Antigen and checkpoint receptor engagement recalibrates T cell receptor signal strength.

How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3-Tocky mice, we characterized early quantitative and qualitative changes that occur in CD4+ T cells in relation to TCR signaling strength. We captured how dose- and time-dependent programming of distinct co-inhibitory receptors rapidly recalibrates T cell activation thresholds and visualized the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 immunotherapy leads to an increased TCR signal strength. We defined a strong TCR signal metric of five genes upregulated by anti-PD1 in T cells (TCR.strong), which was superior to a canonical T cell activation gene signature in stratifying melanoma patient outcomes to anti-PD1 therapy. Our study therefore reveals how analysis of TCR signal strength-and its manipulation-can provide powerful metrics for monitoring outcomes to immunotherapy.

The murine meninges acquire lymphoid tissue properties and harbour autoreactive B cells during chronic Trypanosoma brucei infection.

The meningeal space is a critical brain structure providing immunosurveillance for the central nervous system (CNS), but the impact of infections on the meningeal immune landscape is far from being fully understood. The extracellular protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis (HAT) or sleeping sickness, accumulates in the meningeal spaces, ultimately inducing severe meningitis and resulting in death if left untreated. Thus, sleeping sickness represents an attractive model to study immunological dynamics in the meninges during infection. Here, by combining single-cell transcriptomics and mass cytometry by time-of-flight (CyTOF) with in vivo interventions, we found that chronic T. brucei infection triggers the development of ectopic lymphoid aggregates (ELAs) in the murine meninges. These infection-induced ELAs were defined by the presence of ER-TR7+ fibroblastic reticular cells, CD21/35+ follicular dendritic cells (FDCs), CXCR5+ PD1+ T follicular helper-like phenotype, GL7+ CD95+ GC-like B cells, and plasmablasts/plasma cells. Furthermore, the B cells found in the infected meninges produced high-affinity autoantibodies able to recognise mouse brain antigens, in a process dependent on LTß signalling. A mid-throughput screening identified several host factors recognised by these autoantibodies, including myelin basic protein (MBP), coinciding with cortical demyelination and brain pathology. In humans, we identified the presence of autoreactive IgG antibodies in the cerebrospinal fluid (CSF) of second stage HAT patients that recognised human brain lysates and MBP, consistent with our findings in experimental infections. Lastly, we found that the pathological B cell responses we observed in the meninges required the presence of T. brucei in the CNS, as suramin treatment before the onset of the CNS stage prevented the accumulation of GL7+ CD95+ GC-like B cells and brain-specific autoantibody deposition. Taken together, our data provide evidence that the meningeal immune response during chronic T. brucei infection results in the acquisition of lymphoid tissue-like properties, broadening our understanding of meningeal immunity in the context of chronic infections. These findings have wider implications for understanding the mechanisms underlying the formation ELAs during chronic inflammation resulting in autoimmunity in mice and humans, as observed in other autoimmune neurodegenerative disorders, including neuropsychiatric lupus and multiple sclerosis.

A temporally dynamic Foxp3 autoregulatory transcriptional circuit controls the effector Treg programme.

Regulatory T cells (Treg) are negative regulators of the immune response; however, it is poorly understood whether and how Foxp3 transcription is induced and regulated in the periphery during T-cell responses. Using Foxp3-Timer of cell kinetics and activity (Tocky) mice, which report real-time Foxp3 expression, we show that the flux of new Foxp3 expressors and the rate of Foxp3 transcription are increased during inflammation. These persistent dynamics of Foxp3 transcription determine the effector Treg programme and are dependent on a Foxp3 autoregulatory transcriptional circuit. Persistent Foxp3 transcriptional activity controls the expression of coinhibitory molecules, including CTLA-4 and effector Treg signature genes. Using RNA-seq, we identify two groups of surface proteins based on their relationship to the temporal dynamics of Foxp3 transcription, and we show proof of principle for the manipulation of Foxp3 dynamics by immunotherapy: new Foxp3 flux is promoted by anti-TNFRII antibody, and high-frequency Foxp3 expressors are targeted by anti-OX40 antibody. Collectively, our study dissects time-dependent mechanisms behind Foxp3-driven T-cell regulation and establishes the Foxp3-Tocky system as a tool to investigate the mechanisms behind T-cell immunotherapies.

