ABSTRACT
T cells dynamically interact with multiple, distinct cellular subsets to determine effector and memory differentiation. Here, we developed a platform to quantify cell location in three dimensions to determine the spatial requirements that direct T cell fate. After viral infection, we demonstrated that CD8+ effector T cell differentiation is associated with positioning at the lymph node periphery. This was instructed by CXCR3 signaling since, in its absence, T cells are confined to the lymph node center and alternatively differentiate into stem-like memory cell precursors. By mapping the cellular sources of CXCR3 ligands, we demonstrated that CXCL9 and CXCL10 are expressed by spatially distinct dendritic and stromal cell subsets. Unlike effector cells, retention of stem-like memory precursors in the paracortex is associated with CCR7 expression. Finally, we demonstrated that T cell location can be tuned, through deficiency in CXCL10 or type I interferon signaling, to promote effector or stem-like memory fates.
Subject(s)
Arenaviridae Infections/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation , Chemokine CXCL10/metabolism , Chemokine CXCL9/metabolism , Immunologic Memory , Lymph Nodes/metabolism , Precursor Cells, T-Lymphoid/metabolism , Receptors, CXCR3/metabolism , Animals , Arenaviridae Infections/genetics , Arenaviridae Infections/immunology , Arenaviridae Infections/virology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , Cell Lineage , Cells, Cultured , Chemokine CXCL10/genetics , Chemokine CXCL9/genetics , Chemotaxis, Leukocyte , Dendritic Cells/immunology , Dendritic Cells/metabolism , Disease Models, Animal , Host-Pathogen Interactions , Interferon Type I/metabolism , Ligands , Lymph Nodes/immunology , Lymph Nodes/virology , Lymphocytic choriomeningitis virus/immunology , Lymphocytic choriomeningitis virus/pathogenicity , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Precursor Cells, T-Lymphoid/immunology , Precursor Cells, T-Lymphoid/virology , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/metabolism , Receptors, CCR7/metabolism , Receptors, CXCR3/genetics , Signal Transduction , Stem Cell Niche , Stromal Cells/immunology , Stromal Cells/metabolismABSTRACT
Tissue-resident memory T (TRM) cells are non-recirculating cells that exist throughout the body. Although TRM cells in various organs rely on common transcriptional networks to establish tissue residency, location-specific factors adapt these cells to their tissue of lodgment. Here we analyze TRM cell heterogeneity between organs and find that the different environments in which these cells differentiate dictate TRM cell function, durability and malleability. We find that unequal responsiveness to TGFß is a major driver of this diversity. Notably, dampened TGFß signaling results in CD103- TRM cells with increased proliferative potential, enhanced function and reduced longevity compared with their TGFß-responsive CD103+ TRM counterparts. Furthermore, whereas CD103- TRM cells readily modified their phenotype upon relocation, CD103+ TRM cells were comparatively resistant to transdifferentiation. Thus, despite common requirements for TRM cell development, tissue adaptation of these cells confers discrete functional properties such that TRM cells exist along a spectrum of differentiation potential that is governed by their local tissue microenvironment.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Cell Plasticity/immunology , Cellular Microenvironment/immunology , Immunologic Memory/immunology , Animals , Antigens, CD/immunology , CD8-Positive T-Lymphocytes/cytology , Female , Integrin alpha Chains/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction/immunology , Transforming Growth Factor beta1/metabolismABSTRACT
Although tissue-resident memory T cells (TRM cells) are critical in fighting infection, their fate after local pathogen re-encounter is unknown. Here we found that skin TRM cells engaged virus-infected cells, proliferated in situ in response to local antigen encounter and did not migrate out of the epidermis, where they exclusively reside. As a consequence, secondary TRM cells formed from pre-existing TRM cells, as well as from precursors recruited from the circulation. Newly recruited antigen-specific or bystander TRM cells were generated in the skin without displacement of the pre-existing TRM cell pool. Thus, pre-existing skin TRM cell populations are not displaced after subsequent infections, which enables multiple TRM cell specificities to be stably maintained within the tissue.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunologic Memory/immunology , Skin/immunology , Animals , Cell Proliferation/physiology , Herpes Simplex/immunology , Mice , Mice, Inbred C57BL , Mice, TransgenicABSTRACT
The sympathetic nervous system (SNS) controls various physiological functions via the neurotransmitter noradrenaline. Activation of the SNS in response to psychological or physical stress is frequently associated with weakened immunity. Here, we investigated how adrenoceptor signaling influences leukocyte behavior. Intravital two-photon imaging after injection of noradrenaline revealed transient inhibition of CD8+ and CD4+ T cell locomotion in tissues. Expression of ß-adrenergic receptor in hematopoietic cells was not required for NA-mediated inhibition of motility. Rather, chemogenetic activation of the SNS or treatment with adrenergic receptor agonists induced vasoconstriction and decreased local blood flow, resulting in abrupt hypoxia that triggered rapid calcium signaling in leukocytes and halted cell motility. Oxygen supplementation reversed these effects. Treatment with adrenergic receptor agonists impaired T cell responses induced in response to viral and parasitic infections, as well as anti-tumor responses. Thus, stimulation of the SNS impairs leukocyte mobility, providing a mechanistic understanding of the link between adrenergic receptors and compromised immunity.
