategy to further increase delivery of an antigen to the tolerogenic microenvironment of the liver via conjugation to a synthetic mannose polymer (p(Man)). This delivery leads to reduced antigen-specific T follicular helper cell and B cell responses resulting in diminished ADA production which is maintained throughout subsequent administrations of the native biologic. We found that p(Man) treatment impairs the antibody response against recombinant uricase, a highly immunogenic biologic, without a dependence on hapten-skewing or control by Tregs. Through transcriptomic analysis we identified increased TCR signaling and increased apoptosis and exhaustion in T cells as effects of p(Man)-antigen treatment, which may inform the underlying mechanism driving the reduction in ADAs. This modular platform may enhance tolerance to current and future biologics, enabling long-term therapeutic solutions for an ever-increasing healthcare problem.
Design and characterization of a tumor-retained interleukin-2 cytokine/antibody fusion protein for cancer immunotherapy
Authors: Silver Aliyah Brianne , Spangler Jamie B
Published: August 2023

Interleukin-2 (IL-2) is a pleiotropic cytokine with clinical utility in stimulating anti-cancer immunity. However, its efficacy is limited by its short serum half-life, systemic toxicity, and concurrent activation of immunostimulatory and immunosuppressive immune cells. IL-2 can signal down an intermediate-affinity receptor consisting of IL-2 receptor β (IL-2Rβ) and the common gamma chain (γc) or a high-affinity receptor that also includes IL-2Rα. Regulatory T (Treg) cells express high levels of IL-2Rα making them more responsive to IL-2 than natural killer (NK), naïve/resting effector T (Teff), and memory T cells. To extend the half-life and selectively bias the immunostimulatory functions of IL-2 in a unimolecular format, we engineered a single-chain fusion protein, F10 immunocytokine (F10 IC), that intramolecularly fuses IL-2 to an anti-IL-2 antibody that sterically blocks IL-2Rα binding and allosterically enhances IL-2Rβ binding. However, preclinical data suggested a narrow therapeutic index (TI) for F10 IC. To improve the TI of F10 IC we designed a format of F10 IC poised for intratumoral delivery and retention. We fused collagen binding domains (CBD) to F10 IC in various topographies (F10 IC-CBD) to exploit the abundance and exposure of collagen in the tumor microenvironment. With intratumoral delivery, F10 IC-CBD is retained in the tumor and exhibits a therapeutically favorable pharmacokinetic profile. Furthermore, F10 IC-CBD increases and prolongs intratumoral immune cell infiltration, significantly slows tumor progression, minimizes F10 IC toxicity and synergizes with anti-PD-1 blockade in syngeneic mouse tumor models. We have illustrated the potential of F10 IC-CBD as a potent tumor-agnostic cancer therapy.

Supported by grants from the NIH (R01 EB029341, T32 AI007417), Melanoma Research Alliance, Willowcroft Foundation and a Sanofi iAward.
Antigen loaded monocytes prime CD4+ T cell responses through Notch2 dependent cDC2
Authors: Lueck Benjamin D , D’Anniballe Vincent M , Gunn Michael D
Published: August 2023

Cellular vaccines, primarily monocyte-derived dendritic cells (MoDC), have recently been realized as therapeutic cancer vaccines with modest clinical efficacy. Understanding the mechanism by which these vaccines lead to antigen presentation is key to improving their potency. A significant advancement was the discovery that MoDC vaccines do not directly present antigen, but instead rely on endogenous dendritic cells for their antigen presenting cell (APC) function. Subsequent research has also demonstrated that primary monocytes can be loaded with antigen and used as cellular vaccines, outperforming MoDC vaccines in mouse models of melanoma and glioblastoma. In this study, we characterized how monocyte vaccines generate CD4+ T cell responses. We utilized the CD11c-Cre RBPJfl/fl mouse model, which lack a subset of Notch2 dependent cDC2 characterized by expression of Endothelial cell-selective adhesion molecule (ESAMhi). We demonstrate that the ESAMhicDC2 subset is required for effective CD4+ OT-II T cell priming after vaccination with ovalbumin (OVA) loaded monocytes. Interestingly, depletion of the ESAMhi DC population does not affect monocyte vaccine-induced CD8+ T cell priming, a process previously shown to be dependent on cDC1. This indicates that monocyte vaccines generate T cell responses by independently transferring antigen to both ESAMhi and CD8+ DCs, leveraging their APC function to prime CD4+ and CD8+ T cell responses, respectively. This work furthers the mechanistic understanding of monocyte vaccines, and will inform the design of future monocyte vaccines as they enter clinical trials as therapeutic cancer vaccines.

