Generation of Tumor-Reactive T Cells by Co-culture of Peripheral Blood Lymphocytes and Tumor Organoids.

Cancer immunotherapies have shown substantial clinical activity for a subset of patients with epithelial cancers. Still, technological platforms to study cancer T-cell interactions for individual patients and understand determinants of responsiveness are presently lacking. Here, we establish and validate a platform to induce and analyze tumor-specific T cell responses to epithelial cancers in a personalized manner. We demonstrate that co-cultures of autologous tumor organoids and peripheral blood lymphocytes can be used to enrich tumor-reactive T cells from peripheral blood of patients with mismatch repair-deficient colorectal cancer and non-small-cell lung cancer. Furthermore, we demonstrate that these T cells can be used to assess the efficiency of killing of matched tumor organoids. This platform provides an unbiased strategy for the isolation of tumor-reactive T cells and provides a means by which to assess the sensitivity of tumor cells to T cell-mediated attack at the level of the individual patient.

Translational repression of pre-formed cytokine-encoding mRNA prevents chronic activation of memory T cells.

Memory T cells are critical for the immune response to recurring infections. Their instantaneous reactivity to pathogens is empowered by the persistent expression of cytokine-encoding mRNAs. How the translation of proteins from pre-formed cytokine-encoding mRNAs is prevented in the absence of infection has remained unclear. Here we found that protein production in memory T cells was blocked via a 3' untranslated region (3' UTR)-mediated process. Germline deletion of AU-rich elements (AREs) in the Ifng-3' UTR led to chronic cytokine production in memory T cells. This aberrant protein production did not result from increased expression and/or half-life of the mRNA. Instead, AREs blocked the recruitment of cytokine-encoding mRNA to ribosomes; this block depended on the ARE-binding protein ZFP36L2. Thus, AREs mediate repression of translation in mouse and human memory T cells by preventing undesirable protein production from pre-formed cytokine-encoding mRNAs in the absence of infection.

IL-27 maintains cytotoxic Ly6C+ γδ T cells that arise from immature precursors.

In mice, γδ-T lymphocytes that express the co-stimulatory molecule, CD27, are committed to the IFNγ-producing lineage during thymic development. In the periphery, these cells play a critical role in host defense and anti-tumor immunity. Unlike αß-T cells that rely on MHC-presented peptides to drive their terminal differentiation, it is unclear whether MHC-unrestricted γδ-T cells undergo further functional maturation after exiting the thymus. Here, we provide evidence of phenotypic and functional diversity within peripheral IFNγ-producing γδ T cells. We found that CD27+ Ly6C- cells convert into CD27+Ly6C+ cells, and these CD27+Ly6C+ cells control cancer progression in mice, while the CD27+Ly6C- cells cannot. The gene signatures of these two subsets were highly analogous to human immature and mature γδ-T cells, indicative of conservation across species. We show that IL-27 supports the cytotoxic phenotype and function of mouse CD27+Ly6C+ cells and human Vδ2+ cells, while IL-27 is dispensable for mouse CD27+Ly6C- cell and human Vδ1+ cell functions. These data reveal increased complexity within IFNγ-producing γδ-T cells, comprising immature and terminally differentiated subsets, that offer new insights into unconventional T-cell biology.

Regulation of IFN-γ production by ZFP36L2 in T cells is time-dependent.

CD8+ T cells kill target cells by releasing cytotoxic molecules and proinflammatory cytokines, such as TNF and IFN-γ. The magnitude and duration of cytokine production are defined by posttranscriptional regulation, and critical regulator herein are RNA-binding proteins (RBPs). Although the functional importance of RBPs in regulating cytokine production is established, the kinetics and mode of action through which RBPs control cytokine production are not well understood. Previously, we showed that the RBP ZFP36L2 blocks the translation of preformed cytokine encoding mRNA in quiescent memory T cells. Here, we uncover that ZFP36L2 regulates cytokine production in a time-dependent manner. T cell-specific deletion of ZFP36L2 (CD4-cre) had no effect on T-cell development or cytokine production during early time points (2-6 h) of T-cell activation. In contrast, ZFP36L2 specifically dampened the production of IFN-γ during prolonged T-cell activation (20-48 h). ZFP36L2 deficiency also resulted in increased production of IFN-γ production in tumor-infiltrating T cells that are chronically exposed to antigens. Mechanistically, ZFP36L2 regulates IFN-γ production at late time points of activation by destabilizing Ifng mRNA in an AU-rich element-dependent manner. Together, our results reveal that ZFP36L2 employs different regulatory nodules in effector and memory T cells to regulate cytokine production.