Subject(s)
Adrenergic Agents/immunology , Cell Movement/immunology , Immunity/immunology , Leukocytes/immunology , Sympathetic Nervous System/immunology , Animals , Calcium Signaling/immunology , Cell Line, Tumor , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Adrenergic/immunology , Signal Transduction/immunology , T-Lymphocytes/immunologyABSTRACT
CD169+ macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169+ macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTß) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTßR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTßR revealed that both receptors contribute equally to LN CD169+ macrophage differentiation. In the spleen, the Cd169-directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8+ T cells. Taken together, the data provide evidence that CD169+ macrophage differentiation in LN and spleen requires dual signals from LTßR and RANK with implications for the immune response.
Subject(s)
Lymph Nodes/immunology , Lymphotoxin beta Receptor/metabolism , Macrophages/immunology , Macrophages/metabolism , Receptor Activator of Nuclear Factor-kappa B/metabolism , Sialic Acid Binding Ig-like Lectin 1/metabolism , Signal Transduction , Spleen/immunology , B-Lymphocytes/immunology , RANK Ligand/metabolism , Stromal Cells/metabolismABSTRACT
The lymph node (LN) is home to resident macrophage populations that are essential for immune function and homeostasis, but key factors controlling this niche are undefined. Here, we show that fibroblastic reticular cells (FRCs) are an essential component of the LN macrophage niche. Genetic ablation of FRCs caused rapid loss of macrophages and monocytes from LNs across two in vivo models. Macrophages co-localized with FRCs in human LNs, and murine single-cell RNA-sequencing revealed that FRC subsets broadly expressed master macrophage regulator CSF1. Functional assays containing purified FRCs and monocytes showed that CSF1R signaling was sufficient to support macrophage development. These effects were conserved between mouse and human systems. These data indicate an important role for FRCs in maintaining the LN parenchymal macrophage niche.
Subject(s)
Fibroblasts , Signal Transduction , Mice , Humans , Animals , Macrophages , Lymph NodesABSTRACT
Fibroblastic reticular cells (FRCs) construct microanatomical niches that support lymph node (LN) homeostasis and coordination of immune responses. Transcription factors regulating the functionality of FRCs remain poorly understood. Here, we investigated the role of the transcription factor SpiB that is expressed in LN FRCs. Conditional ablation of SpiB in FRCs impaired the FRC network in the T-cell zone of LNs, leading to reduced numbers of FRCs and altered homeostatic functions including reduced CCL21 and interleukin-7 expression. The size and cellularity of LNs remained intact in the absence of SpiB but the space between the reticular network increased, indicating that although FRCs were reduced in number they stretched to maintain network integrity. Following virus infection, antiviral CD8+ T-cell responses were impaired, suggesting a role for SpiB expression in FRCs in orchestrating immune responses. Together, our findings reveal a new role for SpiB as an important regulator of FRC functions and immunity in LNs.