Synovial Regulatory T Cells Occupy a Discrete TCR Niche in Human Arthritis and Require Local Signals To Stabilize FOXP3 Protein Expression.

Although there is great interest in harnessing the immunosuppressive potential of FOXP3(+) regulatory T cells (Tregs) for treating autoimmunity, a sizeable knowledge gap exists regarding Treg fate in human disease. In juvenile idiopathic arthritis (JIA) patients, we have previously reported that atypical CD25(+)FOXP3(-) Treg-like cells uniquely populate the inflamed site. Intriguingly, their proportions relative to CD25(+)FOXP3(+) Tregs associate with arthritis course, suggesting a role in disease. The ontogeny of these FOXP3(-) Treg-like cells is, however, unknown. In this study, we interrogated clonal relationships between CD4(+) T cell subsets in JIA, using high-throughput TCR repertoire analysis. We reveal that FOXP3(+) Tregs possess highly exclusive TCRß usage from conventional T cells, in blood, and also at the inflamed site, where they are clonally expanded. Intriguingly, the repertoires of FOXP3(+) Tregs in synovial fluid are highly overlapping with CD25(+)FOXP3(-) Treg-like cells, indicating fluctuations in FOXP3 expression in the inflamed joint. Furthermore, cultured synovial Tregs rapidly downregulated FOXP3 protein (but not mRNA), and this process was prevented by addition of synovial fluid from JIA patients, through an IL-6-independent mechanism. Our findings suggest that most Tregs arise from a separate lineage from conventional T cells, and that this repertoire divergence is largely maintained under chronic inflammatory conditions. We propose that subsequent Treg expansions at the inflamed site creates an environment that leads to competition for limited resources within the synovium, resulting in the destabilization of FOXP3 expression in some Tregs.

Hypomethylation at the regulatory T cell-specific demethylated region in CD25hi T cells is decoupled from FOXP3 expression at the inflamed site in childhood arthritis.

The maintenance of FOXP3 expression in CD25(hi) regulatory T cells (Tregs) is crucial to the control of inflammation and essential for successful Treg transfer therapies. Coexpression of CD25 and FOXP3 in combination with a hypomethylated region within the FOXP3 gene, called the Treg-specific demethylated region (TSDR), is considered the hallmark of stable Tregs. The TSDR is an epigenetic motif that is important for stable FOXP3 expression and is used as a biomarker to measure Treg lineage commitment. In this study, we report that, unlike in peripheral blood, CD4(+) T cell expression of CD25 and FOXP3 is frequently dissociated at the inflamed site in patients with juvenile idiopathic arthritis, which led us to question the stability of human Tregs in chronic inflammatory environments. We describe a novel CD4(+)CD127(lo)CD25(hi) human T cell population that exhibits extensive TSDR and promoter demethylation in the absence of stable FOXP3 expression. This population expresses high levels of CTLA-4 and can suppress T conventional cell proliferation in vitro. These data collectively suggest that this population may represent a chronically activated FOXP3(lo) Treg population. We show that these cells have defects in IL-2 signaling and reduced expression of a deubiquitinase important for FOXP3 stability. Clinically, the proportions of these cells within the CD25(hi) T cell subset are increased in patients with the more severe courses of disease. Our study demonstrates, therefore, that hypomethylation at the TSDR can be decoupled from FOXP3 expression in human T cells and that environment-specific breakdown in FOXP3 stability may compromise the resolution of inflammation in juvenile idiopathic arthritis.

CD161 defines the subset of FoxP3+ T cells capable of producing proinflammatory cytokines.

Regulatory FoxP3+CD4+ T cells (Treg) are vital for maintaining the balance between tolerance, adequate immune response, and autoimmunity. Despite this immunoregulatory role, it has been shown that Treg may also produce proinflammatory cytokines. Here we present a distinct population of Treg, defined by CD161 expression, as the major source of FoxP3+ Treg-derived proinflammatory cytokines. CD161+ Treg can be followed throughout development, from thymus and cord blood to healthy child and adult samples. CD161+ Treg display anergy, are suppressive in cocultures with conventional T cells (Tconv), and possess a predominantly demethylated Treg-specific demethylated region of the FOXP3 locus. In addition to the production of interleukin (IL) 17A, interferon γ, and IL-2, CD161+FoxP3+ cells share markers with Tconv, including expression of the transcription factors retinoic acid-related orphan receptor Cv2 (RORCv2) and T-cell-specific T-box transcription factor (Tbet). Expression of CD161 and enrichment for cytokine production are stable characteristics of CD161+ Treg upon both short- and longer-term culture in vitro. Additionally, CD161+ Treg are highly enriched within the inflammatory environment of childhood arthritis, suggesting a role in disease. Our data therefore demonstrate that CD161+FoxP3+ T cells are a novel Treg subset, found in health and disease, which display high proinflammatory potential but also exhibit hallmark Treg characteristics.

