Transcription Factor IRF3 Research Articles

Tyrosine phosphorylation of IRF3 by BLK facilitates its sufficient activation and innate antiviral response.

Viral infection triggers the activation of transcription factor IRF3, and its activity is precisely regulated for robust antiviral immune response and effective pathogen clearance. However, how full activation of IRF3 is achieved has not been well defined. Herein, we identified BLK as a key kinase that positively modulates IRF3-dependent signaling cascades and executes a pre-eminent antiviral effect. BLK deficiency attenuates RNA or DNA virus-induced ISRE activation, interferon production and the cellular antiviral response in human and murine cells, whereas overexpression of BLK has the opposite effects. BLK-deficient mice exhibit lower serum cytokine levels and higher lethality after VSV infection. Moreover, BLK deficiency impairs the secretion of downstream antiviral cytokines and promotes Senecavirus A (SVA) proliferation, thereby supporting SVA-induced oncolysis in an in vivo xenograft tumor model. Mechanistically, viral infection triggers BLK autophosphorylation at tyrosine 309. Subsequently, activated BLK directly binds and phosphorylates IRF3 at tyrosine 107, which further promotes TBK1-induced IRF3 S386 and S396 phosphorylation, facilitating sufficient IRF3 activation and downstream antiviral response. Collectively, our findings suggest that targeting BLK enhances viral clearance via specifically regulating IRF3 phosphorylation by a previously undefined mechanism.

C-type lectin receptor 2d forms homodimers and heterodimers with TLR2 to negatively regulate IRF5-mediated antifungal immunity

Dimerization of C-type lectin receptors (CLRs) or Toll-like receptors (TLRs) can alter their ligand binding ability, thereby modulating immune responses. However, the possibilities and roles of dimerization between CLRs and TLRs remain unclear. Here we show that C-type lectin receptor-2d (CLEC2D) forms homodimers, as well as heterodimers with TLR2. Quantitative ligand binding assays reveal that both CLEC2D homodimers and CLEC2D/TLR2 heterodimers have a higher binding ability to fungi-derived β-glucans than TLR2 homodimers. Moreover, homo- or hetero-dimeric CLEC2D mediates β-glucan-induced ubiquitination and degradation of MyD88 to inhibit the activation of transcription factor IRF5 and subsequent IL-12 production. Clec2d-deficient female mice are resistant to infection with Candida albicans, a human fungal pathogen, owing to the increase of IL-12 production and subsequent generation of IFN-γ-producing NK cells. Together, these data indicate that CLEC2D forms homodimers or heterodimers with TLR2, which negatively regulate antifungal immunity through suppression of IRF5-mediated IL-12 production. These homo- and hetero-dimers of CLEC2D and TLR2 provide an example of receptor dimerization to regulate host innate immunity against microbial infections.

In-vitro immunomodulatory responses and antiviral activities of antimicrobial peptide octominin against fish pathogenic viruses

Antimicrobial peptides (AMPs) are considered a novel approach to stimulate fish antiviral mechanisms for defense against a broad range of viral infections by enhancing immunomodulatory activities. Octominin is an AMP derived from the defense proteins of Octopus minor. In this study, preliminary screening of octominin against viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and infectious pancreatic necrosis virus (IPNV) was carried out. Moreover, immune responses upon octominin treatment and IHNV challenge were investigated using fathead minnow (FHM) cells. The CC50s of octominin for FHM and Chinook salmon embryo-214 (CHSE-214) cells were 2146.2 and 1865.2 μg/mL, respectively. With octominin treatment, EC50 resulted in 732.8, 435.1, and 925.9 μg/mL for VHSV, IHNV, and IPNV, respectively. The selectivity indices were 2.9, 4.9, and 2.0, respectively. The transcriptional analysis results demonstrated the induced transcription factors (Irf3; 143-fold, Irf7; 105-fold, and NF-κB; 8-fold), stress response gene (HspB8; 2-fold), and apoptosis functional gene (p53; 3-fold) in octominin treated (500 μg/mL) FHM cells for 48 h. Moreover, IHNV viral copy number was slightly decreased with the octominin treatment (500 μg/mL) in FHM cells. Overall results suggest that octominin could be a potential antiviral agent, although further studies are necessary to understand its mode of action and the mechanism of its antiviral activity.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2.

