Memory CD8+ T cells upregulate glycolysis and effector functions under limiting oxygen conditions.

Memory CD8+ T cells are indispensable for maintaining long-term immunity against intracellular pathogens and tumors. Despite their presence at oxygen-deprived infected tissue sites or in tumors, the impact of local oxygen pressure on memory CD8+ T cells remains largely unclear. We sought to elucidate how oxygen pressure impacts memory CD8+ T cells arising after infection with Listeria monocytogenes-OVA. Our data revealed that reduced oxygen pressure during in vitro culture switched CD8+ T cell metabolism from oxidative phosphorylation to a glycolytic phenotype. Quantitative proteomic analysis showed that limiting oxygen conditions increased the expression of glucose transporters and components of the glycolytic pathway, while decreasing TCA cycle and mitochondrial respiratory chain proteins. The altered CD8+ T cell metabolism did not affect the expansion potential, but enhanced the granzyme B and IFN-γ production capacity. In vivo, memory CD8+ T cells cultured under low oxygen pressure provided protection against bacterial rechallenge. Taken together, our study indicates that strategies of cellular immune therapy may benefit from reducing oxygen during culture to develop memory CD8+ T cells with superior effector functions.

Age is just a number: long-lasting T-cell immunity can span several lifetimes.

T-cell memory is considered long-lasting, but new evidence recently published in Nature by Soerens et al. shows that, under certain conditions, mouse memory T-cell immunity can span several mouse lifetimes.

Post-transcriptional control of T-cell cytokine production: Implications for cancer therapy.

As part of the adaptive immune system, T cells are vital for the eradication of infected and malignantly transformed cells. To perform their protective function, T cells produce effector molecules that are either directly cytotoxic, such as granzymes, perforin, interferon-γ and tumour necrosis factor α, or attract and stimulate (immune) cells, such as interleukin-2. As these molecules can also induce immunopathology, tight control of their production is required. Indeed, inflammatory cytokine production is regulated on multiple levels. Firstly, locus accessibility and transcription factor availability and activity determine the amount of mRNA produced. Secondly, post-transcriptional mechanisms, influencing mRNA splicing/codon usage, stability, decay, localization and translation rate subsequently determine the amount of protein that is produced. In the immune suppressive environments of tumours, T cells gradually lose the capacity to produce effector molecules, resulting in tumour immune escape. Recently, the role of post-transcriptional regulation in fine-tuning T-cell effector function has become more appreciated. Furthermore, several groups have shown that exhausted or dysfunctional T cells from cancer patients or murine models possess mRNA for inflammatory mediators, but fail to produce effector molecules, hinting that post-transcriptional events also play a role in hampering tumour-infiltrating lymphocyte effector function. Here, the post-transcriptional regulatory events governing T-cell cytokine production are reviewed, with a specific focus on the importance of post-transcriptional regulation in anti-tumour responses. Furthermore, potential approaches to circumvent tumour-mediated dampening of T-cell effector function through the (dis)engagement of post-transcriptional events are explored, such as CRISPR/Cas9-mediated genome editing or chimeric antigen receptors.

Human T cells employ conserved AU-rich elements to fine-tune IFN-γ production.

Long-lasting CD8+ T cell responses are critical in combatting infections and tumors. The pro-inflammatory cytokine IFN-γ is a key effector molecule herein. We recently showed that in murine T cells the production of IFN-γ is tightly regulated through adenylate uridylate-rich elements (AREs) that are located in the 3' untranslated region (UTR) of the Ifng mRNA molecule. Loss of AREs resulted in prolonged cytokine production in activated T cells and boosted anti-tumoral T cell responses. Here, we investigated whether these findings can be translated to primary human T cells. Utilizing CRISPR-Cas9 technology, we deleted the ARE region from the IFNG 3' UTR in peripheral blood-derived human T cells. Loss of AREs stabilized the IFNG mRNA in T cells and supported a higher proportion of IFN-γ protein-producing T cells. Importantly, combining MART-1 T cell receptor engineering with ARE-Del gene editing showed that this was also true for antigen-specific activation of T cells. MART-1-specific ARE-Del T cells showed higher percentages of IFN-γ producing T cells in response to MART-1 expressing tumor cells. Combined, our study reveals that ARE-mediated posttranscriptional regulation is conserved between murine and human T cells. Furthermore, generating antigen-specific ARE-Del T cells is feasible, a feature that could potentially be used for therapeutical purposes.