Inflammation and type one diabetes.

Type one diabetes (T1D) is a complex T cell-mediated autoimmune disease, the defining feature of which is the destruction of the insulin-secreting beta- (ß)- cell. Both genetic and environmental factors combine to precipitate disease, and the outcome of the pathological process is dependent on multiple inter-related factors. In this review, the mechanisms behind the initiation and propagation of the autoimmune response are analysed, and the contribution of differing T-helper (T(h)) subsets--in particular T(h)1- and T(h)17-related cytokines--to the disease process are discussed. An argument is then synthesized that proposes that the ß-cell's response to stress and inflammation is the critical determinant in predicting disease outcome and that, immunologically, a delicate balance exists between regulation and inflammation at the site of islet infiltration. Strategies for disease intervention, therefore, will not only require the induction of T-cell tolerance by tipping the balance towards regulation but will also need to contain approaches that result in the scavenging of inflammatory mediators, in order to facilitate repair. Ultimately, given that clinical diabetes presents late in the autoimmune process, strategies for ß-cell regeneration must now be addressed. There is thus a requirement for an increased, collaborative effort between stem cell biologists and immunologists in order to tailor an optimal therapeutic strategy for the treatment of this debilitating disease.

Epigenetic changes at Il12rb2 and Tbx21 in relation to plasticity behavior of Th17 cells.

Plasticity within Th cell populations may play a role in enabling site-specific immune responses to infections while limiting tissue destruction. Epigenetic processes are fundamental to such plasticity; however, to date, most investigations have focused on in vitro-generated T cells. In this study, we have examined the molecular mechanisms underpinning murine Th17 plasticity in vivo by assessing H3K4 and H3K27 trimethylation marks at Tbx21, Rorc, Il17a, Ifng, and Il12rb2 loci in purified ex vivo-isolated and in vitro-generated Th17 cells. Although both populations had largely comparable epigenetic signatures, including bivalent marks at Tbx21, freshly isolated ex vivo Th17 cells displayed restricted expression from Il12rb2 due to the presence of repressive chromatin modifications. This receptor, however, could be upregulated on isolated ex vivo Th17 cells after in vitro activation or by in vivo immunization and was augmented by the presence of IFN-γ. Such activated cells could then be deviated toward a Th1-like profile. We show that IL-12 stimulation removes H3K27 trimethylation modifications at Tbx21/T-bet leading to enhanced T-bet expression with in vitro Th17 cells. Our study reveals important potential phenotypic differences between ex vivo- and in vitro-generated Th17 cells and provides mechanistic insight into Th17 cell plasticity.

Nr4a nuclear receptors: markers and modulators of antigen receptor signaling.

Nr4a1-3 encode a small family of orphan nuclear hormone receptors with transcriptional activity. Their expression reflects both acute and chronic antigen-receptor signaling in T and B-cells, and they have been implicated in critical aspects of lymphocyte development, tolerance, and function. These include roles in regulatory T-cell (Treg), thymic-negative selection, humoral responses, anergy, and exhaustion. Here, we review recent advances in this field such as functional roles in B-cells, transcriptional targets, and mechanism of action. We highlight recurrent themes, including integration of antigen-receptor signaling with costimulatory input, as well as unanswered questions and translational applications of this work.

T cell biomarkers come to the fore in cancer immunotherapy.

A comprehensive study by van der Sluis et al.1 demonstrates immunotherapeutic targeting of OX40 and PD-L1 results in enhanced tumor clearance, which is linked to the dynamic emergence of distinct subsets of CD8+ T cells.

T-cell response to checkpoint blockade immunotherapies: from fundamental mechanisms to treatment signatures.