Subject(s)
Fibroblasts , Transcription Factors , Transcription Factors/metabolism , Fibroblasts/metabolism , CD8-Positive T-Lymphocytes , Lymph NodesABSTRACT
Autoimmune diseases are often treated by glucocorticoids and immunosuppressive drugs that could increase the risk for infection, which in turn deteriorate disease and cause mortality. Low-dose IL-2 (Ld-IL2) therapy emerges as a new treatment for a wide range of autoimmune diseases. To examine its influence on infection, we retrospectively studied 665 patients with systemic lupus erythematosus (SLE) including about one third receiving Ld-IL2 therapy, where Ld-IL2 therapy was found beneficial in reducing the incidence of infections. In line with this clinical observation, IL-2 treatment accelerated viral clearance in mice infected with influenza A virus or lymphocytic choriomeningitis virus (LCMV). Noticeably, despite enhancing anti-viral immunity in LCMV infection, IL-2 treatment exacerbated CD8+ T cell-mediated immunopathology. In summary, Ld-IL2 therapy reduced the risk of infections in SLE patients and enhanced the control of viral infection, but caution should be taken to avoid potential CD8+ T cell-mediated immunopathology.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunosuppressive Agents/pharmacology , Interleukin-2/pharmacology , Lupus Erythematosus, Systemic/immunology , Opportunistic Infections/immunology , Animals , CD8-Positive T-Lymphocytes/drug effects , Cohort Studies , Female , Humans , Immunocompromised Host/immunology , Male , Mice , Mice, Inbred C57BL , Retrospective StudiesABSTRACT
Secondary lymphoid organs (SLO), including the spleen and lymph nodes (LN) are a meeting place for immune cells to initiate adaptive immune responses. Lymphocytes constantly circulate between SLO through the blood and lymph in search of their cognate antigen and are activated within the organized microarchitecture of SLO. Lymphoid stromal cells (LSC) of mesenchymal and endothelial origin construct and support the microarchitecture of SLO by defining distinct compartments and providing signals that can either promote or inhibit immune responses. Here, we discuss recent studies indicating that LSC, including fibroblastic reticular cells (FRC), contribute substantially to immune responses and may tune responses to secondary challenge.
Subject(s)
Cell Communication/immunology , Immunity , Stromal Cells/immunology , Stromal Cells/metabolism , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Homeostasis , Humans , Immune System/cytology , Immune System/immunology , Immune System/metabolism , Immunologic Memory , Immunomodulation , Lymphocyte Activation/immunology , Lymphoid Tissue/cytology , Lymphoid Tissue/immunology , Lymphoid Tissue/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Naive CD4+ T cells must differentiate in order to orchestrate immunity to Plasmodium, yet understanding of their emerging phenotypes, clonality, spatial distributions, and cellular interactions remains incomplete. Here, we observe that splenic polyclonal CD4+ T cells differentiate toward T helper 1 (Th1) and T follicular helper (Tfh)-like states and exhibit rarer phenotypes not elicited among T cell receptor (TCR) transgenic counterparts. TCR clones present at higher frequencies exhibit Th1 skewing, suggesting that variation in major histocompatibility complex class II (MHC-II) interaction influences proliferation and Th1 differentiation. To characterize CD4+ T cell interactions, we map splenic microarchitecture, cellular locations, and molecular interactions using spatial transcriptomics at near single-cell resolution. Tfh-like cells co-locate with stromal cells in B cell follicles, while Th1 cells in red pulp co-locate with activated monocytes expressing multiple chemokines and MHC-II. Spatial mapping of individual transcriptomes suggests that proximity to chemokine-expressing monocytes correlates with stronger effector phenotypes in Th1 cells. Finally, CRISPR-Cas9 gene disruption reveals a role for CCR5 in promoting clonal expansion and Th1 differentiation. A database of cellular locations and interactions is presented: https://haquelab.mdhs.unimelb.edu.au/spatial_gui/.