Shock drives a STAT3 and JunB-mediated coordinated transcriptional and DNA methylation response in the endothelium.

Endothelial dysfunction is a crucial factor in promoting organ failure during septic shock. However, the underlying mechanisms are unknown. Here, we show that kidney injury after lipopolysaccharide (LPS) insult leads to strong endothelial transcriptional and epigenetic responses. Furthermore, SOCS3 loss leads to an aggravation of the responses, demonstrating a causal role for the STAT3-SOCS3 signaling axis in the acute endothelial response to LPS. Experiments in cultured endothelial cells demonstrate that IL-6 mediates this response. Furthermore, bioinformatics analysis of in vivo and in vitro transcriptomics and epigenetics suggests a role for STAT, AP1 and interferon regulatory family (IRF) transcription factors. Knockdown of STAT3 or the AP1 member JunB partially prevents the changes in gene expression, demonstrating a role for these transcription factors. In conclusion, endothelial cells respond with a coordinated response that depends on overactivated IL-6 signaling via STAT3, JunB and possibly other transcription factors. Our findings provide evidence for a critical role of IL-6 signaling in regulating shock-induced epigenetic changes and sustained endothelial activation, offering a new therapeutic target to limit vascular dysfunction.

Control of nutrient uptake by IRF4 orchestrates innate immune memory.

Natural killer (NK) cells are innate cytotoxic lymphocytes with adaptive immune features, including antigen specificity, clonal expansion and memory. As such, NK cells share many transcriptional and epigenetic programs with their adaptive CD8+ T cell siblings. Various signals ranging from antigen, co-stimulation and proinflammatory cytokines are required for optimal NK cell responses in mice and humans during virus infection; however, the integration of these signals remains unclear. In this study, we identified that the transcription factor IRF4 integrates signals to coordinate the NK cell response during mouse cytomegalovirus infection. Loss of IRF4 was detrimental to the expansion and differentiation of virus-specific NK cells. This defect was partially attributed to the inability of IRF4-deficient NK cells to uptake nutrients required for survival and memory generation. Altogether, these data suggest that IRF4 is a signal integrator that acts as a secondary metabolic checkpoint to orchestrate the adaptive response of NK cells during viral infection.

SARS-CoV-2 infection induces epigenetic changes in the LTR69 subfamily of endogenous retroviruses

Accumulating evidence suggests that endogenous retroviruses (ERVs) play an important role in the host response to infection and the development of disease. By analyzing ChIP-sequencing data sets, we show that SARS-CoV-2 infection induces H3K27 acetylation of several loci within the LTR69 subfamily of ERVs. Using functional assays, we identified one SARS-CoV-2-activated LTR69 locus, termed Dup69, which exhibits regulatory activity and is responsive to the transcription factors IRF3 and p65/RELA. LTR69_Dup69 is located about 500 bp upstream of a long non-coding RNA gene (ENSG00000289418) and within the PTPRN2 gene encoding a diabetes-associated autoantigen. Both ENSG00000289418 and PTPRN2 showed a significant increase in expression upon SARS-CoV-2 infection. Thus, our study sheds light on the interplay of exogenous with endogenous viruses and helps to understand how ERVs regulate gene expression during infection.

Interferon induction by STING requires its translocation to the late endosomes.

To combat microbial infections, mammalian cells use a variety of innate immune response pathways to induce synthesis of anti-microbial proteins. The cGAS/STING pathway recognizes cytoplasmic viral or cellular DNA to elicit signals that lead to type I interferon and other cytokine synthesis. cGAMP, synthesized by DNA-activated cGAS, activates the ER-associated protein, STING, which oligomerizes and translocates to other intracellular membrane compartments to trigger different branches of signaling. We have reported that, in the ER, EGFR-mediated phosphorylation of Tyr245 of STING is required for its transit to the late endosomes, where it recruits and activates the transcription factor IRF3 required for IFN induction. In the current study, we inquired whether STING Tyr245 phosphorylation per se or STING's location in the late endosomes was critical for its ability to recruit IRF3 and induce IFN. Using pharmacological inhibitors or genetic ablation of proteins that are essential for specific steps of STING trafficking, we demonstrated that the presence of STING in the late endosomal membranes, even without Tyr245 phosphorylation, was sufficient for IRF3-mediated IFN induction.