Sequence determinants as key regulators in gene expression of T cells.

T cell homeostasis, T cell differentiation, and T cell effector function rely on the constant fine-tuning of gene expression. To alter the T cell state, substantial remodeling of the proteome is required. This remodeling depends on the intricate interplay of regulatory mechanisms, including post-transcriptional gene regulation. In this review, we discuss how the sequence of a transcript influences these post-transcriptional events. In particular, we review how sequence determinants such as sequence conservation, GC content, and chemical modifications define the levels of the mRNA and the protein in a T cell. We describe the effect of different forms of alternative splicing on mRNA expression and protein production, and their effect on subcellular localization. In addition, we discuss the role of sequences and structures as binding hubs for miRNAs and RNA-binding proteins in T cells. The review thus highlights how the intimate interplay of post-transcriptional mechanisms dictate cellular fate decisions in T cells.

Circular RNAs exhibit limited evidence for translation, or translation regulation of the mRNA counterpart in terminal hematopoiesis.

Each day, about 1012 erythrocytes and platelets are released into the bloodstream. This substantial output from hematopoietic stem cells is tightly regulated by transcriptional and epigenetic factors. Whether and how circular RNAs (circRNAs) contribute to the differentiation and/or identity of hematopoietic cells is to date not known. We recently reported that erythrocytes and platelets contain the highest levels and numbers of circRNAs among hematopoietic cells. Here, we provide the first detailed analysis of circRNA expression during erythroid and megakaryoid differentiation. CircRNA expression not only significantly increased upon enucleation, but also had limited overlap between progenitor cells and mature cells, suggesting that circRNA expression stems from regulated processes rather than resulting from mere accumulation. To study circRNA function in hematopoiesis, we first compared the expression levels of circRNAs with the translation efficiency of their mRNA counterpart. We found that only one out of 2531 (0.04%) circRNAs associated with mRNA-translation regulation. Furthermore, irrespective of thousands of identified putative open reading frames, deep ribosome-footprinting sequencing, and mass spectrometry analysis provided little evidence for translation of endogenously expressed circRNAs. In conclusion, circRNAs alter their expression profile during terminal hematopoietic differentiation, yet their contribution to regulate cellular processes remains enigmatic.

Dynamic Post-Transcriptional Events Governing CD8+ T Cell Homeostasis and Effector Function.

Effective T cell responses against infections and tumors require a swift and ample production of cytokines, chemokines, and cytotoxic molecules. The production of these effector molecules relies on rapid changes of gene expression, determined by cell-intrinsic signals and environmental cues. Here, we review our current understanding of gene-specific regulatory networks that define the magnitude and timing of cytokine production in CD8+ T cells. We discuss the dynamic features of post-transcriptional control during CD8+ T cell homeostasis and activation, and focus on the crosstalk between cell signaling and RNA-binding proteins. Elucidating gene-specific regulatory circuits may help in the future to rectify dysfunctional T cell responses.

CD29 Enriches for Cytotoxic Human CD4+ T Cells.

