Same yet different - how lymph node heterogeneity affects immune responses.

Lymph nodes are secondary lymphoid organs in which immune responses of the adaptive immune system are initiated and regulated. Distributed throughout the body and embedded in the lymphatic system, local lymph nodes are continuously informed about the state of the organs owing to a constant drainage of lymph. The tissue-derived lymph carries products of cell metabolism, proteins, carbohydrates, lipids, pathogens and circulating immune cells. Notably, there is a growing body of evidence that individual lymph nodes differ from each other in their capacity to generate immune responses. Here, we review the structure and function of the lymphatic system and then focus on the factors that lead to functional heterogeneity among different lymph nodes. We will discuss how lymph node heterogeneity impacts on cellular and humoral immune responses and the implications for vaccination, tumour development and tumour control by immunotherapy.

Mitochondrial dysfunction promotes the transition of precursor to terminally exhausted T cells through HIF-1α-mediated glycolytic reprogramming.

T cell exhaustion is a hallmark of cancer and persistent infections, marked by inhibitory receptor upregulation, diminished cytokine secretion, and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex), but the metabolic principles governing Tpex maintenance and the regulatory circuits that control their exhaustion remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics, and metabolomic analyses, we show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the functional exhaustion of T cells. At the molecular level, we find that mitochondrial dysfunction causes redox stress, which inhibits the proteasomal degradation of hypoxia-inducible factor 1α (HIF-1α) and promotes the transcriptional and metabolic reprogramming of Tpex cells into terminally exhausted T cells. Our findings also bear clinical significance, as metabolic engineering of chimeric antigen receptor (CAR) T cells is a promising strategy to enhance the stemness and functionality of Tpex cells for cancer immunotherapy.

Lymph node medulla regulates the spatiotemporal unfolding of resident dendritic cell networks.

Unlike macrophage networks composed of long-lived tissue-resident cells within specific niches, conventional dendritic cells (cDCs) that generate a 3D network in lymph nodes (LNs) are short lived and continuously replaced by DC precursors (preDCs) from the bone marrow (BM). Here, we examined whether specific anatomical niches exist within which preDCs differentiate toward immature cDCs. In situ photoconversion and Prtn3-based fate-tracking revealed that the LN medullary cords are preferential entry sites for preDCs, serving as specific differentiation niches. Repopulation and fate-tracking approaches demonstrated that the cDC1 network unfolded from the medulla along the vascular tree toward the paracortex. During inflammation, collective maturation and migration of resident cDC1s to the paracortex created discontinuity in the medullary cDC1 network and temporarily impaired responsiveness. The decrease in local cDC1 density resulted in higher Flt3L availability in the medullary niche, which accelerated cDC1 development to restore the network. Thus, the spatiotemporal development of the cDC1 network is locally regulated in dedicated LN niches via sensing of cDC1 densities.

CD4+ T cell-induced inflammatory cell death controls immune-evasive tumours.

Most clinically applied cancer immunotherapies rely on the ability of CD8+ cytolytic T cells to directly recognize and kill tumour cells1-3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment4-6. The ability of CD4+ effector cells to contribute to antitumour immunity independently of CD8+ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified7-10. Here, we describe a mechanism whereby a small number of CD4+ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8+ T cell targeting. The CD4+ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II+CD11c+ antigen-presenting cells. We show that T helper type 1 cell-directed CD4+ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4+ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4+ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8+ T cells and natural killer cells and advance cancer immunotherapies.

CD4+ T cell calibration of antigen-presenting cells optimizes antiviral CD8+ T cell immunity.

Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-ß (IFNα/ß)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/ß or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.

MYB orchestrates T cell exhaustion and response to checkpoint inhibition.