Supported by NCI P50 CA 190991 and funding from the Stead Scholarship Program of the Duke University Department of Medicine.
Synergy between oxaliplatin and adenovirus initiates immunogenic cell death in human colorectal carcinoma cells
Authors: Jessup J Milburn , Hewitt Stephen M , Fuerst Thomas R
Published: August 2023

Externalization of Calreticulin (CALR) is the critical first step in the initiation of Immunogenic Cell Death. Since Oxaliplatin (OX) cooperates with an oncolytic chimeric human adenovirus Ad5/3-NP2.ADP (ADP) to induce adaptive immunity as well as regression of established cancer, we sought to determine whether the cooperation was additive or synergistic. Methods include immunohistochemistry (IHC) for Calreticulin expression in mice 11 days after intratumoral injection of the OX-ADP mixture. In vitro assays with limiting dilutions of OX and ADP on human Clone A colorectal carcinoma cell monolayers were cultured for 3 days and cytotoxicity, viral infectivity by GFP, and the expression of externalized CALR (ectoCALR) on Clone A cells measured by static cytometry. Synergy was measured using SynergyFinder 3.0 (Ianevski et al., PMC9252834) that creates 2-D interactive surfaces to measure synergy and antagonism. Areas of synergy have mean scores > 10. IHC revealed strong CALR expression in OX-ADP treated tumors, weak expression in tumors injected with OX or ADP alone and minimal in PBS treated controls. Mean Viral Infectivity synergy score was 27.2 for the area of (Ox μM, ADP MOI): (0.12μM, 4 MOI) to (12.5μM, 100 MOI) while mean synergy score for ectoCALR expression was 29.2 for OX/ADP (0.01μM, 0.16) to (0.12μM, 20 MOI). The role of oxidative and endoplasmic reticulum stress as the basis of synergy is under study. Oxaliplatin and adenovirus are synergistic in their ability to initiate ICD which is important for vaccine development.
Chimeric antigen receptor-T cell efficacy can be evaluated on an Organ-Chip model system
Authors: Woodham Andrew W , Marianne Marianne , Cespedes Adriana V , Heng Anthony R , Frischmann Amanda S , Fortes Gabrielle M , Kajula Ville J , Ewart Lorna , Carman Christopher V
Published: August 2023

Objective

The need for human-centric model systems that can test the efficacy of chimeric antigen receptor (CAR) therapies is expanding rapidly, as these hold great promise for cancer treatment. We recently developed a system for inflammatory immune cell recruitment on the human Colon Intestine-Chip as a model for inflammatory bowel disease (IBD). The goal of the current study was to develop a novel system for measuring the recruitment and killing capacity of CAR-T cells in an Organ-Chip system.

Methods

We seeded a representative human HER2+ non-small cell lung carcinoma cell line (A549) into the top channel and human lung microvascular endothelial cells into the vascular (bottom) channel of Emulate’s S1 Organ-Chips. HER2-specific or CD19-specific (negative control) CAR-T cells were perfused through the vascular channel. Total CAR-T cell recruitment from the vasculature channel to the carcinoma-containing (top) channel and CAR-T-mediated killing (caspase activity, and IFN-g production) were measured over 72 hours. This system is further being used to monitor CAR-T cell exhaustion and efficacy of co-therapies (e.g., that might support CAR-T recruitment and killing efficacy).

Results

HER2 CAR-T cells were recruited to the top channel, which was supported by the addition of known cancer chemokines (e.g. CXCL10), and induced significant killing and response to co-therapies as measured by caspase activity compared to controls.

Conclusion

These early findings suggest that the human-centric Organ-Chip model can evaluate the efficacy of CAR-T cell therapies, and in particular, provide a system that integrates both the recruitment and killing aspects of CAR-T function.
Fourth generation Glypican-3–specific CAR-NK cells co-secreting IL-15 and IFN-α have increased anti-tumor function versus hepatocellular carcinoma
Authors: Badami Ester , Busà Rosalia , Gallina Anna , Iannolo Gioacchin , Amico Giandomenico , Douradinha Bruno , Conaldi Pier Giulio
Published: August 2023