Targeting PI3Kα increases the efficacy of anti-PD-1 antibody in cervical cancer.

Targeting programmed death 1(PD-1) has been approved for relapsed cervical cancer with unsatisfactory clinical efficacy. This study aims to analyse the impact of PI3K pathway activation on tumour immune microenvironment and evaluates the immune sensitization effect by PI3K inhibition in cervical cancer. The effect of PIK3CA mutation on PD-L1 expression and CD8+ T cells differentiation was determined in cervical cancer tissues. Luciferase and ChIP-qPCR/PCR assays were used to determine the transcriptional regulation of PD-L1 by PIK3CA-E545K. The effects of PI3K inhibitor treatment on immune environment in vitro and in vivo were evaluated by RNA sequencing (RNA-seq) and flow cytometry. The efficacy of PI3K inhibitor and anti-PD-1 therapy was assessed in cell-derived xenografts (CDX) and patients-derived xenografts (PDX). PD-L1 overexpression is more frequently observed in elder women with squamous cervical carcinoma. It predicts longer progress-free survival and overall survival. PIK3CA mutation results in increased mRNA and protein levels of PD-L1, the repression of CD8+ T cell differentiation in cervical cancer. Here, we report a case that continuous pembrolizumab monotherapy treatment induced complete remission of a recurrent cervical cancer patient with systemic metastasis and PIK3CA-E545K mutation, implying that PIK3CA mutation is potentially a biomarker for pembrolizumab treatment in cervical cancer. Specifically, this mutation promotes the expression of PD-L1 by upregulating the transcription factor IRF1. PI3Kα-specific inhibitor markedly activates immune microenvironment by regulating the PD-1/L1-related pathways and promoting CD8+ T cell differentiation and proliferation in Caski-CDXs with PIK3CA-E545K mutation. PI3Kα inhibitor significantly enhances the anti-tumour efficacy of PD-1 blockade in CDXs and PDXs. PIK3CA mutations may predict the response of cervical cancer to PD-1 blockade. The efficacy of PI3Kα inhibitors combined with PD-1 antibodies is promising in cervical cancer and warrants additional clinical and mechanistic investigations.

CRISPR-Cas- induced IRF3 and MAVS knockouts in a salmonid cell line disrupt PRR signaling and affect viral replication.

Interferon (IFN) responses are critical in the resolution of viral infections and are actively targeted by many viruses. They also play a role in inducing protective responses after vaccination and have been successfully tested as vaccine adjuvants. IFN responses are well conserved and function very similar in teleosts and mammals. Like in mammals, IFN responses in piscine cells are initiated by intracellular detection of the viral infection by different pattern recognition receptors. Upon the recognition of viral components, IFN responses are rapidly induced to combat the infection. However, many viruses may still replicate and be able to inhibit or circumvent the IFN response by different means. By employing CRISPR Cas9 technology, we have disrupted proteins that are central for IFN signaling in the salmonid cell line CHSE-214. We successfully generated KO clones for the mitochondrial antiviral signaling protein MAVS, the transcription factors IRF3 and IRF7-1, as well as a double KO for IRF7-1/3 using an optimized protocol for delivery of CRISPR-Cas ribonucleoproteins through nucleofection. We found that MAVS and IRF3 KOs inhibited IFN and IFN-stimulated gene induction after intracellular poly I:C stimulation as determined through gene expression and promoter activation assays. In contrast, the IRF7-1 KO had no clear effect. This shows that MAVS and IRF3 are essential for initiation of intracellular RNA-induced IFN responses in CHSE-214 cells. To elucidate viral interference with IFN induction pathways, the KOs were infected with Salmon alphavirus 3 (SAV3) and infectious pancreatic necrosis virus (IPNV). SAV3 infection in control and IRF7-1 KO cells yielded similar titers and no cytopathic effect, while IRF3 and MAVS KOs presented with severe cytopathic effect and increased titers 6 days after SAV 3 infection. In contrast, IPNV yields were reduced in IRF3 and MAVS KOs, suggesting a dependency on interactions between viral proteins and pattern recognition receptor signaling components during viral replication. Aside from more insight in this signaling in salmonids, our results indicate a possible method to increase viral titers in salmonid cells.

