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Highly motile and functionally diverse immune cells orchestrate effective immune responses through complex and dynamic cooperative behavior. Multiphoton intravital microscopy (MP-IVM) presents a unique and powerful tool to study the coordinated action of immune cell interactions in situ. Here, we review the current state of intravital microscopy in deepening our understanding of the immune system and discuss its fundamental limitations. In addition, we draw insights from recent technical advances in multiplex static tissue-imaging methods and propose an approach that could enable simultaneous visualization of cellular dynamics, deep phenotyping, and transcriptional states through a new type of correlative microscopy that combines these imaging technologies with advances in complex data analysis.

A central question in immunology is what features allow the immune system to respond in a timely manner to a variety of pathogens encountered at unanticipated times and diverse body sites. Two decades of advanced and static dynamic imaging methods have now revealed several major principles facilitating host defense. Suborgan spatial prepositioning of distinct cells promotes time-efficient interactions upon pathogen sensing. Such pre-organization also provides an effective barrier to movement of pathogens from parenchymal tissues into the blood circulation. Various molecular mechanisms maintain effective intercellular communication among otherwise rapidly moving cells. These and related discoveries have benefited from recent increases in the number of parameters that can be measured simultaneously in a single tissue section and the extension of such multiplex analyses to 3D tissue volumes. The application of new computational methods to such imaging data has provided a quantitative, in vivo context for cell trafficking and signaling pathways traditionally explored in vitro or with dissociated cell preparations. Here, we summarize our efforts to devise and employ diverse imaging tools to probe immune system organization and function, concluding with a commentary on future developments, which we believe will reveal even more about how the immune system operates in health and disease.

Natural Killer T (NKT) cells and Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells that express semi-invariant αβ T cell receptors (TCRs) through which they recognise CD1d and MR1 molecules, respectively, in complex with specific ligands. These cells play important roles in health and disease in many organs, but their precise intra-organ location is not well established. Here, using CD1d and MR1 tetramer staining techniques, we describe the precise location of NKT and MAIT cells in lymphoid and peripheral organs. Within the thymus, NKT cells were concentrated in the medullary side of the corticomedullary junction. In spleen and lymph nodes, NKT cells were mainly localised within T cell zones (TCZ), although following in vivo activation with the potent NKT-cell ligand α-GalCer, they expanded throughout the spleen. MAIT cells were clearly detectable in Vα19 TCR transgenic mice and were rare but detectable in lymphoid tissue of non-transgenic mice. In contrast to NKT cells, MAIT cells were more closely associated with the B cell zone (BCZ) and red pulp (RP) of the spleen. Accordingly, we have provided an extensive analysis of the in situ localisation of NKT and MAIT cells and suggest differences between the intra-organ location of these two cell types.

8531 Background: The lung intratumor microbiome influences lung cancer tumorigenesis and treatment responses, but detailed data on the extent, location, and effects of microbes within lung tumors is missing, information needed to improve treatment outcomes and prognosis. Methods: To address this gap, we developed a novel spatial meta-transcriptomic method simultaneously detecting the expression level of 1,811 host genes and three microbe targets (16S rRNA, 28S rRNA and CMV). After rigorous validation, we analyzed the spatial meta-transcriptomic profiles of tumor cells, T cells, macrophages, other immune cells, and stroma in tumor samples from 12 patients with early-stage lung cancer. Results: Bacterial burden was significantly higher in tumor cells compared to T cells, macrophages, other immune cells, and stroma. This burden increased from tumor-adjacent normal lung and tertiary lymphoid structures to tumor cells to the airways, suggesting that lung intratumor bacteria derive from the latter route of entry. Expression of oncogenic β-catenin and epithelial-mesenchymal transition pathway genes was strongly correlated with bacterial burden, as were tumor subtypes, mutation profile, histology and smoking history. Conclusions: Intratumor bacteria were enriched with tumor cells and associated with multiple oncogenic pathways, supporting a rationale for reducing the local intratumor microbiome in lung cancer to optimize clinical outcomes. This research was supported in part by the Intramural Research Programs of the NCI and NIAID. Other funding sources included ASCO Young Investigator Award, SITC-AstraZeneca Immunotherapy in Lung Cancer (Early Stage NSCLC) Clinical Fellowship Award, NIH Bench-to-Bedside and Back Program (BtB), NCI R00 award (CA226400), Emerson Collective Cancer Research fund, Lung Cancer Research Foundation (LCRF) pilot grant and W.W. Smith Trust Foundation award. This study was approved by NCI institutional review board (NCT00242723 and NCT02146170) and Animal Use and Care Committee at the University of Pennsylvania (#806875).