Hepatocellular carcinoma (HCC) ranks as the third most frequent cause of cancer-related mortality, indicating the urgent need for effective therapies. Glypican-3 (GPC3) is up-regulated in HCC and not expressed in normal liver cells, representing a strategic target. Chimeric antigen receptor (CAR)-modified natural killer (NK) cells are a promising immunotherapeutic approach for cancer treatment. However, their potential applications have not been fully explored in HCC. We have patented a protocol of purification and expansion of clinically relevant numbers of NK cells starting from the liver perfusate from deceased donors. Further, we have demonstrated that activating NK cells with interferon-alpha (IFN-α) potentiates the immune response in vitro and in vivo both in a murine model of hepatocarcinoma and of viral hepatitis. In this study, we engineered primary CAR-NK cells to acquire specificity for GPC3. The construct consists of the intracellular signaling region CD28, 4–1BB, CD3ζ and CD8α hinge domain and of the truncated form of human epidermal growth factor receptor (tEGFR) as suicide gene. The fourth-generation construct is designed to secrete the transgenic cytokine IL-15, essential for NK cells function and survival. A second plasmid was also generated differing for the additional secretion of IFN-α. Here we show that we could achieve a transduction efficiency of above 50%. Exposure of GPC3+ HCC cells to CAR-NK cells resulted in enhanced specific cytotoxicity and cytokine production such al IL-2 and IFN-γ; moreover IFN-α secretion significantly increased anti-GPC3 function. Our data encourage preclinical proof-of-concept studies to clinically translate GPC3-specific NK cells as a treatment option for HCC patients.

This work was supported by Fondazione Ri.MED (Fondo Generale, ID 10019) and ISMETT (ATMP-P A105412 project, ID I00000219).
PD-L1 Expression on Circulating Antigen Presenting Macrophages in Blood Predicts PFS & OS in an Array of Metastatic Cancer Types Treated with PD-L1/PD-1 Immunotherapies
Authors: Adams Daniel L , Lopez Pablo , Lin Steven H , Ho Thai H , Gardner Kirby P , Tang Cha Mei
Published: August 2023

Cancer Associated Macrophage Like cells (CAMLs) are antigen presenting circulating stromal cells found in the blood of cancer patients (pts). Recently, PD-L1 expression on CAMLs was described in breast (BC), lung (LC) and renal cell carcinomas (RCC), appearing to parallel the inflammatory PD-L1 state of the tumor microenvironment, and acting as a possible predictive biomarker for PD-L1/PD-1 immunotherapeutics (IMTs). We monitored CAML PD-L1 from 48 pts in an array of solid tumors, prior PD-L1/PD-1 IMTs induction, to evaluate CAML’s ability to predict response by progression free survival (PFS) and overall survival (OS).

Three single blind multi-year prospective studies were run to test PD-L1 CAML expression against PFS & OS, prior to induction of therapy in metastatic (m) LC (n=24), mBC (n=16) or mRCC (n=8). We recruited pts with pathologically confirmed progressing disease after at least one failed prior line of therapy. Blood samples (7.5 mL) were taken prior to start of new therapeutic regimes, i.e. pembrolizumab (n=36) or nivolumab (n=12). Whole blood was filtered by CellSieve microfilters to collect CAMLs, stain for PD-L1, and then scored as a binary high or low, to evaluate PFS & OS hazard ratios (HRs) by censored univariate & multivariate analysis at 24 months.

CAMLs were found in 98% of all tested samples. Overall, pts with high CAML PD-L1 expression treated with IMT had significantly better PFS (HR=2.9, 95%CI=1.4–6.1, p=0.0067) and better OS (HR=2.7, 95%CI=1.2–6.1, p=0.0317) vs patients with low CAML PD-L1.

These data suggests that PD-L1 expression in circulating CAMLs appears to predict pts with increased benefit to PD-L1/PD-1 IMTs in an array of tumor subtypes, though larger studies are needed to validate these findings.

Private Funding by Creatv MicroTech, Inc. and supported grants by NCI-NIH (R43CA206840), and the Defense Advanced Research Projects Agency (DARPA)(W911NF-14-C-0098).
First dose infusion reaction during anti-CD20 monoclonal antibody therapy in chronic lymphocytic leukemia associates with high IP-10 levels amid cytokine release syndrome
Authors: Chu Charles C , Moore Jeremiah E , Bloom Paige C , Bruno Jennifer E , Quataert Sally A , Mosmann Tim R , Jaimes-Delgadillo Nydia , Herne Christine Ann , VanDerMeid Karl R , Baran Andrea M , Peterson Derick R , Taylor Ronald P , Elliott Michael Rusty , Wallace Danielle S , Barr Paul M , Zent Clive S
Published: August 2023

Intravenous (IV) anti-CD20 monoclonal antibody (mAb) therapy for chronic lymphocytic leukemia (CLL) patients often produces a first dose infusion reaction (FDIR) within the first 2 h that requires careful monitoring to avoid serious complications and fatalities. To better understand FDIR, we studied blood samples collected from 37 treatment-naïve CLL patients in our clinical trial NCT03788291 undergoing IV treatment with 50 mg of anti-CD20 mAb (rituximab). We analyzed four time points: baseline (prior to infusion), 1 h later (during infusion), at the end of infusion (~2.5 h) and at 48 h. Infusion reactions (CTCAE (v5) grade ≥2 events) were managed by interrupting the infusion and with symptomatic treatment. 24 (65%) patients had a FDIR and all patients completed the infusion.

