A Metabolism-Based Quorum Sensing Mechanism Contributes to Termination of Inflammatory Responses.

Recruitment of immune cells with antimicrobial activities is essential to fight local infections but has the potential to trigger immunopathology. Whether the immune system has the ability to sense inflammation intensity and self-adjust accordingly to limit tissue damage remains to be fully established. During local infection with an intracellular pathogen, we have shown that nitric oxide (NO) produced by recruited monocyte-derived cells was essential to limit inflammation and cell recruitment. Mechanistically, we have provided evidence that NO dampened monocyte-derived cell cytokine and chemokine production by inhibiting cellular respiration and reducing cellular ATP:ADP ratio. Such metabolic control operated at the tissue level but only when a sufficient number of NO-producing cells reached the site of infection. Thus, NO production and activity act as a quorum sensing mechanism to help terminate the inflammatory response.

SPICE-Met: profiling and imaging energy metabolism at the single-cell level using a fluorescent reporter mouse.

The regulation of cellular energy metabolism is central to most physiological and pathophysiological processes. However, most current methods have limited ability to functionally probe metabolic pathways in individual cells. Here, we describe SPICE-Met (Single-cell Profiling and Imaging of Cell Energy Metabolism), a method for profiling energy metabolism in single cells using flow cytometry or imaging. We generated a transgenic mouse expressing PercevalHR, a fluorescent reporter for cellular ATP:ADP ratio. Modulation of PercevalHR fluorescence with metabolic inhibitors was used to infer the dependence of energy metabolism on oxidative phosphorylation and glycolysis in defined cell populations identified by flow cytometry. We applied SPICE-Met to analyze T-cell memory development during vaccination. Finally, we used SPICE-Met in combination with real-time imaging to dissect the heterogeneity and plasticity of energy metabolism in single macrophages ex vivo and identify three distinct metabolic patterns. Functional probing of energy metabolism with single-cell resolution should greatly facilitate the study of immunometabolism at a steady state, during disease pathogenesis or in response to therapy.

Quorum sensing governs collective dendritic cell activation in vivo.

Dendritic cell (DC) activation by viral RNA sensors such as TLR3 and MDA-5 is critical for initiating antiviral immunity. Optimal DC activation is promoted by type I interferon (IFN) signaling which is believed to occur in either autocrine or paracrine fashion. Here, we show that neither autocrine nor paracrine type I IFN signaling can fully account for DC activation by poly(I:C) in vitro and in vivo. By controlling the density of type I IFN-producing cells in vivo, we establish that instead a quorum of type I IFN-producing cells is required for optimal DC activation and that this process proceeds at the level of an entire lymph node. This collective behavior, governed by type I IFN diffusion, is favored by the requirement for prolonged cytokine exposure to achieve DC activation. Furthermore, collective DC activation was found essential for the development of innate and adaptive immunity in lymph nodes. Our results establish how collective rather than cell-autonomous processes can govern the initiation of immune responses.

Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti-tumor activity in vivo.

Cytotoxic T cells (CTLs) can eliminate tumor cells through the delivery of lethal hits, but the actual efficiency of this process in the tumor microenvironment is unclear. Here, we visualized the capacity of single CTLs to attack tumor cells in vitro and in vivo using genetically encoded reporters that monitor cell damage and apoptosis. Using two distinct malignant B-cell lines, we found that the majority of cytotoxic hits delivered by CTLs in vitro were sublethal despite proper immunological synapse formation, and associated with reversible calcium elevation and membrane damage in the targets. Through intravital imaging in the bone marrow, we established that the majority of CTL interactions with lymphoma B cells were either unproductive or sublethal. Functional heterogeneity of CTLs contributed to diverse outcomes during CTL-tumor contacts in vivo. In the therapeutic settings of anti-CD19 CAR T cells, the majority of CAR T cell-tumor interactions were also not associated with lethal hit delivery. Thus, differences in CTL lytic potential together with tumor cell resistance to cytotoxic hits represent two important bottlenecks for anti-tumor responses in vivo.

Spatiotemporal dynamics of calcium signals during neutrophil cluster formation.