CD8+ T cells that respond to chronic viral infections or cancer are characterized by the expression of inhibitory receptors such as programmed cell death protein 1 (PD-1) and by the impaired production of cytokines. This state of restrained functionality-which is referred to as T cell exhaustion1,2-is maintained by precursors of exhausted T (TPEX) cells that express the transcription factor T cell factor 1 (TCF1), self-renew and give rise to TCF1- exhausted effector T cells3-6. Here we show that the long-term proliferative potential, multipotency and repopulation capacity of exhausted T cells during chronic infection are selectively preserved in a small population of transcriptionally distinct CD62L+ TPEX cells. The transcription factor MYB is not only essential for the development of CD62L+ TPEX cells and maintenance of the antiviral CD8+ T cell response, but also induces functional exhaustion and thereby prevents lethal immunopathology. Furthermore, the proliferative burst in response to PD-1 checkpoint inhibition originates exclusively from CD62L+ TPEX cells and depends on MYB. Our findings identify CD62L+ TPEX cells as a stem-like population that is central to the maintenance of long-term antiviral immunity and responsiveness to immunotherapy. Moreover, they show that MYB is a transcriptional orchestrator of two fundamental aspects of exhausted T cell responses: the downregulation of effector function and the long-term preservation of self-renewal capacity.

Translation of Collagen Ultrastructure to Biomaterial Fabrication for Material-Independent but Highly Efficient Topographic Immunomodulation.

Supplement-free induction of cellular differentiation and polarization solely through the topography of materials is an auspicious strategy but has so far significantly lagged behind the efficiency and intensity of media-supplementation-based protocols. Consistent with the idea that 3D structural motifs in the extracellular matrix possess immunomodulatory capacity as part of the natural healing process, it is found in this study that human-monocyte-derived macrophages show a strong M2a-like prohealing polarization when cultured on type I rat-tail collagen fibers but not on collagen I films. Therefore, it is hypothesized that highly aligned nanofibrils also of synthetic polymers, if packed into larger bundles in 3D topographical biomimetic similarity to native collagen I, would induce a localized macrophage polarization. For the automated fabrication of such bundles in a 3D printing manner, the strategy of "melt electrofibrillation" is pioneered by the integration of flow-directed polymer phase separation into melt electrowriting and subsequent selective dissolution of the matrix polymer postprocessing. This process yields nanofiber bundles with a remarkable structural similarity to native collagen I fibers, particularly for medical-grade poly(ε-caprolactone). These biomimetic fibrillar structures indeed induce a pronounced elongation of human-monocyte-derived macrophages and unprecedentedly trigger their M2-like polarization similar in efficacy as interleukin-4 treatment.

Spatiotemporal regulation of type I interferon expression determines the antiviral polarization of CD4+ T cells.

Differentiation of CD4+ T cells into either follicular helper T (TFH) or type 1 helper T (TH1) cells influences the balance between humoral and cellular adaptive immunity, but the mechanisms whereby pathogens elicit distinct effector cells are incompletely understood. Here we analyzed the spatiotemporal dynamics of CD4+ T cells during infection with recombinant vesicular stomatitis virus (VSV), which induces early, potent neutralizing antibodies, or recombinant lymphocytic choriomeningitis virus (LCMV), which induces a vigorous cellular response but inefficient neutralizing antibodies, expressing the same T cell epitope. Early exposure of dendritic cells to type I interferon (IFN), which occurred during infection with VSV, induced production of the cytokine IL-6 and drove TFH cell polarization, whereas late exposure to type I IFN, which occurred during infection with LCMV, did not induce IL-6 and allowed differentiation into TH1 cells. Thus, tight spatiotemporal regulation of type I IFN shapes antiviral CD4+ T cell differentiation and might instruct vaccine design strategies.

A Triad of Immune Cells Promotes Infection.

The intracellular pathogen L. monocytogenes takes advantage of several myeloid cell populations to establish infection in the spleen. In this issue, Liu et al. (2019) reveal how marginal zone B cells, dendritic cells, and marginal metallophilic macrophages act together with IL-10 to promote L. monocytogenes infection, while simultaneously enabling adaptive CD8+ T cell responses.

Lymphatic Endothelial Cells Are Essential Components of the Subcapsular Sinus Macrophage Niche.