Patient and CLL characteristics including measures of disease severity (Rai stage, IGHV mutation status, cytogenetic defects) did not correlate with FDIR. High levels of CLL cells and/or CD20 and their subsequent depletion resulting in decreased serum mAb and/or complement are thought to be risks for FDIR. However, measurements of these parameters did not correlate. Induction of cytokine release syndrome (CRS) may correlate with FDIR. However, CRS was observed in all samples after mAb infusion. The only significant associations with FDIR were higher levels of IP-10, IL-6, and IL-8, with IP-10 most significant. Anti-CD20 mAb activated cytotoxic effector cells, such as tissue-resident macrophages that ingest CLL cells via antibody-dependent cellular phagocytosis (ADCP), may be responsible for these events leading to FDIR. A mouse model of anti-CD20 mAb FDIR and in vitro ADCP cell systems will be useful to further understand the biology of FDIR.

Supported by funding from Acerta/AstraZeneca, the Cadregari Foundation, a generous donation by Elizabeth Aaron, and the NIH NCI grant number R21CA267040.
Mitochondrial translation inhibition in CD4+T cells selectively sensitizes the Th2 subset to increased cell death
Authors: Jawla Neha , Arimbasseri Aneeshkumar
Published: August 2023

Upon stimulation with antigen, resting T cells undergo extensive proliferation followed by differentiation into effector cells and long-lived memory cells. Upregulation of mitochondrial oxidative phosphorylation (OXPHOS) is essential for the activation and proliferation of resting T cells. Mitochondrial DNA encodes 13 proteins, which are integral parts of the complexes that form the mitochondrial respiratory chain. Since mitochondrial ribosomes are evolutionarily similar to bacterial ribosomes, antibiotics targeting bacterial ribosomes inhibit mitochondrial protein synthesis as well. But how these antibiotics affect T cell proliferation and differentiation is not well understood. To address this, we used chloramphenicol to inhibit mitochondrial translation during the activation of naive murine CD4+T cells in Th1 or Th2 polarizing conditions. Cell proliferation and effector functions were reduced in both Th1 and Th2 cells when activated in presence of chloramphenicol. Moreover, chloramphenicol significantly altered metabolic reprogramming in Th1 and Th2 cells indicated by a lower rate of OXPHOS and upregulation of glycolysis to maximum levels. Interestingly, inhibition of mitochondrial translation resulted in increased apoptosis following activation specifically in the Th2 effector subset. Th2 cells displayed a significant increase in caspase 3/7 activity upon mitochondrial translation inhibition. RNA sequencing analysis identified the upregulation of Bim and Puma in Th2 cells, two pro-apoptotic proteins that are known to be upregulated in response to metabolic stress. These results show that Th2 cells specifically undergo apoptosis in response to antibiotics that target mitochondrial translation.

Support from ICMR, NII-DBT
Translation inhibitor PDCD4 promotes CD8+ T cell proliferation during antiviral response
Authors: Xie Xiangfei , Araki Koichi
Published: August 2023

CD8+ T cell clonal expansion is critical for controlling intracellular infections and tumors. Thus, unravelling the mechanisms that regulate CD8+ T cell proliferation are imperative to improve vaccine design and immunotherapy. However, the molecular mechanisms that govern CD8+ T cell proliferation are still not fully understood. PDCD4 (Programmed cell death 4) was shown to inhibit translation by sequestering mRNA helicase eIF4A, which leads to translation inhibition of genes involved in cell proliferation. Despite the well-characterized anti-proliferative role of PDCD4, our studies suggest that PDCD4 promotes CD8+ T cell proliferation during the expansion phase of acute LCMV infection. We find that PDCD4 over-expressed Ag-specific CD8+ T cells have higher proliferation rate which leads to increased cell number compared to control Ag-specific CD8+ T cells. Consistent with the over-expression results, PDCD4 knocking-down Ag-specific CD8+ T cells undergo less cell proliferation, resulting in reduced clonal expansion compared to controls. Interestingly, contrary to the paradigm that increased proliferation generates more short-lived effector cells, CD8+ T cell proliferation mediated by PDCD4 does not skew the differentiation of short-lived and memory precursor effector cells. This PDCD4 mediated CD8+ T cell proliferation is also confirmed by over-expression of a constitutively active form of PDCD4 which resists degradation by CD8+ T cell endogenous ubiquitin pathway. In conclusion, our work has identified a novel pro-proliferative role of PDCD4 during CD8+ T cell response.