Neutrophils form cellular clusters or swarms in response to injury or pathogen intrusion. Yet, intracellular signaling events favoring this coordinated response remain to be fully characterized. Here, we show that calcium signals play a critical role during mouse neutrophil clustering around particles of zymosan, a structural fungal component. Pioneer neutrophils recognizing zymosan or live Candida albicans displayed elevated calcium levels. Subsequently, a transient wave of calcium signals in neighboring cells was observed followed by the attraction of neutrophils that exhibited more persistent calcium signals as they reached zymosan particles. Calcium signals promoted LTB4 production while the blocking of extracellular calcium entry or LTB4 signaling abrogated cluster formation. Finally, using optogenetics to manipulate calcium influx in primary neutrophils, we show that calcium signals could initiate recruitment of neighboring neutrophils in an LTB4-dependent manner. Thus, sustained calcium responses at the center of the cluster are necessary and sufficient for the generation of chemoattractive gradients that attract neutrophils in a self-reinforcing process.

Dynamic in situ cytometry uncovers T cell receptor signaling during immunological synapses and kinapses in vivo.

Upon antigen recognition, T cells form either static (synapses) or migratory (kinapses) contacts with antigen-presenting cells. Addressing whether synapses and kinapses result in distinct T cell receptor (TCR) signals has been hampered by the inability to simultaneously assess T cell phenotype and behavior. Here, we introduced dynamic in situ cytometry (DISC), a combination of intravital multiphoton imaging and flow cytometry-like phenotypic analysis. Taking advantage of CD62L shedding as a marker of early TCR signaling, we examined how T cells sense TCR ligands of varying affinities in vivo. We uncovered three modes of antigen recognition: synapses with the strongest TCR signals, kinapses with robust signaling, and kinapses with weak signaling. As illustrated here, the DISC approach should provide unique opportunities to link immune cell behavior to phenotype and function in vivo.

Intravital imaging reveals distinct dynamics for natural killer and CD8(+) T cells during tumor regression.

Recognition of NKG2D ligands by natural killer (NK) cells plays an important role during antitumoral responses. To address how NKG2D engagement affects intratumoral NK cell dynamics, we performed intravital microscopy in a Rae-1ß-expressing solid tumor. This NKG2D ligand drove NK cell accumulation, activation, and motility within the tumor. NK cells established mainly dynamic contacts with their targets during tumor regression. In sharp contrast, cytotoxic T lymphocytes (CTLs) formed stable contacts in tumors expressing their cognate antigen. Similar behaviors were observed during effector functions in lymph nodes. In vitro, contacts between NK cells and their targets were cytotoxic but did not elicit sustained calcium influx nor adhesion, whereas CTL contact stability was critically dependent on extracellular calcium entry. Altogether, our results offer mechanistic insight into how NK cells and CTLs can exert cytotoxic activity with remarkably different contact dynamics.

Visualizing the functional diversification of CD8+ T cell responses in lymph nodes.

CD8(+) T cell responses generate effector cells endowed with distinct functional potentials but the contribution of early events in this process is unclear. Here, we have imaged T cells expressing a fluorescent reporter for the activation of the interferon-γ (IFN-γ) locus during priming in lymph nodes. We have demonstrated marked differences in the efficiency of gene activation during stable T cell-dentritic cell (DC) contacts, influenced in part by signal strength. Imaging the first cell division, we have demonstrated that heterogeneity in T cell functional potential was largely apparent as T cells initiated clonal expansion. Moreover, by analyzing the fate of single activated T cells ex vivo, we have provided evidence that these early differences resulted in clonal progenies with distinct functional properties. Thus, the early set of T cell-DC interactions in lymph nodes largely contribute to the heterogeneity of T cell responses through the generation of functionally divergent clonal progenies.

Signal strength regulates antigen-mediated T-cell deceleration by distinct mechanisms to promote local exploration or arrest.

T lymphocytes are highly motile cells that decelerate upon antigen recognition. These cells can either completely stop or maintain a low level of motility, forming contacts referred to as synapses or kinapses, respectively. Whether similar or distinct molecular mechanisms regulate T-cell deceleration during synapses or kinapses is unclear. Here, we used microfabricated channels and intravital imaging to observe and manipulate T-cell kinapses and synapses. We report that high-affinity antigen induced a pronounced deceleration selectively dependent on Ca(2+) signals and actin-related protein 2/3 complex (Arp2/3) activity. In contrast, low-affinity antigens induced a switch of migration mode that promotes T-cell exploratory behavior, characterized by partial deceleration and frequent direction changes. This switch depended on T-cell receptor binding but was largely independent of downstream signaling. We propose that distinct mechanisms of T-cell deceleration can be triggered during antigenic recognition to favor local exploration and signal integration upon suboptimal stimulus and complete arrest on the best antigen-presenting cells.