2D multiplexed antibody-based imaging provides a framework to study cell-cell interactions in thin tissue sections but lacks the ability to interrogate spatial relationships in larger 3D anatomical structures. Several methods for 3D imaging of optically cleared tissue exist, but current approaches do not provide the marker depth afforded by high content 2D imaging. To overcome this constraint, we combined a fast hydrophilic tissue clearing technique, clearing-enhanced 3D (Ce3D), with the recently developed Iterative Bleaching Extends Multiplexity (IBEX) technique, and created 3D-IBEX. 3D-IBEX offers a high-resolution multiplex 3D imaging method. It has been used to analyze murine lung, lymph nodes, retina, and cornea as well as human retina and jejunum, yielding seamless views of large-scale tissue structures while obtaining single-cell resolution of multiple markers, permitting the identification of discrete cell subsets and structures. To date, up to 15 markers have been visualized in a single sample using 3 iterative staining and imaging cycles. This represents a major advance in the emerging field of multiplexed high-resolution 3D imaging but requires further refinements to address issues such as slow antibody penetration, the need for new computational algorithms to process large datasets, and implementation of methods for optimal registration of iterative images. When fully realized, 3D-IBEX will open new avenues for characterization of rare cell subsets missed in thin tissue sections, visualization of structures like nerves and vessels whose trajectories in a tissue are disrupted by sectioning, and delineation of mesoscale tissue domains that contribute to homeostasis and are often the substrates for pathology. Intramural NIAID Research Opportunities (INRO) program.

The immune system can suppress tumour development both by eliminating malignant cells and by preventing the outgrowth and spread of cancer cells that resist eradication1. Clinical and experimental data suggest that the latter mode of control-termed cancer-immune equilibrium1-can be maintained for prolonged periods of time, possibly up to several decades2-4. Although cancers most frequently originate in epithelial layers, the nature and spatiotemporal dynamics of immune responses that maintain cancer-immune equilibrium in these tissue compartments remain unclear. Here, using a mouse model of transplantable cutaneous melanoma5, we show that tissue-resident memory CD8+ T cells (TRM cells) promote a durable melanoma-immune equilibrium that is confined to the epidermal layer of the skin. A proportion of mice (~40%) transplanted with melanoma cells remained free of macroscopic skin lesions long after epicutaneous inoculation, and generation of tumour-specific epidermal CD69+ CD103+ TRM cells correlated with this spontaneous disease control. By contrast, mice deficient in TRM formation were more susceptible to tumour development. Despite being tumour-free at the macroscopic level, mice frequently harboured melanoma cells in the epidermal layer of the skin long after inoculation, and intravital imaging revealed that these cells were dynamically surveyed by TRM cells. Consistent with their role in melanoma surveillance, tumour-specific TRM cells that were generated before melanoma inoculation conferred profound protection from tumour development independently of recirculating T cells. Finally, depletion of TRM cells triggered tumour outgrowth in a proportion (~20%) of mice with occult melanomas, demonstrating that TRM cells can actively suppress cancer progression. Our results show that TRM cells have a fundamental role in the surveillance of subclinical melanomas in the skin by maintaining cancer-immune equilibrium. As such, they provide strong impetus for exploring these cells as targets of future anticancer immunotherapies.