Subject(s)
CD4-Positive T-Lymphocytes , Cell Differentiation , Malaria , Animals , Mice , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Malaria/immunology , Malaria/parasitology , Mice, Inbred C57BL , Phenotype , Receptors, Antigen, T-Cell/metabolism , Receptors, CCR5/metabolism , Receptors, CCR5/genetics , Spleen/immunology , Th1 Cells/immunologyABSTRACT
The spleen is a gatekeeper of systemic immunity where immune responses against blood-borne pathogens are initiated and sustained. Non-haematopoietic stromal cells construct microanatomical niches in the spleen that make diverse contributions to physiological spleen functions and regulate the homeostasis of immune cells. Additional signals from spleen autonomic nerves also modify immune responses. Recent insight into the diversity of the splenic fibroblastic stromal cells has revised our understanding of how these cells help to orchestrate splenic responses to infection and contribute to immune responses. In this Review, we examine our current understanding of how stromal niches and neuroimmune circuits direct the immunological functions of the spleen, with a focus on T cell immunity.
Subject(s)
Spleen , T-Lymphocytes , Humans , Homeostasis , Stromal CellsABSTRACT
Skin-resident CD8+ T cells include distinct interferon-γ-producing [tissue-resident memory T type 1 (TRM1)] and interleukin-17 (IL-17)-producing (TRM17) subsets that differentially contribute to immune responses. However, whether these populations use common mechanisms to establish tissue residence is unknown. In this work, we show that TRM1 and TRM17 cells navigate divergent trajectories to acquire tissue residency in the skin. TRM1 cells depend on a T-bet-Hobit-IL-15 axis, whereas TRM17 cells develop independently of these factors. Instead, c-Maf commands a tissue-resident program in TRM17 cells parallel to that induced by Hobit in TRM1 cells, with an ICOS-c-Maf-IL-7 axis pivotal to TRM17 cell commitment. Accordingly, by targeting this pathway, skin TRM17 cells can be ablated without compromising their TRM1 counterparts. Thus, skin-resident T cells rely on distinct molecular circuitries, which can be exploited to strategically modulate local immunity.
Subject(s)
CD8-Positive T-Lymphocytes , Immunologic Memory , Memory T Cells , Skin , CD8-Positive T-Lymphocytes/immunology , Memory T Cells/immunology , Skin/immunology , Humans , Th17 Cells/immunology , Inducible T-Cell Co-Stimulator Ligand/metabolism , Proto-Oncogene Proteins c-maf/metabolism , Interleukin-7/metabolismABSTRACT
Lymphoid tissue stromal cells are important regulators of spleen homeostasis and immune responses. Here, we present an optimized protocol that describes the digestion and enrichment steps for the isolation and analysis of rare populations of stromal cells, including fibroblastic reticular cells, perivascular cells, and glial cells found in the spleen. This protocol is suitable for subsequent analysis of spleen stromal cells by flow cytometry or single-cell RNA sequencing and to analyze different disease models. For complete details on the use and execution of this protocol, please refer to Alexandre et al. (2022).1.
Subject(s)
Neuroglia , Spleen , Animals , Mice , Flow Cytometry , Homeostasis , Stromal CellsABSTRACT
The eye is considered immune privileged such that immune responses are dampened to protect vision. As the most anterior compartment of the eye, the cornea is exposed to pathogens and can mount immune responses that recruit effector T cells. However, presence of immune memory in the cornea is not defined. Here, we use intravital 2-photon microscopy to examine T cell responses in the cornea in mice. We show that recruitment of CD8+ T cells in response to ocular virus infection results in the formation of tissue-resident memory T (TRM) cells. Motile corneal TRM cells patrol the cornea and rapidly respond in situ to antigen rechallenge. CD103+ TRM cell generation requires antigen and transforming growth factor ß. In vivo imaging in humans also reveals highly motile cells that patrol the healthy cornea. Our study finds that TRM cells form in the cornea where they can provide local protective immunity.