CD4+ T cells are key contributors in the induction of adaptive immune responses against pathogens. Even though CD4+ T cells are primarily classified as noncytotoxic helper T cells, it has become appreciated that a subset of CD4+ T cells is cytotoxic. However, tools to identify these cytotoxic CD4+ T cells are lacking. We recently showed that CD29 (integrin ß1, ITGB1) expression on human CD8+ T cells enriches for the most potent cytotoxic T cells. In this study, we questioned whether CD29 expression also associates with cytotoxic CD4+ T cells. We show that human peripheral blood-derived CD29hiCD4+ T cells display a cytotoxic gene expression profile, which closely resembles that of CD29hi cytotoxic CD8+ T cells. This CD29hi cytotoxic phenotype was observed ex vivo and was maintained in in vitro cultures. CD29 expression enriched for CD4+ T cells, which effectively produced the proinflammatory cytokines IFN-γ, IL-2, and TNF-α, and cytotoxic molecules. Lastly, CD29-expressing CD4+ T cells transduced with a MART1-specific TCR showed target cell killing in vitro. In conclusion, we demonstrate in this study that CD29 can be employed to enrich for cytotoxic human CD4+ T cells.

CD29 identifies IFN-γ-producing human CD8+ T cells with an increased cytotoxic potential.

Cytotoxic CD8+ T cells can effectively kill target cells by producing cytokines, chemokines, and granzymes. Expression of these effector molecules is however highly divergent, and tools that identify and preselect CD8+ T cells with a cytotoxic expression profile are lacking. Human CD8+ T cells can be divided into IFN-γ- and IL-2-producing cells. Unbiased transcriptomics and proteomics analysis on cytokine-producing fixed CD8+ T cells revealed that IL-2+ cells produce helper cytokines, and that IFN-γ+ cells produce cytotoxic molecules. IFN-γ+ T cells expressed the surface marker CD29 already prior to stimulation. CD29 also marked T cells with cytotoxic gene expression from different tissues in single-cell RNA-sequencing data. Notably, CD29+ T cells maintained the cytotoxic phenotype during cell culture, suggesting a stable phenotype. Preselecting CD29-expressing MART1 TCR-engineered T cells potentiated the killing of target cells. We therefore propose that CD29 expression can help evaluate and select for potent therapeutic T cell products.

T cells at work: How post-transcriptional mechanisms control T cell homeostasis and activation.

T cells are central players of the adaptive immune system by protecting us from recurring infections and by killing malignant cells. Protective T cell responses rely on the concerted production of effector molecules such as cytolytic mediators, granzymes, and perforins, as well as pro-inflammatory cytokines and chemokines. Once activated, T cells drastically change their gene expression and rapidly respond to insults by producing ample amounts of effector molecules. In the absence of antigen, T cells remain in a quiescent state and survey our body for possible pathogenic insults. Resting T cells are, however, not inert, but continuously regulate their protein production to survive and to be prepared for possible re-infections. Here, we review our current knowledge on the regulation of gene expression in activated and quiescent T cells. We specifically focus on post-transcriptional mechanisms that define the protein output and that allow dormant cells to undergo active signaling and selective translation, keeping them poised for activation. Finally, we discuss which signals drive T cell survival and their preparedness to respond to insults and which mechanisms are involved in these processes.

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.

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.

Peripheral and systemic antigens elicit an expandable pool of resident memory CD8+ T cells in the bone marrow.

BM has been put forward as a major reservoir for memory CD8+  T cells. In order to fulfill that function, BM should "store" memory CD8+ T cells, which in biological terms would require these "stored" memory cells to be in disequilibrium with the circulatory pool. This issue is a matter of ongoing debate. Here, we unequivocally demonstrate that murine and human BM harbors a population of tissue-resident memory CD8+ T (TRM ) cells. These cells develop against various pathogens, independently of BM infection or local antigen recognition. BM CD8+ TRM cells share a transcriptional program with resident lymphoid cells in other tissues; they are polyfunctional cytokine producers and dependent on IL-15, Blimp-1, and Hobit. CD8+ TRM cells reside in the BM parenchyma, but are in close contact with the circulation. Moreover, this pool of resident T cells is not size-restricted and expands upon peripheral antigenic re-challenge. This works extends the role of the BM in the maintenance of CD8+ T cell memory to include the preservation of an expandable reservoir of functional, non-recirculating memory CD8+ T cells, which develop in response to a large variety of peripheral antigens.