The TLR7/IRF-5 axis sensitizes memory CD4+ T cells to Fas-mediated apoptosis during HIV-1 infection.

HIV-1 infection is characterized by inflammation and a progressive decline in CD4+ T cell count. Despite treatment with antiretroviral therapy (ART), the majority of people living with HIV (PLWH) maintain residual levels of inflammation, a low degree of immune activation, and higher sensitivity to cell death in their memory CD4+ T cell compartment. To date, the mechanisms responsible for this high sensitivity remain elusive. We have identified the transcription factor IRF-5 to be involved in impairing the maintenance of murine CD4+ T cells during chronic infection. Here, we investigate whether IRF-5 also contributes to memory CD4+ T cell loss during HIV-1 infection. We show that TLR7 and IRF-5 were upregulated in memory CD4+ T cells from PLWH, when compared with naturally protected elite controllers and HIVfree participants. TLR7 was upstream of IRF-5, promoting Caspase 8 expression in CD4+ T cells from ART HIV-1+ but not from HIVfree donors. Interestingly, the TLR7/IRF-5 axis acted synergistically with the Fas/FasL pathway, suggesting that TLR7 and IRF-5 expression in ART HIV-1+ memory CD4+ T cells represents an imprint that predisposes cells to Fas-mediated apoptosis. This predisposition could be blocked using IRF-5 inhibitory peptides, suggesting IRF-5 blockade as a possible therapy to prevent memory CD4+ T cell loss in PLWH.

The underlying mechanism of transcription factor IRF1, PRDM1, and ZNF263 involved in the regulation of NPPB rs3753581 on pulse pressure hypertension

To investigate the correlation between NPPB gene variants and pulse pressure hypertension and the underlying regulatory mechanisms and try to confirm that NPPB may be a potential molecular target of gene therapy for pulse pressure hypertension. A total of 898 participants were recruited from the First Affiliated Hospital of Fujian Medical University and the plasmids with differential expression of NPPB were constructed. Genotype distribution of NPPB(rs3753581, rs198388, and rs198389)was analyzed and the expression of N-terminal pro-B-type natriuretic peptide(NT-proBNP) and renin-angiotensin -aldosterone system(RAAS) related indicators were identified in the groups studied. According to a genotype analysis, there was a significant difference in the genotype distribution of NPPB rs3753581 among the groups (P = 0.034). In logistic regression analysis, NPPB rs3753581 TT was associated with a 1.8-fold greater risk of pulse pressure hypertension than NPPB rs3753581 GG (odds ratio = 1.801; 95% confidence interval: 1.070–3.032; P = 0.027). The expression of NT-proBNP and RAAS related indicators in clinical and laboratory samples showed striking differences. The activity of firefly and Renilla luciferase in pGL-3-NPPB-luc (-1299G) was higher than pGL-3-NPPBmut-luc(-1299 T)(P < 0.05). The binding of NPPB gene promoter rs3753581 (-1299G) with transcription factors IRF1, PRDM1, and ZNF263 was predicted and validated by the bioinformatics software TESS and chromatin immunoprecipitation(P < 0.05). NPPB rs3753581 was correlated with genetic susceptibility to pulse pressure hypertension and the transcription factors IRF1, PRDM1, and ZNF263 may be involved in the regulation of NPPB rs3753581 promoter (-1299G) on the expression of NT-proBNP/RAAS.