Immune checkpoint immunotherapies act to block inhibitory receptors on the surface of T cells and other cells of the immune system. This can increase activation of immune cells and promote tumour clearance. Whilst this is very effective in some types of cancer, significant proportions of patients do not respond to single-agent immunotherapy. To improve patient outcomes, we must first mechanistically understand what drives therapy resistance. Many studies have utilised genetic, transcriptional, and histological signatures to find correlates of effective responses to treatment. It is key that we understand pretreatment predictors of response, but also to understand how the immune system becomes treatment resistant during therapy. Here, we review our understanding of the T-cell signatures that are critical for response, how these immune signatures change during treatment, and how this information can be used to rationally design therapeutic strategies. We highlight how chronic antigen recognition drives heterogeneous T-cell exhaustion and the role of T-cell receptor (TCR) signal strength in exhausted T-cell differentiation and molecular response to therapy. We explore how dynamic changes in negative feedback pathways can promote resistance to single-agent therapy. We speculate that this resistance may be circumvented in the future through identifying the most effective combinations of immunotherapies to promote sustained and durable antitumour responses.

Flow cytometric analysis of CD4+ T cell reactivation following anti-PD1 immunotherapy in a transgenic mouse model.

This protocol uses the Tg4 Nr4a3-Tocky mouse model to recalibrate T cell activation thresholds and reveals the role that immune checkpoints play in controlling T cell activation. The example approach here uses flow cytometry to characterize quantitative and qualitative changes in splenic CD4+ T cells reactivated in the presence of anti-PD1 immunotherapy. The protocol is optimized for studying anti-PD1 pathway blockade only. The protocol is not compatible with cellular fixation, and T cells should be analyzed immediately after staining. For complete details on the use and execution of this protocol, please refer to Elliot et al. (2021).

Succinate uptake by T cells suppresses their effector function via inhibition of mitochondrial glucose oxidation.

Succinate dehydrogenase (SDH) loss-of-function mutations drive succinate accumulation in tumor microenvironments, for example in the neuroendocrine tumors pheochromocytoma (PC) and paraganglioma (PG). Control of innate immune cell activity by succinate is described, but effects on T cells have not been interrogated. Here we report that exposure of human CD4+ and CD8+ T cells to tumor-associated succinate concentrations suppresses degranulation and cytokine secretion, including of the key anti-tumor cytokine interferon-γ (IFN-γ). Mechanistically, this is associated with succinate uptake-partly via the monocarboxylate transporter 1 (MCT1)-inhibition of succinyl coenzyme A synthetase activity and impaired glucose flux through the tricarboxylic acid cycle. Consistently, pharmacological and genetic interventions restoring glucose oxidation rescue T cell function. Tumor RNA-sequencing data from patients with PC and PG reveal profound suppression of IFN-γ-induced genes in SDH-deficient tumors compared with those with other mutations, supporting a role for succinate in modulating the anti-tumor immune response in vivo.

Nur77-Tempo mice reveal T cell steady state antigen recognition.

In lymphocytes, Nr4a gene expression is specifically regulated by antigen receptor signalling, making them ideal targets for use as distal T cell receptor (TCR) reporters. Nr4a3-Timer of cell kinetics and activity (Tocky) mice are a ground-breaking tool to report TCR-driven Nr4a3 expression using Fluorescent Timer protein (FT). FT undergoes a time-dependent shift in its emission spectrum following translation, allowing for the temporal reporting of transcriptional events. Our recent work suggested that Nr4a1/Nur77 may be a more sensitive gene to distal TCR signals compared to Nr4a3, so we, therefore, generated Nur77-Timer-rapidly-expressed-in-lymphocytes (Tempo) mice that express FT under the regulation of Nur77. We validated the ability of Nur77-Tempo mice to report TCR and B cell receptor signals and investigated the signals regulating Nur77-FT expression. We found that Nur77-FT was sensitive to low-strength TCR signals, and its brightness was graded in response to TCR signal strength. Nur77-FT detected positive selection signals in the thymus, and analysis of FT expression revealed that positive selection signals are often persistent in nature, with most thymic Treg expressing FT Blue. We found that active TCR signals in the spleen are low frequency, but CD69+ lymphoid T cells are enriched for FT Blue+ Red+ T cells, suggesting frequent TCR signalling. In non-lymphoid tissue, we saw a dissociation of FT protein from CD69 expression, indicating that tissue residency is not associated with tonic TCR signals. Nur77-Tempo mice, therefore, combine the temporal dynamics from the Tocky innovation with increased sensitivity of Nr4a1 to lower TCR signal strengths.

