Expansion of tumor-associated Treg cells upon disruption of a CTLA-4-dependent feedback loop.

Foxp3+ T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients.

CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment.

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.

CXCL10 chemokine regulates heterogeneity of the CD8+ T cell response and viral set point during chronic infection.

CD8+ T cells responding to chronic infection adapt an altered differentiation program that provides some restraint on pathogen replication yet limits immunopathology. This adaptation is imprinted in stem-like cells and propagated to their progeny. Understanding the molecular control of CD8+ T cell differentiation in chronic infection has important therapeutic implications. Here, we find that the chemokine receptor CXCR3 is highly expressed on viral-specific stem-like CD8+ T cells and that one of its ligands, CXCL10, regulates the persistence and heterogeneity of responding CD8+ T cells in spleens of mice chronically infected with lymphocytic choriomeningitis virus. CXCL10 is produced by inflammatory monocytes and fibroblasts of the splenic red pulp, where it grants stem-like cells access to signals promoting differentiation and limits their exposure to pro-survival niches in the white pulp. Consequently, functional CD8+ T cell responses are greater in Cxcl10-/- mice and are associated with a lower viral set point.

Chemokines and the immune response to cancer.

Chemokines are chemotactic cytokines that regulate the migration of immune cells. Chemokines function as cues for the coordinated recruitment of immune cells into and out of tissue and also guide the spatial organization and cellular interactions of immune cells within tissues. Chemokines are critical in directing immune cell migration necessary to mount and then deliver an effective anti-tumor immune response; however, chemokines also participate in the generation and recruitment of immune cells that contribute to a pro-tumorigenic microenvironment. Here, we review the role of the chemokine system in anti-tumor and pro-tumor immune responses and discuss how malignant cells and the tumor microenvironment regulate the overall chemokine landscape to shape the type and outcome of immune responses to cancer and cancer treatment.

Intratumoral Activity of the CXCR3 Chemokine System Is Required for the Efficacy of Anti-PD-1 Therapy.

Despite compelling rates of durable clinical responses to programmed cell death-1 (PD-1) blockade, advances are needed to extend these benefits to resistant tumors. We found that tumor-bearing mice deficient in the chemokine receptor CXCR3 responded poorly to anti-PD-1 treatment. CXCR3 and its ligand CXCL9 were critical for a productive CD8+ T cell response in tumor-bearing mice treated with anti-PD-1 but were not required for the infiltration of CD8+ T cells into tumors. The anti-PD-1-induced anti-tumor response was facilitated by CXCL9 production from intratumoral CD103+ dendritic cells, suggesting that CXCR3 facilitates dendritic cell-T cell interactions within the tumor microenvironment. CXCR3 ligands in murine tumors and in plasma of melanoma patients were an indicator of clinical response to anti-PD-1, and their induction in non-responsive murine tumors promoted responsiveness to anti-PD-1. Our data suggest that the CXCR3 chemokine system is a biomarker for sensitivity to PD-1 blockade and that augmenting the intratumoral function of this chemokine system could improve clinical outcomes.

A minimum number of autoimmune T cells to induce autoimmunity?

While autoimmune T cells are present in most individuals, only a minority of the population suffers from an autoimmune disease. To better appreciate the limits of T cell tolerance, we carried out experiments to determine how many autoimmune T cells are required to initiate an experimental autoimmune disease. Variable numbers of autoimmune OT-I T cells were transferred into RIP-OVA mice, which were injected with antigen-loaded DCs in a single footpad; this restricted T cell priming to a few OT-I T cells that are present in the draining popliteal lymph node. Using selective plane illumination microscopy (SPIM) we counted the number of OT-I T cells present in the popliteal lymph node at the time of priming. Analysis of our data suggests that a single autoimmune T cell cannot induce an experimental autoimmune disease, but a "quorum" of 2-5 autoimmune T cells clearly has this capacity.