In lymph nodes, subcapsular sinus macrophages (SSMs) form an immunological barrier that monitors lymph drained from peripheral tissues. Upon infection, SSMs activate B and natural killer T (NKT) cells while secreting inflammatory mediators. Here, we investigated the mechanisms regulating development and homeostasis of SSMs. Embryonic SSMs originated from yolk sac hematopoiesis and were replaced by a postnatal wave of bone marrow (BM)-derived monocytes that proliferated to establish the adult SSM network. The SSM network self-maintained by proliferation with minimal BM contribution. Upon pathogen-induced transient deletion, BM-derived cells contributed to restoring the SSM network. Lymphatic endothelial cells (LECs) were the main source of CSF-1 within the lymph node and conditional deletion of Csf1 in adult LECs decreased the network of SSMs and medullary sinus macrophages (MSMs). Thus, SSMs have a dual hematopoietic origin, and LECs are essential to the niche supporting these macrophages.

Charcot-Leyden Crystals Activate the NLRP3 Inflammasome and Cause IL-1ß Inflammation in Human Macrophages.

Charcot-Leyden crystals (CLCs) are Galectin-10 protein crystals that can form after eosinophils degranulate. CLCs can appear and persist in tissues from patients with eosinophilic disorders, such as asthma, allergic reactions, and fungal and helminthic infections. Despite abundant reports of their occurrence in human disease, the inflammatory potential of CLCs has remained unknown. In this article, we show that CLCs induce the release of the proinflammatory cytokine IL-1ß upon their phagocytosis by primary human macrophages in vitro. Chemical inhibition and small interfering RNA knockdown of NLRP3 in primary human macrophages abrogated their IL-1ß response to CLCs. Using C57BL/6 ASC-mCitrine transgenic inflammasome reporter mice, we showed that the instillation of CLCs into the lungs promoted the assembly of ASC complexes in infiltrating immune cells (neutrophils and inflammatory monocytes) and resulted in IL-1ß accumulation into the bronchoalveolar lavage fluid. Our findings reveal that CLCs are recognized by the NLRP3 inflammasome, which may sustain inflammation that follows eosinophilic inflammatory processes.

CD4+ T cell help in cancer immunology and immunotherapy.

Cancer immunotherapy aims to promote the activity of cytotoxic T lymphocytes (CTLs) within a tumour, assist the priming of tumour-specific CTLs in lymphoid organs and establish efficient and durable antitumour immunity. During priming, help signals are relayed from CD4+ T cells to CD8+ T cells by specific dendritic cells to optimize the magnitude and quality of the CTL response. In this Review, we highlight the cellular dynamics and membrane receptors that mediate CD4+ T cell help and the molecular mechanisms of the enhanced antitumour activity of CTLs. We outline how deficient CD4+ T cell help reduces the response of CTLs and how maximizing CD4+ T cell help can improve outcomes in cancer immunotherapy strategies.

In Vivo Labeling by CD73 Marks Multipotent Stromal Cells and Highlights Endothelial Heterogeneity in the Bone Marrow Niche.

Despite much work studying ex vivo multipotent stromal cells (MSCs), the identity and characteristics of MSCs in vivo are not well defined. Here, we generated a CD73-EGFP reporter mouse to address these questions and found EGFP+ MSCs in various organs. In vivo, EGFP+ mesenchymal cells were observed in fetal and adult bones at proliferative ossification sites, while in solid organs EGFP+ cells exhibited a perivascular distribution pattern. EGFP+ cells from the bone compartment could be clonally expanded ex vivo from single cells and displayed trilineage differentiation potential. Moreover, in the central bone marrow CD73-EGFP+ specifically labeled sinusoidal endothelial cells, thought to be a critical component of the hematopoietic stem cell niche. Purification and molecular characterization of this CD73-EGFP+ population revealed an endothelial subtype that also displays a mesenchymal signature, highlighting endothelial cell heterogeneity in the marrow. Thus, the CD73-EGFP mouse is a powerful tool for studying MSCs and sinusoidal endothelium.

Reactive Neutrophil Responses Dependent on the Receptor Tyrosine Kinase c-MET Limit Cancer Immunotherapy.