Circular RNA expression in human hematopoietic cells is widespread and cell-type specific.

Hematopoietic stem cells differentiate into a broad range of specialized blood cells. This process is tightly regulated and depends on transcription factors, micro-RNAs, and long non-coding RNAs. Recently, also circular RNA (circRNA) were found to regulate cellular processes. Their expression pattern and their identity is however less well defined. Here, we provide the first comprehensive analysis of circRNA expression in human hematopoietic progenitors, and in differentiated lymphoid and myeloid cells. We here show that the expression of circRNA is cell-type specific, and increases upon maturation. CircRNA splicing variants can also be cell-type specific. Furthermore, nucleated hematopoietic cells contain circRNA that have higher expression levels than the corresponding linear RNA. Enucleated blood cells, i.e. platelets and erythrocytes, were suggested to use RNA to maintain their function, respond to environmental factors or to transmit signals to other cells via microvesicles. Here we show that platelets and erythrocytes contain the highest number of circRNA of all hematopoietic cells, and that the type and numbers of circRNA changes during maturation. This cell-type specific expression pattern of circRNA in hematopoietic cells suggests a hithero unappreciated role in differentiation and cellular function.

Distinct PKC-mediated posttranscriptional events set cytokine production kinetics in CD8+ T cells.

Effective T cell responses against invading pathogens require the concerted production of three key cytokines: TNF-α, IFN-γ, and IL-2. The cytokines functionally synergize, but their production kinetics widely differ. How the differential timing of expression is regulated remains, however, poorly understood. We compared the relative contribution of transcription, mRNA stability, and translation efficiency on cytokine production in murine effector and memory CD8+ T cells. We show that the immediate and ample production of TNF-α is primarily mediated by translation of preformed mRNA through protein kinase C (PKC)-induced recruitment of mRNA to polyribosomes. Also, the initial production of IFN-γ uses translation of preformed mRNA. However, the magnitude and subsequent expression of IFN-γ, and of IL-2, depends on calcium-induced de novo transcription and PKC-dependent mRNA stabilization. In conclusion, PKC signaling modulates translation efficiency and mRNA stability in a transcript-specific manner. These cytokine-specific regulatory mechanisms guarantee that T cells produce ample amounts of cytokines shortly upon activation and for a limited time.

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.

Combined Single-Cell Measurement of Cytokine mRNA and Protein Identifies T Cells with Persistent Effector Function.

Effective T cell responses entail the coproduction of IFN-γ, TNF-α, and IL-2. Cytokine production is determined by transcriptional and posttranscriptional events. However, increased transcript levels do not always translate into protein production, and therefore simultaneous transcripts and protein measurement are essential for the appropriate analysis of T cell responses. In this study, we optimized flow cytometry-based fluorescence in situ hybridization (Flow-FISH) for IFN-γ to multicolor flow cytometry that allows for single-cell measurement of mRNA and protein levels. This high-throughput analysis detected Ag-specific human T cells of low frequency. We also employed Flow-FISH for single-tube analysis of IFN-γ transcript and protein profile to simultaneously study the responsiveness of different T cell subsets, that is, naive, effector, and memory T cells. Importantly, the simultaneous transcript and protein analysis of IFN-γ and of TNF-α and IL-2 revealed that T cell responses consist of two types: one subtype loses mRNA expression during activation, whereas the other maintains high transcript levels throughout stimulation. High cytokine transcript levels correlated with increased protein production. Intriguingly, this mRNAhi-expressing T cell population also produced higher levels of other cytokines, indicating that Flow-FISH helps identify the best cytokine producers during T cell activation. We conclude that Flow-FISH is a rapid, sensitive, and cost-effective method to determine the quality of T cell responses induced by, for instance, T cell vaccines.

TLR-Mediated Innate Production of IFN-γ by CD8+ T Cells Is Independent of Glycolysis.