Infection or inflammation of the skin recruits effector CD8+ T cells that enter the epidermis and form populations of long-lived tissue-resident memory T (TRM) cells. These skin TRM cells migrate within the constrained epidermal environment by extending multiple dynamic dendritic projections and squeezing between keratinocytes to survey the tissue for pathogens. In this study, we examined the signals required for this distinctive mode of T cell migration by inhibiting key cytoskeletal components and performing intravital two-photon microscopy to visualize TRM cell behavior. We found that TRM cell motility and dendrite formation required an intact actomyosin cytoskeleton and the Rho-associated coiled-coil containing kinases. We also identified an essential role for microtubules for maintaining skin TRM cell shape and cellular integrity. We reveal a role for pertussis toxin–sensitive signaling for TRM cell dendritic morphology and migration that is independent of CXCR3 or CXCR6, or the skin-selective chemokine receptors CCR10 and CCR8. However, we found that CXCR6 and CCR10 expression by CD8+ T cells was required for the optimal formation of memory T cell populations, in particular TRM cell populations in the skin.

Neutrophils rapidly infiltrate sites of inflammation during peripheral infection or tissue injury. In addition to their well described roles as pro-inflammatory phagocytes responsible for pathogen clearance, recent studies have demonstrated a broader functional repertoire including mediating crosstalk between innate and adaptive arms of the immune system. Specifically, neutrophils have been proposed to mediate antigen transport to lymph nodes (LN) to modulate T cell priming and to influence T cell migration to infected tissues. Using a mouse model of cutaneous herpes simplex virus type 1 (HSV-1) infection we explored potential contributions of neutrophils toward anti-viral immunity. While a transient, early influx of neutrophils was triggered by dermal scarification, we did not detect migration of neutrophils from the skin to LN. Furthermore, despite recruitment of neutrophils into LN from the blood, priming and expansion of CD4+ and CD8+ T cells was unaffected following neutrophil depletion. Finally, we found that neutrophils were dispensable for the migration of effector T cells into infected skin. Our study suggests that the immunomodulatory roles of neutrophils toward adaptive immunity may be context-dependent, and are likely determined by the type of pathogen and anatomical site of infection.

Panel j was inadvertently labelled as panel k in the caption to Fig. 4. Similarly, 'Fig. 4k' should have been 'Fig. 4j' in the sentence beginning 'TNF-α-deficient gBT-I cells were…'. In addition, the surname of author Umaimainthan Palendira was misspelled 'Palendria'. These errors have been corrected online.

The dynamics of when and where CD4(+) T cells provide help for CD8(+) T cell priming and which dendritic cells (DCs) activate CD4(+) T cells in vivo after localized infection are poorly understood. By using a cutaneous herpes simplex virus infection model combined with intravital 2-photon imaging of the draining lymph node (LN) to concurrently visualize pathogen-specific CD4(+) and CD8(+) T cells, we found that early priming of CD4(+) T cells involved clustering with migratory skin DCs. CD8(+) T cells did not interact with migratory DCs and their activation was delayed, requiring later clustering interactions with LN-resident XCR1(+) DCs. CD4(+) T cells interacted with these late CD8(+) T cell clusters on resident XCR1(+) DCs. Together, these data reveal asynchronous T cell activation by distinct DC subsets and highlight the key role of XCR1(+) DCs as the central platform for cytotoxic T lymphocyte activation and the delivery of CD4(+) T cell help.

Zhang et al. show that DOCK8-deficient T and NK cells develop cell and nuclear shape abnormalities that do not impair chemotaxis but contribute to a form of cell death they term cytothripsis. Cytothripsis of DOCK8-deficient cells prevents the generation of long-lived skin-resident memory CD8 T cells resulting in impaired immune response to skin infection., DOCK8 mutations result in an inherited combined immunodeficiency characterized by increased susceptibility to skin and other infections. We show that when DOCK8-deficient T and NK cells migrate through confined spaces, they develop cell shape and nuclear deformation abnormalities that do not impair chemotaxis but contribute to a distinct form of catastrophic cell death we term cytothripsis. Such defects arise during lymphocyte migration in collagen-dense tissues when DOCK8, through CDC42 and p21-activated kinase (PAK), is unavailable to coordinate cytoskeletal structures. Cytothripsis of DOCK8-deficient cells prevents the generation of long-lived skin-resident memory CD8 T cells, which in turn impairs control of herpesvirus skin infections. Our results establish that DOCK8-regulated shape integrity of lymphocytes prevents cytothripsis and promotes antiviral immunity in the skin.