Inhibitors of the receptor tyrosine kinase c-MET are currently used in the clinic to target oncogenic signaling in tumor cells. We found that concomitant c-MET inhibition promoted adoptive T cell transfer and checkpoint immunotherapies in murine cancer models by increasing effector T cell infiltration in tumors. This therapeutic effect was independent of tumor cell-intrinsic c-MET dependence. Mechanistically, c-MET inhibition impaired the reactive mobilization and recruitment of neutrophils into tumors and draining lymph nodes in response to cytotoxic immunotherapies. In the absence of c-MET inhibition, neutrophils recruited to T cell-inflamed microenvironments rapidly acquired immunosuppressive properties, restraining T cell expansion and effector functions. In cancer patients, high serum levels of the c-MET ligand HGF correlated with increasing neutrophil counts and poor responses to checkpoint blockade therapies. Our findings reveal a role for the HGF/c-MET pathway in neutrophil recruitment and function and suggest that c-MET inhibitor co-treatment may improve responses to cancer immunotherapy in settings beyond c-MET-dependent tumors.

Moving at the frontline.

The factors that regulate how immune cells called innate-like lymphocytes are localized in the tissue have been identified.

Functional classification of memory CD8(+) T cells by CX3CR1 expression.

Localization of memory CD8(+) T cells to lymphoid or peripheral tissues is believed to correlate with proliferative capacity or effector function. Here we demonstrate that the fractalkine-receptor/CX3CR1 distinguishes memory CD8(+) T cells with cytotoxic effector function from those with proliferative capacity, independent of tissue-homing properties. CX3CR1-based transcriptome and proteome-profiling defines a core signature of memory CD8(+) T cells with effector function. We find CD62L(hi)CX3CR1(+) memory T cells that reside within lymph nodes. This population shows distinct migration patterns and positioning in proximity to pathogen entry sites. Virus-specific CX3CR1(+) memory CD8(+) T cells are scarce during chronic infection in humans and mice but increase when infection is controlled spontaneously or by therapeutic intervention. This CX3CR1-based functional classification will help to resolve the principles of protective CD8(+) T-cell memory.

Dendritic cell-targeted vaccines--hope or hype?

The development of an effective vaccine that elicits a strong and durable T cell response against intracellular pathogens and cancer is a challenge. One strategy to enhance the effectiveness of vaccination is by targeting dendritic cells (DCs). In this Opinion article, we discuss existing DC-targeting approaches that induce adaptive immunity. We highlight the crucial issues that need to be addressed to move the field forward and discuss whether targeting DCs could be better than current vaccine approaches.

IL-6 trans-signaling-dependent rapid development of cytotoxic CD8+ T cell function.

Immune control of infections with viruses or intracellular bacteria relies on cytotoxic CD8(+) T cells that use granzyme B (GzmB) for elimination of infected cells. During inflammation, mature antigen-presenting dendritic cells instruct naive T cells within lymphoid organs to develop into effector T cells. Here, we report a mechanistically distinct and more rapid process of effector T cell development occurring within 18 hr. Such rapid acquisition of effector T cell function occurred through cross-presenting liver sinusoidal endothelial cells (LSECs) in the absence of innate immune stimulation and known costimulatory signaling. Rather, interleukin-6 (IL-6) trans-signaling was required and sufficient for rapid induction of GzmB expression in CD8(+) T cells. Such LSEC-stimulated GzmB-expressing CD8(+) T cells further responded to inflammatory cytokines, eliciting increased and protracted effector functions. Our findings identify a role for IL-6 trans-signaling in rapid generation of effector function in CD8(+) T cells that may be beneficial for vaccination strategies.

Spatiotemporal basis of innate and adaptive immunity in secondary lymphoid tissue.

Secondary lymphoid tissues are the sites of both innate and adaptive host defense. Aside from the relatively static nonhematopoietic stromal elements and some macrophages and dendritic cells, most of the cells in these tissues are in constant movement, but the organs maintain a defined microanatomy with preferred locations for the bulk of T cells, B cells, and other lymphocytes and subsets of myeloid cells. Here we describe both the cell dynamics and spatial organization of lymph nodes and review how both physical features and molecular cues guide cell movement to optimize host defense. We emphasize the role of locality in improving the efficiency of a system requiring rare cells to find each other and interact productively through membrane-bound or short-range secreted mediators and highlight how changes in steady-state cell positioning during an infectious challenge contribute to rapid generation of productive responses.