Toll-like receptor-2/7-mediated T cell activation: An innate potential to augment CD8+ T cell cytokine production.

CD8+ T cells are critical to combat pathogens and eradicate malignantly transformed cells. To exert their effector function and kill target cells, T cells produce copious amounts of effector molecules, including the pro-inflammatory cytokines interferon γ, tumour necrosis factor α and interleukin 2. TCR triggering alone is sufficient to induce cytokine secretion by effector and memory CD8+ T cells. However, T cells can also be directly activated by pathogen-derived molecules, such as through the triggering of Toll-like receptors (TLRs). TLR-mediated pathogen sensing by T cells results in the production of only interferon γ. However, in particular when the antigen load on target cells is low, or when TCR affinity to the antigen is limited, antigen-experienced T cells can benefit from costimulatory signals. TLR stimulation can also function in a costimulatory fashion to enhance TCR triggering. Combined TCR and TLR triggering enhances the proliferation, memory formation and effector function of T cells, resulting in enhanced production of interferon γ, tumour necrosis factor α and interleukin 2. Therefore, TLR ligands or the exploitation of TLR signalling could provide novel opportunities for immunotherapy approaches. In fact, CD19 CAR T cells bearing an intracellular TLR2 costimulatory domain were recently employed to treat cancer patients in a clinical trial. Here, the current knowledge regarding TLR2/7 stimulation-induced cytokine production by T cells is reviewed. Specifically, the transcriptional and post-transcriptional pathways engaged upon TLR2/7 sensing and TLR2/7 signalling are discussed. Finally, the potential uses of TLRs to enhance the anti-tumor effector function of T cells are explored.

Costimulation through TLR2 Drives Polyfunctional CD8+ T Cell Responses.

Optimal T cell activation requires Ag recognition through the TCR, engagement of costimulatory molecules, and cytokines. T cells can also directly recognize danger signals through the expression of TLRs. Whether TLR ligands have the capacity to provide costimulatory signals and enhance Ag-driven T cell activation is not well understood. In this study, we show that TLR2 and TLR7 ligands potently lower the Ag threshold for cytokine production in T cells. To investigate how TLR triggering supports cytokine production, we adapted the protocol for flow cytometry-based fluorescence in situ hybridization to mouse T cells. The simultaneous detection of cytokine mRNA and protein with single-cell resolution revealed that TLR triggering primarily drives de novo mRNA transcription. Ifng mRNA stabilization only occurs when the TCR is engaged. TLR2-, but not TLR7-mediated costimulation, can enhance mRNA stability at low Ag levels. Importantly, TLR2 costimulation increases the percentage of polyfunctional T cells, a hallmark of potent T cell responses. In conclusion, TLR-mediated costimulation effectively potentiates T cell effector function to suboptimal Ag levels.

Measuring T Cell Responses by Flow Cytometry-Based Fluorescence In Situ Hybridization.

T cells produce a wide variety of effector molecules in response to infections, such as cytokines, chemokines, granzymes, and perforins. Because different stimuli promote the production of specific effector molecules, T cell responses come in different flavors. In addition, single-cell analysis of protein production revealed that T cells respond heterogeneously to activation. To unravel the regulatory mechanisms that determine T cell effector function, novel methods were developed that simultaneously measure protein levels with the corresponding mRNA. These flow cytometry-based fluorescence in situ hybridization (Flow-FISH) technologies allow for multiparameter analysis with single-cell resolution of both nucleic acids and proteins. Here, we review the currently available methods of Flow-FISH and describe the possible applications thereof, with a specific focus on T cells.

Posttranscriptional Events Orchestrate Immune Homeostasis of CD8+ T Cells.