GATA-3 promotes T-cell specification by repressing B-cell potential in pro-T cells in mice.

Transcription factors orchestrate T-lineage differentiation in the thymus. One critical checkpoint involves Notch1 signaling that instructs T-cell commitment at the expense of the B-lineage program. While GATA-3 is required for T-cell specification, its mechanism of action is poorly understood. We show that GATA-3 works in concert with Notch1 to commit thymic progenitors to the T-cell lineage via 2 distinct pathways. First, GATA-3 orchestrates a transcriptional "repertoire" that is required for thymocyte maturation up to and beyond the pro-T-cell stage. Second, GATA-3 critically suppresses a latent B-cell potential in pro­T cells. As such, GATA-3 is essential to sealing in Notch-induced T-cell fate in early thymocyte precursors by promoting T-cell identity through the repression of alternative developmental options.

Phenotypic CD8+ T cell diversification occurs before, during, and after the first T cell division.

Effector T cell responses rely on a phenotypically and functionally heterogeneous population of cells. Whether this diversity is programmed before clonal expansion or in later phases as a result of stochastic events or asymmetric cell division is not fully understood. In this study, we first took advantage of a sensitive in vitro assay to analyze the composition of single CD8(+) T cell progenies. Heterogeneity was predominantly observed between progenies of distinct clones, but could also be detected within individual progenies. Furthermore, by physically isolating daughter cells of the first T cell division, we showed that differences in paired daughter cell progenies contributed to intraclonal diversification. Finally, we developed an in vivo limiting dilution assay to compare individual T cell progenies following immunization. We provided evidence for simultaneous intraclonal and interclonal diversification in vivo. Our results support the idea that T cell diversification is a continuous process, initiated before clonal expansion and amplified during the first and subsequent cell divisions.

Subcapsular sinus macrophages promote NK cell accumulation and activation in response to lymph-borne viral particles.

Natural killer (NK) cells become activated during viral infection in response to cytokines or to engagement of NK cell activating receptors. However, the identity of cells sensing viral particles and mediating NK cell activation has not been defined. Here, we show that local administration of a modified vaccinia virus Ankara vaccine in mice results in the accumulation of NK cells in the subcapsular area of the draining lymph node and their activation, a process that is strictly dependent on type I IFN signaling. NK cells located in the subcapsular area exhibited reduced motility and were found associated with CD169(+)-positive subcapsular sinus (SCS) macrophages and collagen fibers. Moreover, depletion of SCS macrophages using clodronate liposomes abolished NK cell accumulation and activation. Our results identify SCS macrophages as primary mediators of NK cell activation in response to lymph-borne viral particles suggesting that they act as early sensors of local infection or delivery of viral-based vaccines.

Cutting edge: tumor-targeting antibodies enhance NKG2D-mediated NK cell cytotoxicity by stabilizing NK cell-tumor cell interactions.

Monoclonal antibodies represent a promising approach to fight a variety of tumors, but their mode of action remains to be fully understood. NK cells can recognize Ab-coated targets, as well as stress ligands, on tumor cells. In this study, we investigated how NK cells integrate both kinds of activating signals. NK cell-mediated killing was maximal with the combined recognition of NKG2D ligands and Ab; surprisingly, only NKG2D engagement substantially enhanced degranulation. Conversely, Ab recognition by NK cells uniquely increased contact stability with tumor cells. Furthermore, using intravital imaging of solid tumors, we showed that Ab recognition favored prolonged interactions between NK cells and targets. Altogether, our results demonstrate that NK cell-mediated killing can be differentially regulated at the level of degranulation and contact stability by distinct activating receptors. Thus, complementary signals mediated by recognition of stress ligands and tumor-specific Abs may contribute to the efficacy of NK cells during mAb therapy.

Anti-PD-1 therapy triggers Tfh cell-dependent IL-4 release to boost CD8 T cell responses in tumor-draining lymph nodes.

