Fibroblastic reticular cells (FRCs) are specialized fibroblasts that construct secondary lymphoid organs where they provide crucial signals for immune cell homeostasis and migration. While splenic FRCs are thought to support antiviral T cell responses, their role remains unclear. Here, we found that ablation of splenic FRCs impaired virus-specific CD8+ T cell responses during lymphocytic choriomeningitis virus (LCMV) infection. Immunofluorescence imaging revealed that FRCs promote CD8+ T cell clustering with type 1 conventional dendritic cells (cDC1) in the T cell zone before migration to the infected marginal zone. Without FRCs, T cells instead clustered with cDC1 and virus-infected cells in the marginal zone, leading to suboptimal priming. Mechanistically, FRCs coordinated early viral replication and the inflammatory milieu for optimal DC activation, and an intact FRC network was crucial for generating effector T cells and maintaining protective memory T cells. Thus, splenic FRCs provide essential lymphoid niches for antiviral T cell responses.

With the ever-increasing complexity of microscopy modalities, it is imperative to have computational workflows that enable researchers to process and perform in-depth quantitative analysis of the resulting images. However, workflows that allow flexible, interactive and intuitive analysis from raw images to analysed data are lacking for many experimental use-cases. Notably, integrated software solutions for analysis of complex 3D and live cell images are sorely needed. To address this, we present Cecelia, a toolbox that integrates various open-source packages into a coherent data management suite to make quantitative multidimensional image analysis accessible for non-specialists. We describe the application of Cecelia to several immunologically relevant scenarios and the development of an unbiased approach to distinguish dynamic cell behaviours from live imaging data. Cecelia is available as a software package with a Shiny app interface ( https://github.com/schienstockd/cecelia ). We envision that this framework and its approaches will be of broad use for biological researchers.

Exhausted CD8+ T (TEX) cell responses are maintained by precursors of exhausted T (TPEX) cells that possess high self-renewal and developmental potential. TPEX cells also drive the proliferative burst of effector T cells upon therapeutic immune checkpoint blockade (ICB). However, the spatial context and signals that regulate their differentiation and function are not well defined. Here we identify developmental and functional compartmentalization of TPEX and TEX cells across secondary lymphoid organs during chronic infection. We show that stem-like CD62L+ TPEX and effector-like CX3CR1+ TEX cells constitute a distinct developmental lineage that is promoted by the lymph node (LN) microenvironment and dependent on the transcription factor KLF2. LNs act as a niche in which migratory dendritic cells provide antigen and costimulatory signals to maintain the proliferative fitness of CD62L+ TPEX cells and generation of CX3CR1+ TEX cells. Moreover, LNs exclusively drive the proliferative burst and systemic dissemination of CX3CR1+ TEX cells during ICB. Thus, our findings identify a unique role for LNs in the maintenance of T cell differentiation and function during systemic chronic infection and ICB therapy. Here the authors show a function for lymph nodes in the maintenance of effector T cell differentiation and function during chronic infection and checkpoint blockade, identifying a spatial component in the regulation of exhausted T cell fitness.

CD8 + T cells are a key weapon in the therapeutic armamentarium against cancer. While CD8 + CD103 + T cells with a tissue-resident memory T (T RM ) cell phenotype have been favourably correlated with patient prognoses 1-6 , the tumour microenvironment also contains dysfunctional exhausted T (T EX ) cells that exhibit a myriad of T RM -like features, leading to conflation of these two populations. Here, we deconvolute T RM and T EX cells within the intratumoural CD8 + CD103 + T cell pool across human cancers, ascribing markers and gene signatures that distinguish these CD8 + populations and enable their functional distinction. We found that while T RM cells exhibit superior functionality and are associated with long-term survival post-tumour resection, they are not associated with responsiveness to immune checkpoint blockade. Deconvolution of the two populations showed that tumour-associated T EX and T RM cells are clonally distinct, with the latter comprising both tumour-independent bystanders and tumour-specific cells segregated from their cognate antigen. Intratumoural T RM cells can be forced towards an exhausted fate when chronic antigen stimulation occurs, arguing that the presence or absence of continuous antigen exposure within the microenvironment is the key distinction between respective tumour-associated T EX and T RM populations. These results suggest unique roles for T RM and T EX cells in tumour control, underscoring the need for distinct strategies to harness these T cell populations in novel cancer therapies.