Efficient infection control requires potent T-cell responses at sites of pathogen replication. However, the regulation of T-cell effector function in situ remains poorly understood. Here, we show key differences in the regulation of effector activity between CD4+ and CD8+ T-cells during skin infection with HSV-1. IFN-γ-producing CD4+ T cells disseminated widely throughout the skin and draining lymph nodes (LN), clearly exceeding the epithelial distribution of infectious virus. By contrast, IFN-γ-producing CD8+ T cells were only found within the infected epidermal layer of the skin and associated hair follicles. Mechanistically, while various subsets of lymphoid- and skin-derived dendritic cells (DC) elicited IFN-γ production by CD4+ T cells, CD8+ T cells responded exclusively to infected epidermal cells directly presenting viral antigen. Notably, uninfected cross-presenting DCs from both skin and LNs failed to trigger IFN-γ production by CD8+ T-cells. Thus, we describe a previously unappreciated complexity in the regulation of CD4+ and CD8+ T-cell effector activity that is subset-specific, microanatomically distinct and involves largely non-overlapping types of antigen-presenting cells (APC)., Author Summary HSV-1 is a widely distributed pathogen causing a life-long latent infection associated with periodic bouts of reactivation and severe clinical complications. Adaptive immune responses encompassing CD4+ and CD8+ T-cell activities are key to both the clearance of infectious virus and the control of latent infection. However, precisely how such T-cell responses are regulated, particularly within acutely infected peripheral tissues, remains poorly understood. Using a mouse model of HSV-1 skin infection, we describe a complex regulation of T-cell responses at the site of acute infection. These responses were subset-specific and anatomically distinct, with CD4+ and CD8+ T-cell activities being directed to distinct anatomical compartments within the skin. While IFN-γ-producing CD4+ T cells were broadly distributed, including skin regions a considerable distance away from infected cells, CD8+ T-cell activity was strictly confined to directly infected epithelial compartments. This unexpected spatial segregation was a direct consequence of the involvement of largely non-overlapping types of antigen-presenting cells in driving CD4+ and CD8+ T-cell effector activity. Our results provide novel insights into the cellular regulation of T-cell immunity within peripheral tissues and have the potential to guide the development of T-cell subset-specific approaches for therapeutic and prophylactic intervention in antimicrobial immunity and autoimmunity.

The in vivo dynamics of CD4 and CD8 T cell priming in draining lymph nodes (dLN) following infection and the types of interactions that occur with different dendritic cell (DC) subsets remains unclear. Immune responses to pathogens that remain localized to peripheral tissues and do not directly infect the lymphoid organs critically rely upon migratory DC to traffic antigen to dLN. Following Herpes Simplex virus infection (HSV-1) migratory CD11b+ DC transport viral antigens from the skin to the dLN and then transfer antigen to LN-resident CD8a+ DC for CD8 T cell priming. Although migratory DC can present antigens to CD4 T cells ex vivo, which DC subset(s) activate CD4 T cells in vivo, when and where CD4 T cells provide ‘help’ for CD8 T cell priming and the dynamics of these interactions are not known. Using a HSV-1 flank infection model combined with intravital 2-photon imaging of the draining inguinal LN to concurrently visualize pathogen-specific CD4 and CD8 T cells we find temporally separated priming of CD4 versus CD8 T cells. Early priming of CD4 T cells involved clustering with migratory DC and more rapid responses by these T cells, followed by later interactions of CD8 T cells with LN-resident DC that resulting in priming. Together, these data suggest unexpected temporal differences in T cell activation by DC subsets following localized viral infection.