Subject(s)
CD8-Positive T-Lymphocytes , Immunologic Memory , Animals , Antigens , Cornea , Memory T Cells , MiceABSTRACT
The spleen is a compartmentalized organ that serves as a blood filter and safeguard of systemic immune surveillance. Labyrinthine networks of fibroblastic stromal cells construct complex niches within the white pulp and red pulp that are important for tissue homeostasis and immune activation. However, the identity and roles of the global splenic fibroblastic stromal cells in homeostasis and immune responses are poorly defined. Here, we performed a cellular and molecular dissection of the splenic reticular stromal cell landscape. We found that white pulp fibroblastic reticular cells (FRCs) responded robustly during acute viral infection, but this program of gene regulation was suppressed during persistent viral infection. Single-cell transcriptomic analyses in mice revealed diverse fibroblast cell niches and unexpected heterogeneity among podoplanin-expressing cells that include glial, mesothelial, and adventitial cells in addition to FRCs. We found analogous fibroblastic stromal cell diversity in the human spleen. In addition, we identify the transcription factor SpiB as a critical regulator required to support white pulp FRC differentiation, homeostatic chemokine expression, and antiviral T cell responses. Together, our study provides a comprehensive map of fibroblastic stromal cell types in the spleen and defines roles for red and white pulp fibroblasts for splenic function and orchestration of immune responses.
Subject(s)
Fibroblasts/immunology , Homeostasis/immunology , Spleen/immunology , Stromal Cells/immunology , Animals , Cell Differentiation , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , T-Lymphocytes/immunologyABSTRACT
Concurrent infection with multiple pathogens occurs frequently in individuals and can result in exacerbated infections and altered immunity. However, the impact of such coinfections on immune responses remains poorly understood. Here, we reveal that systemic infection results in an inflammation-induced suppression of local immunity. During localized infection or vaccination in barrier tissues including the skin or respiratory tract, concurrent systemic infection induces a type I interferon-dependent lymphopenia that impairs lymphocyte recruitment to the draining lymph node (dLN) and induces sequestration of lymphocytes in non-draining LN. This contributes to suppressed fibroblastic reticular cell and endothelial cell expansion and dLN remodeling and impairs induction of B cell responses and antibody production. Our data suggest that contemporaneous systemic inflammation constrains the induction of regional immunity.
Subject(s)
Coinfection/immunology , Herpes Simplex/immunology , Inflammation/immunology , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/immunology , Lymphoid Tissue/immunology , Simplexvirus/immunology , Animals , Antibody Formation , Fibroblasts/immunology , Herpes Simplex/virology , Interferon Type I/metabolism , Lymph Nodes/immunology , Lymph Nodes/metabolism , Lymphocytes/immunology , Lymphocytes/metabolism , Lymphocytic Choriomeningitis/virology , Lymphoid Tissue/metabolism , Lymphopenia/immunology , Mice , Mice, Inbred C57BL , Stromal Cells/immunology , Stromal Cells/metabolismABSTRACT
Type 1 conventional DCs (cDC1) excel in the cross-priming of CD8+ T cells, which is crucial for orchestrating efficient immune responses against viruses or tumors. However, our understanding of their physiological functions and molecular regulation has been limited by the lack of proper mutant mouse models allowing their conditional genetic targeting. Because the Xcr1 and A530099j19rik (Karma/Gpr141b) genes belong to the core transcriptomic fingerprint of mouse cDC1, we used them to engineer two novel Cre-driver lines, the Xcr1Cre and KarmaCre mice, by knocking in an IRES-Cre expression cassette into their 3'-UTR. We used genetic tracing to characterize the specificity and efficiency of these new models in several lymphoid and non-lymphoid tissues, and compared them to the Clec9aCre mouse model, which targets the immediate precursors of cDCs. Amongst the three Cre-driver mouse models examined, the Xcr1Cre model was the most efficient and specific for the fate mapping of all cDC1, regardless of the tissues examined. The KarmaCre model was rather specific for cDC1 when compared with the Clec9aCre mouse, but less efficient than the Xcr1Cre model. Unexpectedly, the Xcr1Cre model targeted a small fraction of CD4+ T cells, and the KarmaCre model a significant proportion of mast cells in the skin. Importantly, the targeting specificity of these two mouse models was not changed upon inflammation. A high frequency of germline recombination was observed solely in the Xcr1Cre mouse model when both the Cre and the floxed alleles were brought by the same gamete irrespective of its gender. Xcr1, Karma, and Clec9a being differentially expressed within the cDC1 population, the three CRE-driver lines examined showed distinct recombination patterns in cDC1 phenotypic subsets. This advances our understanding of cDC1 subset heterogeneity and the differentiation trajectory of these cells. Therefore, to the best of our knowledge, upon informed use, the Xcr1Cre and KarmaCre mouse models represent the best tools currently reported to specifically and faithfully target cDC1 in vivo, both at steady state and upon inflammation. Future use of these mutant mouse models will undoubtedly boost our understanding of the biology of cDC1.