STAT1 is required to establish but not maintain interferon‐γ‐induced transcriptional memory

Exposure of human cells to interferon‐γ (IFNγ) results in a mitotically heritable yet reversible state called long‐term transcriptional memory. We previously identified the clustered GBP genes as strongly primed by IFNγ. Here, we discovered that in primed cells, both interferon‐responsive transcription factors STAT1 and IRF1 target chromatin with accelerated kinetics upon re‐exposure to IFNγ, specifically at promotors of primed genes. Priming does not alter the degree of IFNγ‐induced STAT1 activation or nuclear import, indicating that memory does not alter upstream JAK–STAT signaling. We found STAT1 to be critical to establish transcriptional memory but in a manner that is independent of mere transcription activation. Interestingly, while Serine 727 phosphorylation of STAT1 was maintained during the primed state, STAT1 is not required for the heritability of GBP gene memory. Our results suggest that the memory of interferon exposure constitutes a STAT1‐mediated, heritable state that is established during priming. This renders GBP genes poised for subsequent STAT1 and IRF1 binding and accelerated gene activation upon a secondary interferon exposure.

Tumor-derived prostaglandin E2 programs cDC1 dysfunction to impair intratumoral orchestration of anti-cancer T cell responses

Type 1 conventional dendritic cells (cDC1s) are critical for anti-cancer immunity. Protective anti-cancer immunity is thought to require cDC1s to sustain Tcell responses within tumors, but it is poorly understood how this function is regulated and whether its subversion contributes to immune evasion. Here, we show that tumor-derived prostaglandin E2 (PGE2) programmed a dysfunctional state in intratumoral cDC1s, disabling their ability to locally orchestrate anti-cancer CD8+ Tcell responses. Mechanistically, cAMP signaling downstream of the PGE2-receptors EP2 and EP4 was responsible for the programming of cDC1 dysfunction, which depended on the loss of the transcription factor IRF8. Blockade of the PGE2-EP2/EP4-cDC1 axis prevented cDC1 dysfunction in tumors, locally reinvigorated anti-cancer CD8+ Tcell responses, and achieved cancer immune control. In human cDC1s, PGE2-induced dysfunction is conserved and associated with poor cancer patient prognosis. Our findings reveal a cDC1-dependent intratumoral checkpoint for anti-cancer immunity that is targeted by PGE2 for immune evasion.

Gene Set Enrichment Analysis Reveals That Fucoidan Induces Type I IFN Pathways in BMDC

Abstract Fucoidan, a sulfated polysaccharide extracted from brown seaweed, has been proposed to effectively treat and prevent various viral infections. However, the mechanisms behind its antiviral activity are not completely understood. We investigate here the global transcriptional changes in bone marrow-derived dendritic cells (BMDCs) using RNA-Seq technology. Through both analysis of differentially expressed genes (DEG) and gene set enrichment analysis (GSEA), we found that fucoidan-treated BMDCs were enriched in virus-specific response pathways, including that of SARS-CoV-2, as well as pathways associated with nucleic acid-sensing receptors (RLR, TLR, NLR, STING), and type I interferon (IFN) production. We show that these transcriptome changes are driven by well-known regulators of the inflammatory response against viruses, including IRF, NF-κB, and STAT family transcription factors. Furthermore, 435 of the 950 upregulated DEGs are classified as type I IFN-stimulated genes (ISGs). Flow cytometric analysis additionally showed that fucoidan increased MHCII, CD80, and CD40 surface markers in BMDCs, indicative of greater antigen presentation and co-stimulation functionality. Our current study suggests that fucoidan transcriptionally activates PRR signaling, type I IFN production and signaling, ISGs production, and DC maturation, highlighting a potential mechanism of fucoidan-induced antiviral activity. 이 연구는 충남대학교 연구비와 교육부 조성 한국연구재단(NRF-2019R1I1A3A01060331 to J.K.) 또한, 과학기술정보통신부가 지원하는 한국연구재단(NRF)을 통한 브레인풀 프로그램(NRF-2020H1D3A2A02048437, J.G.K.)과 과학기술부(NRF-2021M3H9A1030267, S.Y.K.)의 지원으로 진행됐다.