Purpose:To characterize corneal immune cell morphodynamics and nerve features, and define the in vivo immune landscape in older adults with human immunodeficiency virus (HIV) receiving antiretroviral therapy (ART), relative to healthy age-matched adults. Methods:In this cross-sectional study, 16 HIV-positive individuals receiving ART and 15 age-matched controls underwent ocular surface examinations and functional in vivo confocal microscopy (Fun-IVCM). Time-lapsed videos were created to analyze corneal immune cells (T cells, dendritic cells [DCs], macrophages). Subclinical indicators of corneal health (sensory nerve and endothelial cell features), clinical ocular surface findings, and tear cytokines (analyzed using multiplex bead-based immunoassay) were compared between groups. Results:Participants comprised mostly males (HIV 71 ± 5 years; male:female 15:1; controls 67 ± 6 years; 14:1). The HIV-positive group showed less T-cell motility at the corneal whorl relative to the control group (P = 0.01), and region-dependent differences in T-cell speed (P = 0.001) and DC area (P < 0.001). The HIV-positive group showed greater central corneal nerve fiber width (P = 0.004) and larger endothelial cells (P = 0.02). Clinical findings, corneal immune cell densities, and tear cytokine profiles were similar between groups. Conclusions:Among older individuals with well-controlled HIV infection and clinically-normal ocular surface health, this study identifies subclinical group differences in corneal immune cells (potentially indicative of a heightened, pro-inflammatory activation state in the peripheral cornea) and corneal endothelial cell morphology that parallel those in chronic inflammatory disease. This study demonstrates the utility of Fun-IVCM to evaluate subclinical immune cell features in systemic disease, which could inform the future identification of biomarkers in immune-related conditions.

Balanced immune responses in the eyes are crucial to preserve vision. The ocular immune system has long been considered distinct, owing to the so-called 'immune privilege' of its component tissues. More recently, intravital imaging and transcriptomic techniques have reshaped scientific understanding of the ocular immune landscape, such as revealing the specialization of immune cell populations in the various tissues of the eye. As knowledge of the phenotypes of corneal and retinal immune cells has evolved, links to both the systemic immune system, and the central and peripheral nervous systems, have been identified. Using intravital imaging, T cells have recently been found to reside in, and actively patrol, the healthy human cornea. Disease-associated retinal microglia with links to retinal degeneration have also been identified. This Review provides an updated guide to the ocular immune system, highlighting current knowledge of the immune cells that are present in steady-state and specific diseased ocular tissues, as well as evidence for their relationship to systemic disease. In addition, we discuss emerging intravital imaging techniques that can be used to visualize immune cell morphology and dynamics in living human eyes and how these could be applied to advance understanding of the human immune system.

Regionalized immune surveillance relies on the concerted efforts of diverse memory T cell populations. Of these, tissue-resident memory T (TRM) cells are strategically positioned in barrier tissues, where they enable efficient frontline defense against infections and cancer. However, the long-term persistence of these cells has been implicated in a variety of immune-mediated pathologies. Consequently, modulating TRM cell populations represents an attractive strategy for novel vaccination and therapeutic interventions against tissue-based diseases. Here, we provide an updated overview of TRM cell heterogeneity and function across tissues and disease states. We discuss mechanisms of TRM cell-mediated immune protection and their potential contributions to autoimmune disorders. Finally, we examine how TRM cell responses might be durably boosted or dampened for therapeutic gain.

Neurons that originate from pre-vertebral sympathetic ganglia, the splanchnic-celiac-superior mesenteric ganglion complex (SCSMG) in mouse, have important roles in control of organs of the upper abdomen. Here, we present a protocol for the isolation of the mouse sympathetic SCSMG. We describe steps for surgical incision, ganglia isolation, ganglia fine dissection, and whole-mount SCSMG after clearing-enhanced 3D (Ce3D) clearing method and immunohistochemistry. Given the importance of mice in studies of that control, this protocol aims to assist biomedical researchers in the dissection of the mouse SCSMG.