Targeting Lymph Node Niches Enhances Type 1 Immune Responses to Immunization.

Generating robust CD4+ T-helper cell type 1 (Th1) responses is essential for protective vaccine-induced type 1 immunity. Here, we examine whether immunization formulation associated with enhanced vaccine efficacy promotes antigen targeting and cell recruitment into lymph node (LN) niches associated with optimal type 1 responses. Immunization with antigen and Toll-like receptor agonist emulsified in oil leads to an increased differentiation of IFNγ/TNF-α+ polyfunctional Th1 cells compared to an identical immunization in saline. Oil immunization results in a rapid delivery and persistence of antigen in interfollicular regions (IFRs) of the LN, whereas without oil, antigen is distributed in the medullary region. Following oil immunization, CXCL10-producing inflammatory monocytes accumulate in the IFR, which mobilizes antigen-specific CD4+ T cells into this niche. In this microenvironment, CD4+ T cells are advantageously positioned to encounter arriving IL-12-producing inflammatory dendritic cells (DCs). These data suggest that formulations delivering antigen to the LN IFR create an inflammatory niche that can improve vaccine efficacy.

CXCL10 stabilizes T cell-brain endothelial cell adhesion leading to the induction of cerebral malaria.

Malaria remains one of the world's most significant human infectious diseases and cerebral malaria (CM) is its most deadly complication. CM pathogenesis remains incompletely understood, hindering the development of therapeutics to prevent this lethal complication. Elevated levels of the chemokine CXCL10 are a biomarker for CM, and CXCL10 and its receptor CXCR3 are required for experimental CM (ECM) in mice, but their role has remained unclear. Using multiphoton intravital microscopy, CXCR3 receptor- and ligand-deficient mice and bone marrow chimeric mice, we demonstrate a key role for endothelial cell-produced CXCL10 in inducing the firm adhesion of T cells and preventing their cell detachment from the brain vasculature. Using a CXCL9 and CXCL10 dual-CXCR3-ligand reporter mouse, we found that CXCL10 was strongly induced in the brain endothelium as early as 4 days after infection, while CXCL9 and CXCL10 expression was found in inflammatory monocytes and monocyte-derived DCs within the blood vasculature on day 8. The induction of both CXCL9 and CXCL10 was completely dependent on IFN-γ receptor signaling. These data demonstrate that IFN-γ-induced, endothelium-derived CXCL10 plays a critical role in mediating the T cell-endothelial cell adhesive events that initiate the inflammatory cascade that injures the endothelium and induces the development of ECM.

Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes.

Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues.

pMHC affinity controls duration of CD8+ T cell-DC interactions and imprints timing of effector differentiation versus expansion.

During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation, as low affinity-primed T cells acquired cytotoxic activity earlier than high affinity-primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vessels for egress, whereas high affinity-stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment.

Quantitative measurements in 3-dimensional datasets of mouse lymph nodes resolve organ-wide functional dependencies.

Deep tissue imaging has become state of the art in biology, but now the problem is to quantify spatial information in a global, organ-wide context. Although access to the raw data is no longer a limitation, the computational tools to extract biologically useful information out of these large data sets is still catching up. In many cases, to understand the mechanism behind a biological process, where molecules or cells interact with each other, it is mandatory to know their mutual positions. We illustrate this principle here with the immune system. Although the general functions of lymph nodes as immune sentinels are well described, many cellular and molecular details governing the interactions of lymphocytes and dendritic cells remain unclear to date and prevent an in-depth mechanistic understanding of the immune system. We imaged ex vivo lymph nodes isolated from both wild-type and transgenic mice lacking key factors for dendritic cell positioning and used software written in MATLAB to determine the spatial distances between the dendritic cells and the internal high endothelial vascular network. This allowed us to quantify the spatial localization of the dendritic cells in the lymph node, which is a critical parameter determining the effectiveness of an adaptive immune response.