Anti-PD-1 therapy targets intratumoral CD8+ T cells to promote clinical responses in cancer patients. Recent evidence suggests an additional activity in the periphery, but the underlying mechanism is unclear. Here, we show that anti-PD-1 mAb enhances CD8+ T cell responses in tumor-draining lymph nodes by stimulating cytokine production in follicular helper T cells (Tfh). In two different models, anti-PD-1 mAb increased the activation and proliferation of tumor-specific T cells in lymph nodes. Surprisingly, anti-PD-1 mAb did not primarily target CD8+ T cells but instead stimulated IL-4 production by Tfh cells, the major population bound by anti-PD-1 mAb. Blocking IL-4 or inhibiting the Tfh master transcription factor BCL6 abrogated anti-PD-1 mAb activity in lymph nodes while injection of IL-4 complexes was sufficient to recapitulate anti-PD-1 mAb activity. A similar mechanism was observed in a vaccine model. Finally, nivolumab also boosted human Tfh cells in humanized mice. We propose that Tfh cells and IL-4 play a key role in the peripheral activity of anti-PD-1 mAb.

HIV controllers maintain a population of highly efficient Th1 effector cells in contrast to patients treated in the long term.

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral therapy. To identify parameters of the CD4 response that may contribute to viral control rather than merely reflect a persistently low viremia, we compared the T helper profiles in two groups of patients with more than 10 years of viral suppression: HIV controllers from the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) CO18 cohort (n = 26) and efficiently treated patients (n = 16). Cells specific for immunodominant Gag and cytomegalovirus (CMV) peptides were evaluated for the production of 10 cytokines and cytotoxicity markers and were also directly quantified ex vivo by major histocompatibility complex (MHC) class II tetramer staining. HIV controller CD4(+) T cells were characterized by a higher frequency of gamma interferon (IFN-γ) production, perforin(+)/CD107a(+) expression, and polyfunctionality in response to Gag peptides. While interleukin 4 (IL-4), IL-17, and IL-21 production did not differ between groups, the cells of treated patients produced more IL-10 in response to Gag and CMV peptides, pointing to persistent negative immunoregulation after long-term antiretroviral therapy. Gag293 tetramer-positive cells were detected at a high frequency (0.12%) and correlated positively with IFN-γ-producing CD4(+) T cells in the controller group (R = 0.73; P = 0.003). Tetramer-positive cells were fewer in the highly active antiretroviral therapy (HAART) group (0.04%) and did not correlate with IFN-γ production, supporting the notion of a persistent immune dysfunction in HIV-specific CD4(+) T cells of treated patients. In conclusion, HIV controllers maintained a population of highly efficient Th1 effectors directed against Gag in spite of a persistently low antigenemia, while patients treated in the long term showed a loss of CD4 effector functions.

Subcellular dynamics of T cell immunological synapses and kinapses in lymph nodes.

In vitro studies have revealed that T cell activation occurs during the formation of either dynamic or stable interactions with antigen-presenting cells (APC), and the respective cell junctions have been referred to as immunological kinapses and synapses. However, the relevance and molecular dynamics of kinapses and synapses remain to be established in vivo. Using two-photon imaging, we tracked the distribution of LAT-EGFP molecules during antigen recognition by activated CD4(+) T cells in lymph nodes. At steady state, LAT-EGFP molecules were preferentially found at the uropod of rapidly migrating T cells. In contrast to naïve T cells that fully stopped upon systemic antigen delivery, recently activated T cells decelerated and formed kinapses, characterized by continuous extension of membrane protrusions and by the absence of persistent LAT-EGFP clustering. On the other hand, activated CD4(+) T cells formed stable immunological synapses with antigen-loaded B cells and displayed sustained accumulation of LAT-EGFP fluorescence at the contact zone. Our results show that the state of T cell activation and the type of APC largely influence T cell-APC contact dynamics in lymph nodes. Furthermore, we provide a dynamic look at immunological kinapses and synapses in lymph nodes and suggest the existence of distinct patterns of LAT redistribution during antigen recognition.

Integration of intermittent calcium signals in T cells revealed by temporally patterned optogenetics.