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.

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/.

Tissue-resident memory T (TRM) cells are integral to tissue immunity, persisting in diverse anatomical sites where they adhere to a common transcriptional framework. How these cells integrate distinct local cues to adopt the common TRM cell fate remains poorly understood. Here, we show that whereas skin TRM cells strictly require transforming growth factor β (TGF-β) for tissue residency, those in other locations utilize the metabolite retinoic acid (RA) to drive an alternative differentiation pathway, directing a TGF-β-independent tissue residency program in the liver and synergizing with TGF-β to drive TRM cells in the small intestine. We found that RA was required for the long-term maintenance of intestinal TRM populations, in part by impeding their retrograde migration. Moreover, enhanced RA signaling modulated TRM cell phenotype and function, a phenomenon mirrored in mice with increased microbial diversity. Together, our findings reveal RA as a fundamental component of the host-environment interaction that directs immunosurveillance in tissues.

In vivo confocal microscopy (IVCM) is a widely used technique for imaging the cornea of the eye with a confocal scanning light ophthalmoscope. Cellular resolution and high contrast are achieved without invasive procedures, suiting the study of living humans. However, acquiring useful image data can be challenging due to the incessant motion of the eye, such that images are typically limited by noise and a restricted field of view. These factors affect the degree to which the same cells can be identified and tracked over time. To redress these shortcomings, here we present a data acquisition protocol together with the details of a free, open-source software package written in Matlab. The software package automatically registers and processes IVCM videos to significantly improve contrast, resolution, and field of view. The software also registers scans acquired at progressive time intervals from the same tissue region, producing a time-lapsed video to facilitate visualization and quantification of individual cell dynamics (e.g., motility and dendrite probing). With minimal user intervention, to date, this protocol has been employed to both cross-sectionally and longitudinally assess the dynamics of immune cells in the human corneal epithelium and stroma, using a technique termed functional in vivo confocal microscopy (Fun-IVCM) in 68 eyes from 68 participants. Using the custom software, registration of 'sequence scan' data was successful in 97% of videos acquired from the corneal epithelium and 93% for the corneal stroma. Creation of time-lapsed videos, in which the averages from single videos were registered across time points, was successful in 93% of image series for the epithelium and 75% of image series for the stroma. The reduced success rate for the stroma occurred due to practical difficulties in finding the same tissue between time points, rather than due to errors in image registration. We also present preliminary results showing that the protocol is well suited to in vivo cellular imaging in the retina with adaptive optics scanning laser ophthalmoscopy (AOSLO). Overall, the approach described here substantially improves the efficiency and consistency of time-lapsed video creation to enable non-invasive study of cell dynamics across diverse tissues in the living eye.

The tumour immune microenvironment is shaped by the crosstalk between cancer cells, immune cells, fibroblasts, endothelial cells and other stromal components. Although the immune tumour microenvironment (TME) serves as a source of therapeutic targets, it is also considered a friend or foe to tumour-directed therapies. This is readily illustrated by the importance of T cells in triple-negative breast cancer (TNBC), culminating in the advent of immune checkpoint therapy in combination with cytotoxic chemotherapy as standard of care for both early and advanced-stage TNBC, as well as recent promising signs of efficacy in a subset of hormone receptor-positive disease. In this Review, we discuss the various components of the immune TME in breast cancer and therapies that target or impact the immune TME, as well as the complexity of host physiology. In this Review, Harris et al. summarize the dynamic changes of the immune breast tumour microenvironment (TME) that take place during disease progression and in response to treatment, and outline emerging therapies to target the immune TME in patients with breast cancer.

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.

