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Chronic HCV infection remains a global health concern due to its involvement in hepatic and extrahepatic diseases, including B cell non-Hodgkin lymphoma (BNHL). Clinical and epidemiological evidence support a causal role for HCV in BNHL development, although mechanistic insight is lacking. We performed RNA-sequencing on peripheral B cells from patients with HCV alone, BNHL alone, and HCV-associated BNHL to identify unique and shared transcriptional profiles associated with transformation. In patients with HCV-associated BNHL, we observed the enrichment of an anergic-like gene signature and evidence of clonal expansion that was correlated with the expression of epigenetic regulatory genes. Our data support a role for viral-mediated clonal expansion of anergic-like B cells in HCV-associated BNHL development and suggest epigenetic dysregulation as a potential mechanism driving expansion. We propose epigenetic mechanisms may be involved in both HCV-associated lymphoma and regulation of B cell anergy, representing an attractive target for clinical interventions.

Adoptive T cell therapies (ACT) have been curative for a limited number of cancer patients. The sensitization of cancer cells to T cell killing may expand the benefit of these therapies for more patients. To this end, we use a three-step approach to identify cancer genes that disfavor T cell immunity. First, we profile gene transcripts upregulated by cancer under selection pressure from T cell killing. Second, we identify potential tumor gene targets and pathways that disfavor T cell killing using signaling pathway activation libraries and genome-wide loss-of-function CRISPR-Cas9 screens. Finally, we implement pharmacological perturbation screens to validate these targets and identify BIRC2, ITGAV, DNPEP, BCL2, and ERRα as potential ACT-drug combination candidates. Here, we establish that BIRC2 limits antigen presentation and T cell recognition of tumor cells by suppressing IRF1 activity and provide evidence that BIRC2 inhibition in combination with ACT is an effective strategy to increase efficacy.

Objective Transcriptional profiling of immune cells is an indispensable tool in biomedical research; however, heterogenous sample types routinely used in transcriptomic studies may mask important cell type-specific transcriptional differences. Techniques to isolate desired cell types are used to overcome this limitation. We sought to evaluate the use of immunomagnetic B cell isolation on RNA quality and transcriptional output. Additionally, we aimed to develop a B cell gene signature representative of a freshly isolated B cell population to be used as a tool to verify isolation efficacy and to provide a transcriptional standard for evaluating maintenance or deviation from traditional B cell identity. Results We found RNA quality and RNA-sequencing output to be comparable between donor-matched PBMC, whole blood, and B cells following negative selection by immunomagnetic B cell isolation. Transcriptional analysis enabled the development of an 85 gene B cell signature. This signature effectively clustered isolated B cells from heterogeneous sample types in our study and naïve and memory B cells when applied to transcriptional data from a published source. Additionally, by identifying B cell signature genes whose functional role in B cells is currently unknown, our gene signature has uncovered areas for future investigation.

Lee et al. reveal that administration of IL-1β can alter the host environment to augment the magnitude and functionality of CD8+ T cells, thereby improving the efficacy of adoptively transferred tumor-reactive T cells in treating large, established tumors in mice., Host conditioning has emerged as an important component of effective adoptive cell transfer–based immunotherapy for cancer. High levels of IL-1β are induced by host conditioning, but its impact on the antitumor function of T cells remains unclear. We found that the administration of IL-1β increased the population size and functionality of adoptively transferred T cells within the tumor. Most importantly, IL-1β enhanced the ability of tumor-specific T cells to trigger the regression of large, established B16 melanoma tumors in mice. Mechanistically, we showed that the increase in T cell numbers was associated with superior tissue homing and survival abilities and was largely mediated by IL-1β–stimulated host cells. In addition, IL-1β enhanced T cell functionality indirectly via its actions on radio-resistant host cells in an IL-2– and IL-15–dependent manner. Our findings not only underscore the potential of provoking inflammation to enhance antitumor immunity but also uncover novel host regulations of T cell responses.

Background Characterizing the kinetics of the antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of critical importance to developing strategies that may mitigate the public health burden of coronavirus disease 2019 (COVID-19). We conducted a prospective, longitudinal analysis of COVID-19 convalescent plasma donors at multiple time points over an 11-month period to determine how circulating antibody levels change over time following natural infection. Methods From April 2020 to February 2021, we enrolled 228 donors. At each study visit, subjects either donated plasma or had study samples drawn only. Anti–SARS-CoV-2 donor testing was performed using the VITROS Anti–SARS-CoV-2 Total and IgG assays and an in-house fluorescence reduction neutralization assay. Results Anti–SARS-CoV-2 antibodies were identified in 97% of COVID-19 convalescent donors at initial presentation. In follow-up analyses, of 116 donors presenting at repeat time points, 91.4% had detectable IgG levels up to 11 months after symptom recovery, while 63% had detectable neutralizing titers; however, 25% of donors had neutralizing levels that dropped to an undetectable titer over time. Conclusions Our data suggest that immunological memory is acquired in most individuals infected with SARS-CoV-2 and is sustained in a majority of patients for up to 11 months after recovery. Clinical Trials Registration. NCT04360278.