Supported by grants from NIH (R01 AI139675)
Epigenetic changes program innate immunity during Staphylococcus aureus craniotomy infection
Authors: Van Roy Zachary , Kielian Tammy L
Published: August 2023

A craniotomy is performed to access the brain for the treatment of tumors, epilepsy, or hemorrhage. Despite advances in surgical techniques, infections occur in 1–3% of cases, half of which are caused by Staphylococcus aureus (S. aureus). Since epigenetic mechanisms are critical for regulating leukocyte activation, we examined whether S. aureus drives epigenetic modifications to regulate immune cell function during craniotomy infection. A high-throughput screen of an epigenetic compound library using primary mouse microglia identified BET protein family members and histone deacetylases (HDACs) as critical for regulating TNF, IL-6, and IL-10 production by microglia, macrophages, neutrophils, and granulocytic myeloid-derived suppressor cells (G-MDSCs). Various class I HDACs were increased in these cell types following both in vitro and acute S. aureus infection in vivo. However, at later timepoints in vivo, HDAC2 expression was reduced, illustrating the importance of the brain microenvironment for genomic organization. Microparticle delivery of HDAC and BET inhibitors in vivo significantly increased bacterial burden concomitant with decreased inflammatory mediator production, revealing a critical role for epigenetic mechanisms in bacterial containment. Collectively, our data suggest that inflammatory mediator production is regulated by histone acetylation during craniotomy infection and aberrant epigenetic regulation may be important for promoting bacterial persistence.

Work supported by NINDS R01 NS107369 and T32 NS105594.
Mitochondrial metabolic flexibility is critical for CD8 T cell anti-tumor immunity
Authors: Chen Chao , Qu Cheng-Kui
Published: August 2023

Mitochondria utilize various substrates for energy production and intermediatory metabolism to support cellular activities. Cytotoxic CD8 T cells in different functional states during the life cycle have distinct metabolic requirements, and they experience dynamic changes in energy sources and oxygen levels in different microenvironments. The significance of mitochondrial metabolic flexibility for CD8 cell immune responses remains poorly understood. Here we report that the deletion or pharmacological inhibition of PTPMT1, a mitochondria-based phosphatase, significantly decreased effector cell specification and clonal expansion from activated CD8 cells. In addition, PTPMT1 deletion accelerated CD8 T cell exhaustion and impaired stem-like T cell maintenance, leading to increased tumor growth. PTPMT1 depletion also suppressed CD8 T cell anti-viral responses. Mechanistically, the loss of PTPMT1 critically altered mitochondrial fuel selection – the utilization of pyruvate, a major mitochondrial substrate derived from glucose (the simple carbohydrate), was inhibited whereas fatty acid and glutamate utilizations were enhanced. Persistent mitochondrial substrate shift and metabolic inflexibility induced oxidative stress and apoptosis in PTPMT1 knockout CD8 cells. The impact of the biased mitochondrial fuel selection on CD8 T cells was further verified in mitochondrial pyruvate carrier/transporter (MPC) knockout and pharmacological inhibition models. Collectively, this study reveals an important role of PTPMT1 in facilitating mitochondrial utilization of carbohydrates and that mitochondrial flexibility in energy source selection is critical for CD8 T cell anti-tumor immunity.

National Institutes of Health grants HL162725 and CA275964
Pim2 negatively regulates T-cell immune responses through modulating autophagy
Authors: Tian Linlu , Wu Yongxia , Choi Heejin , Yang Kaipo , Yu Xue-Zhong
Published: August 2023