T cells become activated following one or multiple contacts with antigen-presenting cells. Calcium influx is a key signaling event elicited during these cellular interactions; however, it is unclear whether T cells recall and integrate calcium signals elicited during temporally separated contacts. To study the integration of calcium signals, we designed a programmable, multiplex illumination strategy for temporally patterned optogenetics (TEMPO). We found that a single round of calcium elevation was insufficient to promote nuclear factor of activated T cells (NFAT) activity and cytokine production in a T cell line. However, robust responses were detected after a second identical stimulation even when signals were separated by several hours. Our results suggest the existence of a biochemical memory of calcium signals in T cells that favors signal integration during temporally separated contacts and promote cytokine production. As illustrated here, TEMPO is a versatile approach for dissecting temporal integration in defined signaling pathways.

Tumor-intrinsic sensitivity to the pro-apoptotic effects of IFN-γ is a major determinant of CD4+ CAR T-cell antitumor activity.

CD4+ T cells and CD4+ chimeric antigen receptor (CAR) T cells display highly variable antitumor activity in preclinical models and in patients; however, the mechanisms dictating how and when CD4+ T cells promote tumor regression are incompletely understood. With the help of functional intravital imaging, we report that interferon (IFN)-γ production but not perforin-mediated cytotoxicity was the dominant mechanism for tumor elimination by anti-CD19 CD4+ CAR T cells. Mechanistically, mouse or human CD4+ CAR T-cell-derived IFN-γ diffused extensively to act on tumor cells at distance selectively killing tumors sensitive to cytokine-induced apoptosis, including antigen-negative variants. In anti-CD19 CAR T-cell-treated patients exhibiting elevated CAR CD4:CD8 ratios, strong induction of serum IFN-γ was associated with increased survival. We propose that the sensitivity of tumor cells to the pro-apoptotic activity of IFN-γ is a major determinant of CD4+ CAR T-cell efficacy and may be considered to guide the use of CD4+ T cells during immunotherapy.

HIV controller CD4+ T cells respond to minimal amounts of Gag antigen due to high TCR avidity.

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral treatment. Emerging evidence indicates that HIV control is mediated through very active cellular immune responses, though how such responses can persist over time without immune exhaustion is not yet understood. To investigate the nature of memory CD4+ T cells responsible for long-term anti-HIV responses, we characterized the growth kinetics, Vbeta repertoire, and avidity for antigen of patient-derived primary CD4+ T cell lines. Specific cell lines were obtained at a high rate for both HIV controllers (16/17) and efficiently treated patients (19/20) in response to the immunodominant Gag293 peptide. However, lines from controllers showed faster growth kinetics than those of treated patients. After normalizing for growth rates, IFN-gamma responses directed against the immunodominant Gag293 peptide showed higher functional avidity in HIV controllers, indicating differentiation into highly efficient effector cells. In contrast, responses to Gag161, Gag263, or CMV peptides did not differ between groups. Gag293-specific CD4+ T cells were characterized by a diverse Vbeta repertoire, suggesting that multiple clones contributed to the high avidity CD4+ T cell population in controllers. The high functional avidity of the Gag293-specific response could be explained by a high avidity interaction between the TCR and the peptide-MHC complex, as demonstrated by MHC class II tetramer binding. Thus, HIV controllers harbor a pool of memory CD4+ T cells with the intrinsic ability to recognize minimal amounts of Gag antigen, which may explain how they maintain an active antiviral response in the face of very low viremia.

Enumeration of human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies.

The number of antigen-specific naive CD8(+) T cells is believed to be important in the shaping of adaptive immune responses, and is predictive for the magnitude of priming responses in mouse models. Because of extremely low precursor frequencies, knowledge about these cells comes from indirect techniques and estimations. Here, we present a strategy based on the combination of tetramer staining, magnetic-bead enrichment, and multiparametric cytometry, which permitted direct detection and analysis of CD8(+) T cells reactive for 6 different naive epitopes (MART-1(26-35), HIV-1 Gag p17(77-85), hepatitis C virus [HCV] NS3(1406-1415), HCV Core(132-140), NY-ESO-1(157-165), and cytomegalovirus [CMV] pp65(495-503)). Interestingly, we detected higher than 100-fold differences in precursor frequency across these epitopes (from 0.6 x 10(-6) to 1.3 x 10(-4)), but conserved frequencies among humans. Development of a procedure for direct assessment of T-cell precursor frequency in humans has important implications, with particular relevance to vaccine development and monitoring of tumor and self-reactive T cells.