Context-dependent effects of IL-2 rewire immunity into distinct cellular circuits.

Interleukin 2 (IL-2) is a key homeostatic cytokine, with therapeutic applications in both immunogenic and tolerogenic immune modulation. Clinical use has been hampered by pleiotropic functionality and widespread receptor expression, with unexpected adverse events. Here, we developed a novel mouse strain to divert IL-2 production, allowing identification of contextual outcomes. Network analysis identified priority access for Tregs and a competitive fitness cost of IL-2 production among both Tregs and conventional CD4 T cells. CD8 T and NK cells, by contrast, exhibited a preference for autocrine IL-2 production. IL-2 sourced from dendritic cells amplified Tregs, whereas IL-2 produced by B cells induced two context-dependent circuits: dramatic expansion of CD8+ Tregs and ILC2 cells, the latter driving a downstream, IL-5-mediated, eosinophilic circuit. The source-specific effects demonstrate the contextual influence of IL-2 function and potentially explain adverse effects observed during clinical trials. Targeted IL-2 production therefore has the potential to amplify or quench particular circuits in the IL-2 network, based on clinical desirability.

Long-term antibiotic exposure promotes mortality after systemic fungal infection by driving lymphocyte dysfunction and systemic escape of commensal bacteria.

Antibiotics are a modifiable iatrogenic risk factor for the most common human nosocomial fungal infection, invasive candidiasis, yet the underlying mechanisms remain elusive. We found that antibiotics enhanced the susceptibility to murine invasive candidiasis due to impaired lymphocyte-dependent IL-17A- and GM-CSF-mediated antifungal immunity within the gut. This led to non-inflammatory bacterial escape and systemic bacterial co-infection, which could be ameliorated by IL-17A or GM-CSF immunotherapy. Vancomycin alone similarly enhanced the susceptibility to invasive fungal infection and systemic bacterial co-infection. Mechanistically, vancomycin reduced the frequency of gut Th17 cells associated with impaired proliferation and RORγt expression. Vancomycin's effects on Th17 cells were indirect, manifesting only in vivo in the presence of dysbiosis. In humans, antibiotics were associated with an increased risk of invasive candidiasis and death after invasive candidiasis. Our work highlights the importance of antibiotic stewardship in protecting vulnerable patients from life-threatening infections and provides mechanistic insights into a controllable iatrogenic risk factor for invasive candidiasis.

Application of dual Nr4a1-GFP Nr4a3-Tocky reporter mice to study T cell receptor signaling by flow cytometry.

This protocol uses Nr4a1-GFP Nr4a3-Tocky mice to study T cell receptor (TCR) signaling using flow cytometry. It identifies the optimal mouse transgenic status and fluorochromes compatible with the dual reporter. This protocol has applications in TCR signaling, and we outline how to obtain high-quality datasets. It is not compatible with cellular fixation, and cells should be analyzed immediately after staining. For complete details on the use and execution of this protocol, please refer to Jennings et al., 2020.

Nr4a1 and Nr4a3 Reporter Mice Are Differentially Sensitive to T Cell Receptor Signal Strength and Duration.

Nr4a receptors are activated by T cell receptor (TCR) signaling and play key roles in T cell differentiation. Which TCR signaling pathways regulate Nr4a receptors and their sensitivities to TCR signal strength and duration remains unclear. Using Nr4a1/Nur77-GFP and Nr4a3-Timer of cell kinetics and activity (Tocky) mice, we elucidate the signaling pathways governing Nr4a receptor expression. We reveal that Nr4a1-Nr4a3 are Src family kinase dependent. Moreover, Nr4a2 and Nr4a3 are attenuated by calcineurin inhibitors and bind nuclear factor of activated T cells 1 (NFAT1), highlighting a necessary and sufficient role for NFAT1 in the control of Nr4a2 and Nr4a3, but redundancy for Nr4a1. Nr4a1-GFP is activated by tonic and cognate signals during T cell development, whereas Nr4a3-Tocky requires cognate peptide:major histocompatibility complex (MHC) interactions for expression. Compared to Nr4a3-Tocky, Nr4a1-GFP is approximately 2- to 3-fold more sensitive to TCR signaling and is detectable by shorter periods of TCR signaling. These findings suggest that TCR signal duration may be an underappreciated aspect influencing the developmental fate of T cells in vivo.