CD8(+) T cells can respond to unrelated infections in an Ag-independent manner. This rapid innate-like immune response allows Ag-experienced T cells to alert other immune cell types to pathogenic intruders. In this study, we show that murine CD8(+) T cells can sense TLR2 and TLR7 ligands, resulting in rapid production of IFN-γ but not of TNF-α and IL-2. Importantly, Ag-experienced T cells activated by TLR ligands produce sufficient IFN-γ to augment the activation of macrophages. In contrast to Ag-specific reactivation, TLR-dependent production of IFN-γ by CD8(+) T cells relies exclusively on newly synthesized transcripts without inducing mRNA stability. Furthermore, transcription of IFN-γ upon TLR triggering depends on the activation of PI3K and serine-threonine kinase Akt, and protein synthesis relies on the activation of the mechanistic target of rapamycin. We next investigated which energy source drives the TLR-induced production of IFN-γ. Although Ag-specific cytokine production requires a glycolytic switch for optimal cytokine release, glucose availability does not alter the rate of IFN-γ production upon TLR-mediated activation. Rather, mitochondrial respiration provides sufficient energy for TLR-induced IFN-γ production. To our knowledge, this is the first report describing that TLR-mediated bystander activation elicits a helper phenotype of CD8(+) T cells. It induces a short boost of IFN-γ production that leads to a significant but limited activation of Ag-experienced CD8(+) T cells. This activation suffices to prime macrophages but keeps T cell responses limited to unrelated infections.

GATA1-Deficient Dendritic Cells Display Impaired CCL21-Dependent Migration toward Lymph Nodes Due to Reduced Levels of Polysialic Acid.

Dendritic cells (DCs) play a pivotal role in the regulation of the immune response. DC development and activation is finely orchestrated through transcriptional programs. GATA1 transcription factor is required for murine DC development, and data suggest that it might be involved in the fine-tuning of the life span and function of activated DCs. We generated DC-specific Gata1 knockout mice (Gata1-KODC), which presented a 20% reduction of splenic DCs, partially explained by enhanced apoptosis. RNA sequencing analysis revealed a number of deregulated genes involved in cell survival, migration, and function. DC migration toward peripheral lymph nodes was impaired in Gata1-KODC mice. Migration assays performed in vitro showed that this defect was selective for CCL21, but not CCL19. Interestingly, we show that Gata1-KODC DCs have reduced polysialic acid levels on their surface, which is a known determinant for the proper migration of DCs toward CCL21.

Enhanced CD8 T cell responses through GITR-mediated costimulation resolve chronic viral infection.

Chronic infections are characterized by the inability to eliminate the persisting pathogen and often associated with functional impairment of virus-specific T-cell responses. Costimulation through Glucocorticoid-induced TNFR-related protein (GITR) can increase survival and function of effector T cells. Here, we report that constitutive expression of GITR-ligand (GITRL) confers protection against chronic lymphocytic choriomeningitis virus (LCMV) infection, accelerating recovery without increasing pathology. Rapid viral clearance in GITRL transgenic mice coincided with increased numbers of poly-functional, virus-specific effector CD8+ T cells that expressed more T-bet and reduced levels of the rheostat marker PD-1. GITR triggering also boosted the helper function of virus-specific CD4 T cells already early in the infection, as was evidenced by increased IL-2 and IFNγ production, and more expression of CD40L and T-bet. Importantly, CD4-depletion experiments revealed that the expanded pool of virus-specific effector CD8 T cells and the ensuing viral clearance in LCMV-infected GITRL tg mice was entirely dependent on CD4 T cells. We found no major differences for NK cell and regulatory T cell responses, whereas the humoral response to the virus was increased in GITRL tg mice, but only in the late phase of the infection when the virus was almost eradicated. Based on these findings, we conclude that enhanced GITR-triggering mediates its protective, anti-viral effect on the CD8 T cell compartment by boosting CD4 T cell help. As such, increasing costimulation through GITR may be an attractive strategy to increase anti-viral CTL responses without exacerbating pathology, in particular to persistent viruses such as HIV and HCV.