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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.

International audience; Modulation of cells and molecules of the immune system not only represents a major opportunity to treat a variety of diseases including infections, cancer, autoimmune and inflammatory disorders but could also help understand the intricacies of immune responses. A detailed mechanistic understanding of how a specific immune intervention may provide clinical benefit is essential for the rational design of efficient immunomodulators. Visualizing the impact of immunomodulation in real-time and in vivo has emerged as an important approach to achieve this goal. In this review, we aim to illustrate how multiphoton intravital imaging has helped clarify the mode of action of immunomodulatory strategies such as antibodies or cell therapies. We also discuss how optogenetics combined with imaging will further help manipulate and precisely understand immunomodulatory pathways. Combined with other single-cell technologies, in vivo dynamic imaging has therefore a major potential for guiding preclinical development of immunomodulatory drugs.

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.

International audience; 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.

International audience; 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 antitumor responses in vivo.

We thank M. Guerin for help in image acquisition and the mouse facility and Technology Core of the Center for Translational Science (CRT) at Institut Pasteur for support in conducting the present study; International audience; 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.

Cytotoxic T cells are important components of natural anti-tumor immunity and are harnessed in tumor immunotherapies. Immune responses to tumors and immune therapy outcomes largely vary among individuals, but very few studies examine the contribution of intrinsic behavior of the T cells to this heterogeneity. Here we show the development of a microfluidic-based in vitro method to track the outcome of antigen-specific T cell activity on many individual cancer spheroids simultaneously at high spatiotemporal resolution, which we call Multiscale Immuno-Oncology on-Chip System (MIOCS). By combining parallel measurements of T cell behaviors and tumor fates with probabilistic modeling, we establish that the first recruited T cells initiate a positive feedback loop to accelerate further recruitment to the spheroid. We also provide evidence that cooperation between T cells on the spheroid during the killing phase facilitates tumor destruction. Thus, we propose that both T cell accumulation and killing function rely on collective behaviors rather than simply reflecting the sum of individual T cell activities, and the possibility to track many replicates of immune cell-tumor interactions with the level of detail our system provides may contribute to our understanding of immune response heterogeneity.

International audience; Chimeric antigen receptor (CAR) T cell therapy relies on the activity of a large pool of tumor-targeting cytotoxic effectors. Whether CAR T cells act autonomously or require interactions with the tumor microenvironment (TME) remains incompletely understood. Here, we report an essential cross-talk between CAR T cell subsets and the TME for tumor control in an immunocompetent mouse B cell lymphoma model of anti-CD19 CAR T cell therapy. Using single-cell RNA sequencing, we revealed substantial modification of the TME during CAR T cell therapy. Interferon-γ (IFN-γ) produced by CAR T cells not only enhanced endogenous T and natural killer cell activity but was also essential for sustaining CAR T cell cytotoxicity, as revealed by intravital imaging. CAR T cell-derived IFN-γ facilitated host interleukin-12 production that supported host immune and CAR T cell responses. Compared with CD8+ CAR T cells, CD4+ CAR T cells were more efficient at host immune activation but less capable of direct tumor killing. In summary, CAR T cells do not act independently in vivo but rely instead on cytokine-mediated cross-talk with the TME for optimal activity. Invigorating CAR T cell interplay with the host represents an attractive strategy to prevent relapses after therapy.

By offering the possibility to manipulate cellular functions with spatiotemporal control, optogenetics represents an attractive tool for dissecting immune responses. However, applying these approaches to single cells in vivo remains particularly challenging for immune cells that are typically located in scattering tissues. Here, we introduce an improved calcium actuator with sensitivity allowing for two-photon photoactivation. Furthermore, we identify an actuator/reporter combination that permits the simultaneous manipulation and visualization of calcium signals in individual T cells in vivo. With this strategy, we document the consequences of defined patterns of calcium signals on T cell migration, adhesion, and chemokine release. Manipulation of individual immune cells in vivo should open new avenues for establishing the functional contribution of single immune cells engaged in complex reactions., The ability to manipulate and monitor calcium signaling in cells in vivo would provide insights into signaling in an endogenous context. Here the authors develop a two-photon-responsive calcium actuator and reporter combination to monitor the effect of calcium actuation on T cell migration, adhesion and chemokine release in vivo.

The cytokine interferon (IFN)-γ produced by tumor-reactive T cells is a key effector molecule with pleiotropic effects during anti-tumor immune responses. Although IFN-γ production is targeted at the immunologic synapse, its spatiotemporal activity within the tumor remains elusive. In the present study, we report that, although IFN-γ secretion requires local antigen recognition, IFN-γ diffuses extensively to alter the tumor microenvironment in distant areas. Using intravital imaging and a reporter for STAT1 translocation, we provide evidence that T cells mediate sustained IFN-γ signaling in remote tumor cells. Furthermore, tumor phenotypic alterations required several hours of exposure to IFN-γ, a feature that disfavored local IFN-γ activity over diffusion and bystander activity. Finally, single-cell RNA-sequencing data from melanoma patients also suggested bystander IFN-γ activity in human tumors. Thus, tumor-reactive T cells act collectively to create large cytokine fields that profoundly modify the tumor microenvironment. Two papers by Schumacher and colleagues and Bousso and colleagues show that sensing of IFN-γ by a small number of tumor cells leads to propagation of IFN-γ sensing and response across a larger fraction of the tumor.

International audience; Understanding complex interactions between the immune system and the tumor microenvironment is an essential step towards the rational development and optimization of immunotherapies. Several experimental approaches are available to tackle this complexity but most are not designed to address the dynamic features of immune reactions, including cell migration, cellular interactions, and transient signaling events. By providing a unique means to access these precious parameters, intravital imaging offers a fresh look at intratumoral immune responses at the single-cell level. Here, we discuss how in vivo imaging sheds light on fundamental aspects of tumor immunity and helps elucidate modes of action of immunotherapies. We conclude by discussing future developments that may consolidate the unique contribution of intravital imaging for our understanding of tumor immunity.

Recruitment of immune cells during infection is essential to fueling the immune response but can also trigger immunopathology. A critical question is how the immune system can sense inflammation levels and self-adjust accordingly to limit tissue damage while removing the pathogen. During my Ph.D. I studied the self-resolving cutaneous infection with Leishmania major parasites where tissue damage arises when inflammation is allowed to become excessive. At the site of infection, the immune reaction is driven by recruited monocyte-derived cells that represents the major population of infected cells and are also actively involved in fighting the infection. They secrete pro-inflammatory cytokines but also produce nitric oxide (NO), critical to regulate the outcome of the infection: iNOS KO mice are susceptible and do not control the parasite load, subsequently developing severe tissue damage because of excessive immune cell infiltration. My work demonstrated that monocyte-derived cells at the site of infection are regulated by NO that limits their cellular respiration, lowers their energetic resources and consequently their activity in vivo. This regulation relies on tissue-wide NO diffusion and only exists when a sufficient cell density has been reached, revealing that monocyte-derived cells are endowed with a quorum sensing mechanism that adjusts their population size and activity in time and space to avoid immunopathology.