Pim kinases affect cell survival, cell proliferation, transcriptional activation, and protein translation by phosphorylating various target substrates. We previously showed that Pim2 plays a distinct role from Pim1 and Pim3, and negatively regulates T-cell allogeneic response and anti-tumor immunity. Pim2 was reported to promote the induction of autophagy in cancer cells, a cellular process that critically impacts T-cell effector function, survival and memory formation. We hypothesize that Pim2 constrains T-cell immune responses through modulating autophagy. Using western blot and electron microscopy, we evaluated the effect of Pim2 on autophagic flux in resting and activated T cells. Pim2 deficiency in T cells attenuated LC3 lipidation, P62 degradation as well as autophagosome formation, suggesting that Pim2 facilitates autophagy in T cells. Mechanistically, Pim2 directly bound with P62 and loss of Pim2 led to accumulation of P62 which further blocked autophagic flux in T cells. Furthermore, augmentation of autophagy via overexpressing Atg5 or metformin treatment reduced effector cytokine production in Pim2-deficient T cells in vitro and reversed the heightened ability of Pim2-deficient T cells for inducing graft-versus-host disease and for controlling breast cancer growth in vivo. Taken together, Pim2 is a key promotor of autophagy in T cells and targeting Pim2 to suppress autophagy may represent a promising strategy to improve T-cell effector function in cancer immunotherapy.

The work was supported by NIH grants: R01 CA258440 and R21 CA263140.
Sin3A Controls FoxP3+ Treg Production, Stability and Function
Authors: Christensen Lanette M , Akimova Tatiana , Han Rongxaing , Wang Liqing , Samanta Arabinda , Hancock Wayne W
Published: August 2023

Purpose

Foxp3 T-regulatory (Treg) cells are key to immune homeostasis but the function of paired amphipathic helix protein, Sin3a, is unknown.

Methods

We studied the effects of Sin3a deletion in Treg cells.

Results

Sin3a−/− FoxP3Cre mice died from severe autoimmunity by 2–3 weeks of birth. Mice had enlarged lymph nodes, severe inflammation in lungs, skin, livers and kidneys and autoantibodies to islet cells, striated muscles, keratin, endomysium and gastric parietal cells. Percentages of FoxP3+ Tregs in LNs and spleens were lower (p values ranging from >0.01 to >0.0001) than controls and FoxP3 mRNA expression (>20-fold) and FoxP3 protein per Treg (>3-fold) were also significantly reduced in Sin3a−/− mice. Reduction of FoxP3+ Tregs in the periphery of Sin3a−/− FoxP3cre mice was largely due to the loss of FoxP3, determined by an increase of peripheral ex-Tregs as compared to WT (p<0.001 in sLN and spleens) within Sin3a−/− FoxP3Cre tdTomatoROSA26 lineage tracing mice. Increased mRNA expression of pro-apoptotic genes, decreased mRNA expression of anti-apoptotic genes, and increased presence of Annexin V on the cell surface indicate that apoptosis also contributed to peripheral Treg reduction. Sin3a−/− Tregs produced increased amounts of granzyme B, IFN-γ and IL-2 (12–45% of cells) and had impaired suppressive function (p<0.01). Sin3a was also essential for peripheral iTreg conversion since CD4+CD25− cells from Sin3a−/− FoxP3Cre mice lacked ability to convert to FoxP3+ iTregs ex vivo.

Conclusions

The nuclear co-regulator, Sin3a, helps maintain the unique homeostatic properties of Treg cells. Therapeutic modulation of Treg functions may be possible by pharmacologic targeting of components within the Sin3a complex.
Epigenetic Memory of COVID-19 in Innate Immune Cells and Their Progen-itors--
Authors: Cheong Jin Gyu G
Published: August 2023

Through epigenetic mechanisms, systemic inflammation may trigger innate immune memory and persistent changes in hematopoietic cells; however, it has been difficult to investigate these phenotypes in the context of human disease, such as severe coronavirus disease 2019 (COVID-19). We developed a new workflow to isolate circulating hematopoietic stem and progenitor cells (HSPC) from peripheral blood, combining single-cell RNA/ATAC-seq. With this method, we were able to capture the diversity of circulating HSPC as seen in bone marrow, allowing for the non-invasive investigation of hematopoiesis as well as epigenomic changes after COVID-19. Following severe COVID-19, we observed both transcriptomic and epigenetic changes in HSPC and its progeny innate immune cells, and they persisted for months to a year. Different transcription factor activities (including IRF, AP-1, and CTCF) were associated with these alterations, which altered the regulation of inflammatory programs. Severe COVID-19 patients’ HSPC displayed long-lasting increases in myelopoiesis and granulopoiesis, and circulating monocytes responded more strongly to subsequent stimulation. We found that epigenomic changes in HSPC were retained and passed onto differentiated progeny innate immune cells. In addition, we demonstrate that administration of an anti-IL-6 blockade during acute severe COVID-19 can normalize the persistent phenotypes, suggesting that IL-6 plays a role in the development of epigenetic memory. In conclusion, epigenetic reprogramming of HSPC may underlie altered immune function subsequent to infection and has broad applicability, particularly for millions of COVID-19 survivors with incomplete recovery.
Novel function for gluconeogenic enzyme, FBP1, in T cell activation and expansion
Authors: Kelekar Ameeta , Sha Ruohan , Yang Tingyuan , Bapat Aditi , Park Meeyeon , Simon M Celeste , Yong Jeongsik
Published: August 2023