Summary: Induced pluripotent stem cell (iPSC)-derived T cells may provide future therapies for cancer patients, but those generated by current methods, such as the OP9/DLL1 system, have shown abnormalities that pose major barriers for clinical translation. Our data indicate that these iPSC-derived CD8 single-positive T cells are more like CD4+CD8+ double-positive T cells than mature naive T cells because they display phenotypic markers of developmental arrest and an innate-like phenotype after stimulation. We developed a 3D thymic culture system to avoid these aberrant developmental fates, generating a homogeneous subset of CD8αβ+ antigen-specific T cells, designated iPSC-derived thymic emigrants (iTEs). iTEs exhibit phenotypic and functional similarities to naive T cells both in vitro and in vivo, including the capacity for expansion, memory formation, and tumor suppression. These data illustrate the limitations of current methods and provide a tool to develop the next generation of iPSC-based antigen-specific immunotherapies. : A barrier for clinical application of iPSC-derived CD8 T cells using OP9/DLL1 is their abnormal biology. Vizcardo et al. show that a 3D thymic culture system enables the generation of a homogeneous antigen-specific T cell subset, named iTEs, which closely mimics naive T cells and exhibits potent anti-tumor activity. Keywords: thymopoiesis, T cell differentiation, iPSC differentiation, adoptive cell transfer, naïve T cell, recent rhymic emigrants, fetal thymus organ culture, immunotherapy, 3D culture, tumor antigen specific T cell

T cells play a central role in cancer immunosurveillance and current cancer immunotherapies, including adoptive cell transfer (ACT), T cell receptor or chimeric antigen receptor (CAR) T cell therapies and immune checkpoint blockade. Understanding the factors regulating T cell function is hence critical for improving the success of these immunotherapies. It has been recognized that metabolism can greatly affect different aspects of T cell function, including differentiation, cytokine production, longevity and exhaustion. Here we integrate genome-scale metabolic modeling (GEM) with biological experiments to discover novel metabolic determinants of T cell function. Previously we developed the metabolic transformation algorithm (MTA), a GEM method that was successfully applied to identify driving factors and targets for different diseases. Here applying MTA to data on CAR-T cell gene expression and patient response to anti-CD19 CAR-T therapy, we predicted mitochondrial metabolite transport and specifically proton transport in mitochondrial uncoupling, as key determinants of CAR-T therapy response. Mitochondrial uncoupling is also important for the in vivo persistence of adoptively transferred tumor-infiltrating lymphocytes, as further confirmed by analyzing their gene expressions from a KRAS-targeting ACT dataset. Focusing on the mitochondrial uncoupling protein 2 (Ucp2), which is abundantly expressed in T cells, we experimentally validated that it is required for T cell longevity and anti-tumor function. Specifically, the loss of Ucp2 either via knock-out (KO) or treatment by genipin (a Ucp2 inhibitor) in mice T cells results in accelerated differentiation into “terminal effector cells”, as shown by increased levels of T cell cytotoxicity and exhaustion markers, and decreased levels of central memory and stemness markers. Adoptive transfer of Ucp2-KO Pmel-1 T cells to mice bearing B16 melanomas displayed poorer anti-tumor efficacy and worse survival than the transfer of Ucp2-wildtype T cells. We find that Ucp2 modulates oxidative stress and DNA damage by regulating the levels of mitochondrial superoxide. Reducing mitochondrial reactive oxygen species was sufficient to rescue the loss of Ucp2-mediated effector T cell differentiation, senescence, cytokine production and anti-tumor activity Ucp2-KO Pmel-1 T cells. Tumor-specific CD8+ T cells could be metabolically reprogrammed by Ucp2 overexpression, which improved T cell longevity and anti-tumor function in the Pmel-1/B16 ACT mice model. In sum, our study establishes a novel role of Ucp2 in regulating T cell longevity and anti-tumor activity by repressing increased ROS levels accompanying mitochondrial dysfunction in differentiated and exhausted cells, suggesting that manipulating Ucp2 levels in T cells can be exploited to enhance T cell-based cancer immunotherapies. Citation Format: Madhusudhanan Sukumar, Kuoyuan Cheng, Arunakumar Gangaplara, Yogin Patel, Suman K. Vodnala, Rafiqul Islam, Arash Eidizadeh, Carolyn Subramaniam, Ping Lee, Rigel Kishton, Amanda N. Henning, Michael J. Kruhlak, Zhiya Yu, Ethan M. Shevach, Toren Finkel, Eytan Ruppin, Nicholas P. Restifo. Integrated computational and experimental analysis identifies the mitochondrial uncoupling protein 2 (Ucp2) as a key regulator of T cell anti-tumor function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1527.

Summary T cells are central to all currently effective cancer immunotherapies, but the characteristics defining therapeutically effective anti-tumor T cells have not been comprehensively elucidated. Here, we delineate four phenotypic qualities of effective anti-tumor T cells: cell expansion, differentiation, oxidative stress, and genomic stress. Using a CRISPR-Cas9-based genetic screen of primary T cells we measured the multi-phenotypic impact of disrupting 25 T cell receptor-driven kinases. We identified p38 kinase as a central regulator of all four phenotypes and uncovered transcriptional and antioxidant pathways regulated by p38 in T cells. Pharmacological inhibition of p38 improved the efficacy of mouse anti-tumor T cells and enhanced the functionalities of human tumor-reactive and gene-engineered T cells, paving the way for clinically relevant interventions.