A lncRNA from an inflammatory bowel disease risk locus maintains intestinal host-commensal homeostasis

Inflammatory bowel diseases (IBD) are known to have complex, genetically influenced etiologies, involving dysfunctional interactions between the intestinal immune system and the microbiome. Here, we characterized how the RNA transcript from an IBD-associated long non-coding RNA locus (“CARINH-Colitis Associated IRF1 antisense Regulator of Intestinal Homeostasis”) protects against IBD. We show that CARINH and its neighboring gene coding for the transcription factor IRF1 together form a feedforward loop in host myeloid cells. The loop activation is sustained by microbial factors, and functions to maintain the intestinal host-commensal homeostasis via the induction of the anti-inflammatory factor IL-18BP and anti-microbial factors called guanylate-binding proteins (GBPs). Extending these mechanistic insights back to humans, we demonstrate that the function of the CARINH/IRF1 loop is conserved between mice and humans. Genetically, the T allele of rs2188962, the most probable causal variant of IBD within the CARINH locus from the human genetics study, impairs the inducible expression of the CARINH/IRF1 loop and thus increases genetic predisposition to IBD. Our study thus illustrates how an IBD-associated lncRNA maintains intestinal homeostasis and protects the host against colitis.

Current progress on innate immune evasion mediated by Npro protein of pestiviruses.

Interferon (IFN), the most effective antiviral cytokine, is involved in innate and adaptive immune responses and is essential to the host defense against virus invasion. Once the host was infected by pathogens, the pathogen-associated molecular patterns (PAMPs) were recognized by the host pattern recognition receptors (PRRs), which activates interferon regulatory transcription factors (IRFs) and nuclear factor-kappa B (NF-κB) signal transduction pathway to induce IFN expression. Pathogens have acquired many strategies to escape the IFN-mediated antiviral immune response. Pestiviruses cause massive economic losses in the livestock industry worldwide every year. The immune escape strategies acquired by pestiviruses during evolution are among the major difficulties in its control. Previous experiments indicated that Erns, as an envelope glycoprotein unique to pestiviruses with RNase activity, could cleave viral ss- and dsRNAs, therefore inhibiting the host IFN production induced by viral ss- and dsRNAs. In contrast, Npro, the other envelope glycoprotein unique to pestiviruses, mainly stimulates the degradation of transcription factor IRF-3 to confront the IFN response. This review mainly summarized the current progress on mechanisms mediated by Npro of pestiviruses to antagonize IFN production.

Abstract 1158: Inosine endows CAR T cells with features of increased stemness and anti-tumor potency

Abstract Chimeric antigen receptors (CARs) are synthetic receptors that link an extracellular tumor specific domain to intracellular signaling domains. Despite of remarkable results against refractory B cell malignancies, intense effort is underway to augment the potency of CAR T cells in order to overcome the suppressive tumor microenvironment, which is associated with T cell exhaustion. Adenosine is a major mediator of immune suppression. Ectoenzyme CD39 plays a central role in the generation of adenosine by catalyzing the metabolism of ATP into ADP/AMP. Then CD73 subsequently metabolizes ADP/AMP into adenosine which mediates immune suppression through adenosine associated receptor signaling. CD39 is also expressed by exhausted CD8+ and tumor reactive T cells within the tumor microenvironment, where it is associated with tumor progression, but it remains unclear whether exhausted and/or tumor reactive CD39+CD8+ T cells mediate immune suppression via the purigenic pathway. We developed a high affinity version of the disialoganglioside (GD2)-targeting chimeric antigen receptor (HA-GD2) that spontaneously clusters on the surface of human T cells in the absence of antigen and mimics chronic antigen exposure leading to T cell exhaustion. Using this model, we demonstrate that exhausted CD39+CD8+ CAR T cells actively produce adenosine and mediate immune suppression. In an attempt to generate adenosine resistance and enhance the function of exhausted CAR T cells, we knocked out CD39, CD73, or A2aR (adenosine A2a receptor) but observed only modest changes in phenotype and transcriptomics. In contrast, overexpression of transmembrane-bound adenosine deaminase, which metabolizes adenosine to inosine, induced higher frequency of stem- and central- like memory T cells, and a simultaneous decrease of exhausted T cell subpopulations. Direct exposure of HA-GD2 CAR T cells to high inosine concentration during cell manufacturing process, lead to a higher frequency of central-like memory cells and significant fitness enhancement associated with broad changes at the metabolic level. RNAseq and cyTOF analysis indicated decreased glycolytic flux, increased mitochondrial activity driven by glutamine and polyamine metabolism. Further, inosine altered the epigenetic state of HA-GD2 CAR T cells. We observed significant enrichment of IRF and NF-κB transcription factor motifs and motifs associated with memory differentiation in T cells grown in the presence of inosine. Finally, we showed that production of exhausted HA-GD2 and clinical GD2 CAR T cells in inosine-containing culture media enhances their in vivo efficacy, leading to improved survival in GD2+ tumor-bearing mice. In conclusion, we propose introducing inosine during GMP cell manufacturing as a novel strategy to improve CAR T function and subsequent clinical outcomes of CAR T cell therapy. Citation Format: Dorota Klysz, Katie Freitas, Meena Malipatlolla, Stefanie Maier, Carley Fowler, Louai Labanieh, Lucille Stuani, Bence Daniel, Katalin Sandor, Peng Xu, Jing Huang, Amaury Leruste, Vimal Keerthi, Nikolaos Gkitsas, Shabnum Patel, Justin Arredondo-Guerrero, Chris Fisher, Kyle Asano, Sunny Patel, Ansuman Satpathy, Kara Davis, Elena Sotillo, Steven Feldman, Crystal Mackall. Inosine endows CAR T cells with features of increased stemness and anti-tumor potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1158.