During an immune response, naïve T lymphocytes undergo rapid clonal expansion and must reprogram their metabolism to meet high energetic and biosynthetic demands. The sudden increase in metabolic activity associated with a proliferative burst generates reactive oxygen species (ROS) increasing intracellular oxidative stress, which can disrupt homeostasis or trigger cell death, unless properly regulated. Our studies show that a constitutively active isoform of gluconeogenic enzyme, fructose 1,6-bisphosphatase 1 (FBP1), facilitates re-entry of glucose carbons into the pentose phosphate pathway to ensure the sustained production of NADPH for redox homeostasis and lipid synthesis in stimulated T cells as they transition to a phase of rapid growth, and is essential for their unperturbed clonal expansion and for an effective immune response. Wildtype (wt) FBP1, which catalyzes hydrolysis of fructose-1,6 bisphosphate in an irreversible reaction, has a C-terminal catalytic domain and an inhibitory N-terminal regulatory domain. We demonstrate that a short isoform of FBP1 representing the catalytic domain is induced in human T cells within 24 hours of co-stimulation. The short isoform, FBP1-CD, is the product of an alternatively spliced transcript and is resistant to inhibitors that suppress activity of wt FBP1, which harbors the regulatory domain. FBP1-CD levels and enzyme activity are significantly reduced 5 days post-stimulation, correlating positively with proliferation and inversely with ROS and apoptosis in T cells. In addition to new insights into immune regulation, our studies reveal a broader role for FBP1 in regulating proliferative metabolism and offer a rational explanation for the suppression of FBP1 in solid tumors.

Supported by grant from NIH (R21 AI168777)
Desmosterol plays an atheroprotective role in B cells through B cell subset differentiation and B cell activation
Authors: Ma Shelby , Moriarty Alina , Keeter W Coles , Stahr Natalie Ann , Kirk Cassandra Lee , Waseem Tayab
Published: August 2023

Atherosclerosis is a complex, inflammatory disease associated with lipid accumulation, vascular dysfunction, and inflammatory immune response in the large and medium sized arteries. B cell roles in atherosclerosis are subset specific. Increasing evidence suggests a role for glucose metabolism in shaping the immune response, but less is known about cholesterol metabolism and adaptive immunity. Desmosterol, a precursor of cholesterol in the Bloch cholesterol biosynthesis pathway, serves as a negative regulator of macrophages. To date, the effect of intermediates of cholesterol metabolism on MF functions is an area of intensive study, but little is known about effects of desmosterol in B cell subset-specific functions. We have generated a mouse model with overexpression of 24-dehydrocholesterol reductase (DHCR24), an enzyme that converts desmosterol into cholesterol, specifically in B cells using the cre/flox system crossed with an atherosclerotic mouse model (Dhcr24fl/flCd19cre/+Ldlr−/− mice). The deletion of DHCR24 results in reduction of desmosterol specifically in B cells. After fed a Western diet for 12–16 wks aortas were collected to assess lesion formation and spleens were collected to assess B cell subset distribution, activation, and functions by FACs. We demonstrate that overexpression of DHCR24 in B cells leads accelerated lesion formation. This was accompanied by increased BCR-induced B cell activation via the increased Ca2+ flux along with increased FO B cells and decreased MZ B cells. This suggests that desmosterol plays a suppressive role in B cell activation and plays a role in B cell subset differentiation and activation, highlighting a role of desmosterol as a regulator of B cell functions.
Distinct domains in the transcription factor Runx3 mediate differential antiviral CD8 T cell phenotypes
Authors: Albao Dominic S , Pipkin Matthew E
Published: August 2023