Subject(s)
Cross-Priming/genetics , Dendritic Cells/physiology , Receptors, Chemokine/genetics , 3' Untranslated Regions/genetics , Animals , CD4-Positive T-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/physiology , Cell Differentiation/genetics , Inflammation/genetics , Mice , Mice, Inbred C57BL , Skin/physiopathologyABSTRACT
Our protocol describes a simple procedure for isolating stromal cells from lymph nodes (LN). LN are disrupted then enzymatically digested with collagenase and dispase to produce a single cell suspension that can be stained with fluorescently labelled antibodies and analysed by flow cytometry. This protocol will enable identification of fibroblastic reticular cells (FRC), lymphatic endothelial cells (LEC), blood endothelial cells (BEC) as PNAd+ BEC that form LN high endothelial venules (HEV). This method can be applied to examine LN stromal cell responses during inflammatory events induced by infections or immunologic adjuvants and to subset most leukocytes found in LN.
ABSTRACT
Lymph nodes (LNs) are constructed of intricate networks of endothelial and mesenchymal stromal cells. How these lymphoid stromal cells (LSCs) regulate lymphoid tissue remodeling and contribute to immune responses remains poorly understood. We performed a comprehensive functional and transcriptional analysis of LSC responses to skin viral infection and found that LSC subsets responded robustly, with different kinetics for distinct pathogens. Recruitment of cells to inflamed LNs induced LSC expansion, while B cells sustained stromal responses in an antigen-independent manner. Infection induced rapid transcriptional responses in LSCs. This transcriptional program was transient, returning to homeostasis within 1 month of infection, yet expanded fibroblastic reticular cell networks persisted for more than 3 months after infection, and this altered LN composition reduced the magnitude of LSC responses to subsequent heterologous infection. Our results reveal the complexity of LSC responses during infection and suggest that amplified networks of LN stromal cells support successive immune responses.
Subject(s)
Lymph Nodes/pathology , Virus Diseases/immunology , Virus Diseases/pathology , Animals , Antigens, Viral/immunology , B-Lymphocytes/immunology , Cell Proliferation , Coinfection/immunology , Gene Expression Regulation , Kinetics , Mice, Inbred C57BL , Stromal Cells/pathology , Transcription, Genetic , Virus Diseases/geneticsABSTRACT
Naive CD8(+) T cell priming during tumor development or many primary infections requires cross-presentation by XCR1(+) dendritic cells (DCs). Memory CD8(+) T lymphocytes (mCTLs) harbor a lower activation threshold as compared with naive cells. However, whether their recall responses depend on XCR1(+) DCs is unknown. By using a new mouse model allowing fluorescent tracking and conditional depletion of XCR1(+) DCs, we demonstrate a differential requirement of these cells for mCTL recall during secondary infections by different pathogens. XCR1(+) DCs were instrumental to promote this function upon secondary challenges with Listeria monocytogenes, vesicular stomatitis virus, or Vaccinia virus, but dispensable in the case of mouse cytomegalovirus. We deciphered how XCR1(+) DCs promote mCTL recall upon secondary infections with Listeria. By visualizing for the first time the in vivo choreography of XCR1(+) DCs, NK cells and mCTLs during secondary immune responses, and by neutralizing in vivo candidate molecules, we demonstrate that, very early after infection, mCTLs are activated, and attracted in a CXCR3-dependent manner, by NK cell-boosted, IL-12-, and CXCL9-producing XCR1(+) DCs. Hence, depending on the infectious agent, strong recall of mCTLs during secondary challenges can require cytokine- and chemokine-dependent cross-talk with XCR1(+) DCs and NK cells.