Antigen-specific B cells direct T follicular-like helper cells into lymphoid follicles to mediate Mycobacterium tuberculosis control.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a global cause of death. Granuloma-associated lymphoid tissue (GrALT) correlates with protection during TB, but the mechanisms of protection are not understood. During TB, the transcription factor IRF4 in T cells but not B cells is required for the generation of the TH1 and TH17 subsets of helper T cells and follicular helper T (TFH)-like cellular responses. A population of IRF4+ T cells coexpress the transcription factor BCL6 during Mtb infection, and deletion of Bcl6 (Bcl6fl/fl) in CD4+ T cells (CD4cre) resulted in reduction of TFH-like cells, impaired localization within GrALT and increased Mtb burden. In contrast, the absence of germinal center B cells, MHC class II expression on B cells, antibody-producing plasma cells or interleukin-10-expressing B cells, did not increase Mtb susceptibility. Indeed, antigen-specific B cells enhance cytokine production and strategically localize TFH-like cells within GrALT via interactions between programmed cell death 1 (PD-1) and its ligand PD-L1 and mediate Mtb control in both mice and macaques.

Novel antimyeloma therapeutic option with inhibition of the HDAC1-IRF4 axis and PIM kinase.

Multiple myeloma (MM) preferentially expands and acquires drug resistance in the bone marrow (BM). We herein examined the role of histone deacetylase 1 (HDAC1) in the constitutive activation of the master transcription factor IRF4 and the prosurvival mediator PIM2 kinase in MM cells. The knockdown or inhibition of HDAC1 by the class I HDAC inhibitor MS-275 reduced the basal expression of IRF4 and PIM2 in MM cells. Mechanistically, the inhibition of HDAC1 decreased IRF4 transcription through histone hyperacetylation and inhibiting the recruitment of RNA polymerase II at the IRF4 locus, thereby reducing IRF4-targeting genes, including PIM2. In addition to the transcriptional regulation of PIM2 by the HDAC1-IRF4 axis, PIM2 was markedly upregulated by external stimuli from BM stromal cells and interleukin-6 (IL-6). Upregulated PIM2 contributed to the attenuation of the cytotoxic effects of MS-275. Class I HDAC and PIM kinase inhibitors cooperatively suppressed MM cell growth in the presence of IL-6 and invivo. Therefore, the present results demonstrate the potential of the simultaneous targeting of the intrinsic HDAC1-IRF4 axis plus externally activated PIM2 as an efficient therapeutic option for MM fostered in the BM.