Functional diversity in multicellular organisms is achieved through the differentiation of stem cells. During this process, stem cells must retain both the capacity for self-renewal and the ability to differentiate into highly specialized cell types to produce a diverse array of tissues, each with distinct functions and organization. This plasticity is achieved through alterations to the epigenome, heritable and reversible modifications to DNA and histones that affect chromatin structure and gene transcription without altering the DNA sequence itself. Alterations to the epigenome enable cell type–specific transcriptional control that can change dynamically over the life of a cell. Such flexibility and responsiveness are instrumental in directing gene expression changes throughout cellular differentiation and lineage specification. The acquisition of more specialized functions during differentiation requires not only that the epigenome turn “on” genes involved in lineage commitment, it also necessitates that genes associated with stemness are simultaneously turned “off” ( 1 ). On page 177 of this issue, Pace et al. ( 2 ) demonstrate that this phenomenon exists in CD8+ T cells, in which epigenetic repression of stemness-associated genes by the histone methyltransferase SUV39H1 is required for T cell effector differentiation. Understanding these mechanisms addresses important questions in immunology and is applicable to cancer immunotherapy.

Many biological studies involve either (i) manipulating some aspect of a cell or its environment and then simultaneously measuring the effect on thousands of genes, or (ii) systematically manipulating each gene and then measuring the effect on some response of interest. A common challenge that arises in these studies is to explain how genes identified as relevant in the given experiment are organized into a subnetwork that accounts for the response of interest. The task of inferring a subnetwork is typically dependent on the information available in publicly available, structured databases, which suffer from incompleteness. However, a wealth of potentially relevant information resides in the scientific literature, such as information about genes associated with certain concepts of interest, as well as interactions that occur among various biological entities. We contend that by exploiting this information, we can improve the explanatory power and accuracy of subnetwork inference in multiple applications. Here we propose and investigate several ways in which information extracted from the scientific literature can be used to augment subnetwork inference. We show that we can use literature-extracted information to (i) augment the set of entities identified as being relevant in a subnetwork inference task, (ii) augment the set of interactions used in the process, and (iii) support targeted browsing of a large inferred subnetwork by identifying entities and interactions that are closely related to concepts of interest. We use this approach to uncover the pathways involved in interactions between a virus and a host cell, and the pathways that are regulated by a transcription factor associated with breast cancer. Our experimental results demonstrate that these approaches can provide more accurate and more interpretable subnetworks. Integer program code, background network data, and pathfinding code are available at https://github.com/Craven-Biostat-Lab/subnetwork_inference, Author summary There is a multitude of publicly available databases that contain information about biological entities (i.e., genes, proteins, and other small molecules) as well as information about how these entities interact together. However, these databases are often incomplete. There is a wealth of information present in the text of the scientific literature that is not yet available in these databases. Using tools that mine the scientific literature we are able to extract some of this potentially relevant information. In this work we show how we can use publicly available databases in conjunction with the information extracted from the scientific literature to infer the networks that are involved in specific biological processes, such as viral replication and cancer tumor growth.

Latent Epstein-Barr virus (EBV) infection and cellular hypermethylation are hallmarks of undifferentiated nasopharyngeal carcinoma (NPC). However, EBV infection of normal oral epithelial cells is confined to differentiated cells and is lytic. Here we demonstrate that the EBV genome can become 5-hydroxymethylated and that this DNA modification affects EBV lytic reactivation. We show that global 5-hydroxymethylcytosine (5hmC)-modified DNA accumulates during normal epithelial-cell differentiation, whereas EBV+ NPCs have little if any 5hmC-modified DNA. Furthermore, we find that increasing cellular ten-eleven translocation (TET) activity [which converts methylated cytosine (5mC) to 5hmC] decreases methylation, and increases 5hmC modification, of lytic EBV promoters in EBV-infected cell lines containing highly methylated viral genomes. Conversely, inhibition of endogenous TET activity increases lytic EBV promoter methylation in an EBV-infected telomerase-immortalized normal oral keratinocyte (NOKs) cell line where lytic viral promoters are largely unmethylated. We demonstrate that these cytosine modifications differentially affect the ability of the two EBV immediate-early proteins, BZLF1 (Z) and BRLF1 (R), to induce the lytic form of viral infection. Although methylation of lytic EBV promoters increases Z-mediated and inhibits R-mediated lytic reactivation, 5hmC modification of lytic EBV promoters has the opposite effect. We also identify a specific CpG-containing Z-binding site on the BRLF1 promoter that must be methylated for Z-mediated viral reactivation and show that TET-mediated 5hmC modification of this site in NOKs prevents Z-mediated viral reactivation. Decreased 5-hydroxymethylation of cellular and viral genes may contribute to NPC formation.