Maintaining immune homeostasis is instrumental for host health. Immune cells, such as T cells, are instrumental for the eradication of pathogenic bacteria, fungi and viruses. Furthermore, T cells also play a major role in the fight against cancer. Through the formation of immunological memory, a pool of antigen-experienced T cells remains in the body to rapidly protect the host upon reinfection or retransformation. In order to perform their protective function, T cells produce cytolytic molecules, such as granzymes and perforin, and cytokines such as interferon γ and tumor necrosis factor α. Recently, it has become evident that posttranscriptional regulatory events dictate the kinetics and magnitude of cytokine production by murine and human CD8+ T cells. Here, the recent literature regarding the role posttranscriptional regulation plays in maintaining immune homeostasis of antigen-experienced CD8+ T cells is reviewed.

Employing CRISPR-Cas9 to Enhance T Cell Effector Function.

As part of the adaptive immune system, T cells are critical to maintain immune homeostasis. T cells provide protective immunity by killing infected cells and combatting cancerous cells. To do so, T cells produce and secrete effector molecules, such as granzymes, perforin, and cytokines such as tumor necrosis factor α and interferon γ. However, in immune suppressive environments, such as tumors, T cells gradually lose the capacity to perform their effector function. One way T cell effector function can be enhanced is through genetic engineering with tools such as clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9). This protocol explains in a step-by-step fashion how to perform a controlled electroporation-based CRISPR experiment to enhance human T cell effector function. Of note, these steps are suitable for CRISPR-mediated genome editing in T cells in general and can thus also be used to study proteins of interest that do not influence T cell effector function.

Broad-spectrum defenders: γδ T cells take on a multitude of immune challenges.

A recent article in Proceedings of the National Academy of Sciences investigated γδ T cell antigen specificity in mice and humans, in which the authors show that γδ T cell antigen specificity is not constrained to one epitope. Rather, γδ T cells recognize a broad range of diverse antigens containing similar chemical structures or properties. In this News and Views, the importance of γδ T cell antigen polyspecificity during immune responses is highlighted.

Assessing Antigen-Specific T Cell Responses Through IFN-γ Enzyme-Linked Immune Absorbent Spot (ELISpot).

T cells are instrumental in protecting the host against invading pathogens and the development of cancer. To do so, they produce effector molecules such as granzymes, interleukins, interferons, and perforin. For the development and immunomonitoring of therapeutic applications such as cell-based therapies and vaccines, assessing T cell effector function is paramount. This can be achieved through various methods, such as 51Cr release assays, flow cytometry, and enzyme-linked immune absorbent spot (ELISpot) assays. For T cell ELISpots, plates are coated with antibodies directed against the effector molecule of interest (e.g., IFN-g). Subsequently, peripheral blood mononuclear cells (PBMCs) or isolated T cells are cultured on the plate together with stimuli of choice, and the production of effector molecules is visualized via labeled detection antibodies. For clinical studies, ELISpot is currently the gold standard to determine antigen-specific T cell frequencies. In contrast to 51Cr release assays, ELISpot allows for the exact enumeration of responding T cells, and compared to flow cytometry, ELISpot is more cost-effective and high throughput. Here, we optimize and describe, in a step-by-step fashion, how to perform a controlled IFN-γ ELISpot experiment to determine the frequency of responding or antigen-specific T cells in healthy human volunteers. Of note, this protocol can also be employed to assess the frequency of antigen-specific T cells induced in, e.g., vaccination studies or present in cellular products.

Gene network-based and ensemble modeling-based selection of tumor-associated antigens with a predicted low risk of tissue damage for targeted immunotherapy.

BACKGROUND: Tumor-associated antigens and their derived peptides constitute an opportunity to design off-the-shelf mainline or adjuvant anti-cancer immunotherapies for a broad array of patients. A performant and rational antigen selection pipeline would lay the foundation for immunotherapy trials with the potential to enhance treatment, tremendously benefiting patients suffering from rare, understudied cancers. METHODS: We present an experimentally validated, data-driven computational pipeline that selects and ranks antigens in a multipronged approach. In addition to minimizing the risk of immune-related adverse events by selecting antigens based on their expression profile in tumor biopsies and healthy tissues, we incorporated a network analysis-derived antigen indispensability index based on computational modeling results, and candidate immunogenicity predictions from a machine learning ensemble model relying on peptide physicochemical characteristics. RESULTS: In a model study of uveal melanoma, Human Leukocyte Antigen (HLA) docking simulations and experimental quantification of the peptide-major histocompatibility complex binding affinities confirmed that our approach discriminates between high-binding and low-binding affinity peptides with a performance similar to that of established methodologies. Blinded validation experiments with autologous T-cells yielded peptide stimulation-induced interferon-γ secretion and cytotoxic activity despite high interdonor variability. Dissecting the score contribution of the tested antigens revealed that peptides with the potential to induce cytotoxicity but unsuitable due to potential tissue damage or instability of expression were properly discarded by the computational pipeline. CONCLUSIONS: In this study, we demonstrate the feasibility of the de novo computational selection of antigens with the capacity to induce an anti-tumor immune response and a predicted low risk of tissue damage. On translation to the clinic, our pipeline supports fast turn-around validation, for example, for adoptive T-cell transfer preparations, in both generalized and personalized antigen-directed immunotherapy settings.