Runx3 is one of three Runt-family transcription factors (TF) that control gene expression by modifying chromatin structure. Upon initial T cell receptor stimulation, Runx3 drives chromatin accessibility found in mature circulating and tissue-resident memory CD8 T cells. Runx3-deficient cells preferentially develop a KLRG1hi CD127lo terminal effector (TE) phenotype, whereas Runx3 overexpression promotes formation of KLRG1lo CD127hi classical memory precursor (MP) cells and KLRG1hi CD127hi double positive (DP) effector cells during the effector phase. Runt-family TFs harbor activation, inhibition, and VWRPY domains that interact with distinct chromatin modifying proteins, but their individual contributions to Runx3-driven phenotypes in effector CD8 T cells are unresolved. Here we examined Runx3-deficient CD8 T cells complemented with wildtype or Runx3-domain deleted mutants during viral infection. The activation and VWRPY domains were each required to repress the TE cell phenotype. However, the activation, but not VWRPY domain, was necessary to repress high T-bet expression. Moreover, the larger gene expression pattern resulting from complementation with the VWRPY mutant was generally similar to that of wildtype Runx3. In addition, we found that wildtype Runx3 repressed the TF Tox and upregulated PD-1 and Tim-3, which required its inhibition domain. Runx3 lacking its inhibition domain also generated more MP cells and more highly induced CD127 compared to wildtype Runx3. These results indicate that distinct domains in Runx3 mediate differential control of gene expression in effector CD8 T cells, and might suggest avenues for targeting Runx3 domains to elicit specific memory T cell phenotypes.

Supported by grants from NIAID (R01 AI095634, P01 AI145815)
Glucocorticoids regulate the human non-coding genome
Authors: Kwiat Robert Ernest , Tran Thai , Cao Qilin , Gadkari Manasi , Randazzo Davide , Franco Luis M
Published: August 2023

Glucocorticoids (GCs) are the most commonly used anti-inflammatory and immunosuppressive drugs. However, most of the work on this class of drugs has focused on their relationship to the coding genome. It has been demonstrated in recent years that non-coding genes can play important biological roles. Thus, we performed total RNA-seq and small-RNA-seq in 9 primary human cell types: B cells, CD4+ T cells, monocytes, neutrophils, endothelial cells, fibroblasts, myoblasts, osteoblasts, and preadipocytes, treated in vitro with the GC methylprednisolone. We found that the response of lncRNA genes to GCs appears to be strong and cell type-dependent, with hematopoietic cells being more responsive to GCs than non-hematopoietic cells. Among GC-responsive lncRNAs, the intergenic and RNA host subtypes are overrepresented, while antisense lncRNAs are underrepresented. GC regulation of miRNAs appears to be limited to those that are part of a larger transcript. We generated a global map of GC-responsive lncRNAs and microRNAs in the 9 cell types, and an interactive web application to allow exploration of our results. We identified the lncRNA WAKMAR2 as a GC-induced gene that may play a role in GC action in CD4+ T cells and monocytes. WAKMAR2 has been shown to influence the expression of inflammatory cytokines in non-hematopoietic cells. Single-molecule RNA FISH revealed that, in human monocyte-derived macrophages, WAKMAR2 transcripts are primarily cytoplasmic. Ongoing work is aimed at determining whether WAKMAR2 is in fact a GC-induced negative regulator of inflammatory responses in human primary cells.
An IL-23/STAT4 axis regulates classical dendritic cell expansion and function in CNS autoimmunity
Authors: Alakhras Nada Salem , Kaplan Mark H
Published: August 2023

Signal transducer and activator of transcription 4 (STAT4) is a critical regulator of inflammation in autoimmunity by acting as a messenger for pro-inflammatory cytokines including IL-12 and IL-23. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, germ-line deletion of STAT4 in mice results in resistance to the development of paralysis. Here, we examined STAT4 cell-specific requirements in EAE. Using conditional Stat4 mutant mice, we found that mice lacking Stat4 in CD4+ T cells and CD11c+ cells are resistant to EAE, while Lyz2+ cells are susceptible to EAE demonstrating cell type requirement for STAT4 in EAE. STAT4 is expressed and activated in CD11c+ classical dendritic cells (cDCs) in the CNS at the peak of EAE. Stat4fl/flCD11cCre(Stat4ΔCD11c) mice exhibit a significant decrease of cDC expansion in the CNS, and this correlates with diminished numbers of T cells in the CNS and decreased inflammatory cytokine production. Adoptive transfer of wild-type DCs into Stat4ΔCD11c rescues the development of EAE. In contrast, transferring Il23r−/−DCs to Stat4ΔCD11c did not rescue EAE development. Transferred Il23r−/−DCs were retained in the lymph nodes suggesting that IL-23-STAT4 signaling in cDCs promotes their expansion in the CNS during neuroinflammation. CITE-seq analysis of CNS DCs from control and Stat4ΔCD11c mice identified STAT4-dependent genes in cDCs in EAE. Collectively, our results demonstrate that STAT4 in cDCs is required for their expansion in the CNS, the development of pro-inflammatory T cells, and the clinical symptoms of EAE. Thus, our study reveals previously unrecognized functions of STAT4 in cDCs that could identify novel therapeutic approaches for CNS autoimmunity.