The healthy human cornea is a uniquely transparent sensory tissue where immune responses are tightly controlled to preserve vision. The cornea contains immune cells that are widely presumed to be intraepithelial dendritic cells (DCs). Corneal immune cells have diverse cellular morphologies and morphological alterations are used as a marker of inflammation and injury. Based on our imaging of corneal T cells in mice, we hypothesized that many human corneal immune cells commonly defined as DCs are intraepithelial lymphocytes (IELs). To investigate this, we developed functional in vivo confocal microscopy (Fun-IVCM) to investigate cell dynamics in the human corneal epithelium and stroma. We show that many immune cells resident in the healthy human cornea are T cells. These corneal IELs are characterized by rapid, persistent motility and interact with corneal DCs and sensory nerves. Imaging deeper into the corneal stroma, we show that crawling macrophages and rare motile T cells patrol the tissue. Furthermore, we identify altered immune cell behaviors in response to short-term contact lens wear (acute inflammatory stimulus), as well as in individuals with allergy (chronic inflammatory stimulus) that was modulated by therapeutic intervention. These findings redefine current understanding of immune cell subsets in the human cornea and reveal how resident corneal immune cells respond and adapt to chronic and acute stimuli.

Skin-resident CD8 + T cells include distinct interferon-γ–producing [tissue-resident memory T type 1 (T RM 1)] and interleukin-17 (IL-17)–producing (T RM 17) 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 T RM 1 and T RM 17 cells navigate divergent trajectories to acquire tissue residency in the skin. T RM 1 cells depend on a T-bet–Hobit–IL-15 axis, whereas T RM 17 cells develop independently of these factors. Instead, c-Maf commands a tissue-resident program in T RM 17 cells parallel to that induced by Hobit in T RM 1 cells, with an ICOS–c-Maf–IL-7 axis pivotal to T RM 17 cell commitment. Accordingly, by targeting this pathway, skin T RM 17 cells can be ablated without compromising their T RM 1 counterparts. Thus, skin-resident T cells rely on distinct molecular circuitries, which can be exploited to strategically modulate local immunity.

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.

CD4 + T cells orchestrate adaptive immunity to circulating malaria parasites; yet cellular interactions and molecular mechanisms controlling Th1 and Tfh differentiation in the spleen remain to be fully defined in vivo . Here, using a murine model of CD4-dependent immunity, we tested if Slide-seqV2 , a spatial transcriptomic method with near single-cell resolution, could determine the locations of multiple CD4 + T cell subsets and potentially interacting cellular partners in the spleen during infection. Firstly, Slide-seqV2 readily mapped splenic cellular structure and microanatomical change during infection. Next, computational integration with scRNA-seq reference datasets of splenocytes, stromal cells, and specifically of polyclonal CD4 + T cells and B cells, mapped the relative locations of multiple cell-types within this dense tissue. scRNA-seq of B cells over time mapped emergence of germinal centre B cells, red pulp-located plasmablasts and atypical B cells, and uncovered a prolonged CD4 + T-cell-independent, follicular bystander B cell response marked by Sca-1 and Ly6C upregulation. scRNA-seq of activated, polyclonal CD4 + T cells revealed their similarity to our previous TCR transgenic models. Importantly, spatial analysis revealed polyclonal Th1 cells co-localised with CXCL9/10-producing monocytes in the red pulp, while polyclonal Tfh-like cells were located close to CXCL13-expressing B cell follicles, consistent with our previous CXCR3/CXCR5 competition model of Th1/Tfh bifurcation. CRISPR/Cas9 disruption of either or both CXCR3 and CXCR5 in naïve Plasmodium -specific CD4 + T cells had unexpectedly minor effects on Th1 differentiation in vivo . Instead, CXCR5 was essential for maximising clonal expansion, suggesting a role for splenic CXCL13 + cells in supporting CD4 + T cell proliferation in malaria. Thus, spatial transcriptomics at near single-cell resolution was feasible in densely packed secondary lymphoid tissue, providing multiple insights into mechanisms controlling splenic polyclonal CD4 + T cell and B cell differentiation during infection. Highlights Slide-seqV2 maps splenic microanatomy, including stromal and immune cell location. Bystander activation of all follicular B cells occurs in malaria, marked by Sca-1/Ly6C upregulation. Single naïve polyclonal CD4 + T cells differentiate mostly into Th1 and Tfh cells in malaria. Cell-cell colocalization analysis positions Th1 cells with monocytes in red pulp, and Tfh cells with Cxcl13 + B cell follicles. CXCR5, but not CXCR3, supports parasite-specific CD4 + T cell clonal expansion.