Time-dependent regulation of cytokine production by RNA binding proteins defines T cell effector function.

Potent T cell responses against infections and malignancies require a rapid yet tightly regulated production of toxic effector molecules. Their production level is defined by post-transcriptional events at 3' untranslated regions (3' UTRs). RNA binding proteins (RBPs) are key regulators in this process. With an RNA aptamer-based capture assay, we identify >130 RBPs interacting with IFNG, TNF, and IL2 3' UTRs in human T cells. RBP-RNA interactions show plasticity upon T cell activation. Furthermore, we uncover the intricate and time-dependent regulation of cytokine production by RBPs: whereas HuR supports early cytokine production, ZFP36L1, ATXN2L, and ZC3HAV1 dampen and shorten the production duration, each at different time points. Strikingly, even though ZFP36L1 deletion does not rescue the dysfunctional phenotype, tumor-infiltrating T cells produce more cytokines and cytotoxic molecules, resulting in superior anti-tumoral T cell responses. Our findings thus show that identifying RBP-RNA interactions reveals key modulators of T cell responses in health and disease.

Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV.

Human immunodeficiency virus (HIV) infection can be controlled by anti-retroviral therapy. Suppressing viral replication relies on life-long medication, but anti-retroviral therapy is not without risks to the patient. Therefore, it is important that permanent cures for HIV infection are developed. Three patients have been described to be completely cured from HIV infection in recent years. In all cases, patients received a hematopoietic stem cell (HSC) transplantation due to a hematological malignancy. The HSCs were sourced from autologous donors that expressed a homozygous mutation in the CCR5 gene. This mutation results in a non-functional receptor, and confers resistance to CCR5-tropic HIV strains that rely on CCR5 to enter host cells. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one of the methods of choice for gene editing, and the CRISPR/Cas system has been employed to target loci of interest in the context of HIV. Here, the current literature regarding CRISPR-mediated genome editing to render cells resistant to HIV (re)-infection by knocking out the co-receptors CCR5 and CXCR4 is summarized, and an outlook is provided regarding future (research) directions.

Flow-FISH as a Tool for Studying Bacteria, Fungi and Viruses.

Many techniques are currently in use to study microbes. These can be aimed at detecting, identifying, and characterizing bacterial, fungal, and viral species. One technique that is suitable for high-throughput analysis is flow cytometry-based fluorescence in situ hybridization, or Flow-FISH. This technique employs (fluorescently labeled) probes directed against DNA or (m)RNA, for instance targeting a gene or microorganism of interest and provides information on a single-cell level. Furthermore, by combining Flow-FISH with antibody-based protein detection, proteins of interest can be measured simultaneously with genetic material. Additionally, depending on the type of Flow-FISH assay, Flow-FISH can also be multiplexed, allowing for the simultaneous measurement of multiple gene targets and/or microorganisms. Together, this allows for, e.g., single-cell gene expression analysis or identification of (sub)strains in mixed cultures. Flow-FISH has been used in mammalian cells but has also been extensively employed to study diverse microbial species. Here, the use of Flow-FISH for studying microorganisms is reviewed. Specifically, the detection of (intracellular) pathogens, studying microorganism biology and disease pathogenesis, and identification of bacterial, fungal, and viral strains in mixed cultures is discussed, with a particular focus on the viruses EBV, HIV-1, and SARS-CoV-2.