Th1 cells are critical tissue organizers of myeloid-rich perivascular activation niches.

Unlabelled: Aggregating immune cells within perivascular niches (PVN) can regulate tissue immunity in infection, autoimmunity and cancer. How cells are assembled at PVNs and the activation signals imparted within remain unclear. Here, we integrate dynamic time-resolved imaging with a novel spatially-resolved platform for microanatomical interrogation of transcriptome, immune phenotype and inflammatory mediators in skin PVNs.

Multiphoton fluorescence microscopy for in vivo imaging.

Multiphoton fluorescence microscopy (MPFM) has been a game-changer for optical imaging, particularly for studying biological tissues deep within living organisms. MPFM overcomes the strong scattering of light in heterogeneous tissue by utilizing nonlinear excitation that confines fluorescence emission mostly to the microscope focal volume. This enables high-resolution imaging deep within intact tissue and has opened new avenues for structural and functional studies.

T cells use focal adhesions to pull themselves through confined environments.

Immune cells are highly dynamic and able to migrate through environments with diverse biochemical and mechanical compositions. Their migration has classically been defined as amoeboid under the assumption that it is integrin independent. Here, we show that activated primary Th1 T cells require both confinement and extracellular matrix proteins to migrate efficiently.

T cells Use Focal Adhesions to Pull Themselves Through Confined Environments.

Immune cells are highly dynamic and able to migrate through environments with diverse biochemical and mechanical composition. Their migration has classically been defined as amoeboid under the assumption that it is integrin-independent. Here we show that activated primary Th1 T cells require both confinement and extracellular matrix protein to migrate efficiently.

Ccr2+ Monocyte-Derived Macrophages Influence Trajectories of Acquired Therapy Resistance in Braf-Mutant Melanoma.

Unlabelled: Therapies targeting oncogene addiction have had a tremendous impact on tumor growth and patient outcome, but drug resistance continues to be problematic. One approach to deal with the challenge of resistance entails extending anticancer treatments beyond targeting cancer cells by additionally altering the tumor microenvironment. Understanding how the tumor microenvironment contributes to the evolution of diverse resistance pathways could aid in the design of sequential treatments that can elicit and take advantage of a predictable resistance trajectory.

IL-17-Dependent Dysregulated Cutaneous Immune Homeostasis in the Absence of the Wiskott-Aldrich Syndrome Protein.

Wiskott-Aldrich Syndrome (WAS) is characterized by recurrent infections, thrombocytopenia, and eczema. Here, we show that WASp-deficient mice on a BALB/c background have dysregulated cutaneous immune homeostasis with increased leukocyte accumulation in the skin, 1 week after birth. Increased cutaneous inflammation was associated with epithelial abnormalities, namely, altered keratinization, abnormal epidermal tight junctional morphology and increased trans-epidermal water loss; consistent with epidermal barrier dysfunction.

T cell activation niches-Optimizing T cell effector function in inflamed and infected tissues.

Successful immunity to infection, malignancy, and tissue damage requires the coordinated recruitment of numerous immune cell subsets to target tissues. Once within the target tissue, effector T cells rely on local chemotactic cues and structural cues from the tissue matrix to navigate the tissue, interact with antigen-presenting cells, and release effector cytokines. This highly dynamic process has been "caught on camera" in situ by intravital multiphoton imaging.

CXCL10 peripheral activation niches couple preferred sites of Th1 entry with optimal APC encounter.

Correct positioning of T cells within infected tissues is critical for T cell activation and pathogen control. Upon tissue entry, effector T cells must efficiently locate antigen-presenting cells (APC) for peripheral activation. We reveal that tissue entry and initial peripheral activation of Th1 effector T cells are tightly linked to perivascular positioning of chemokine-expressing APCs.

Hyperspectral multiphoton microscopy for visualization of multiple, spectrally overlapped fluorescent labels.

The insensitivity of multiphoton microscopy to optical scattering enables high-resolution, high-contrast imaging deep into tissue, including in live animals. Scattering does, however, severely limit the use of spectral dispersion techniques to improve spectral resolution. In practice, this limited spectral resolution together with the need for multiple excitation wavelengths to excite different fluorophores limits multiphoton microscopy to imaging a few, spectrally-distinct fluorescent labels at a time, restricting the complexity of biological processes that can be studied.

The spatio-temporal control of effector T cell migration.

Effector T cells leave the lymph nodes armed with specialized functional attributes. Their antigenic targets may be located anywhere in the body, posing the ultimate challenge: how to efficiently identify the target tissue, navigate through a complex tissue matrix and, ultimately, locate the immunological insult. Recent advances in real-time in situ imaging of effector T cell migratory behaviour have revealed a great degree of mechanistic plasticity that enables effector T cells to push and squeeze their way through inflamed tissues.

CD4 T Cell Interstitial Migration Controlled by Fibronectin in the Inflamed Skin.

The extracellular matrix (ECM) is extensively remodeled during inflammation providing essential guidance cues for immune cell migration and signals for cell activation and survival. There is increasing interest in the therapeutic targeting of ECM to mitigate chronic inflammatory diseases and enhance access to the tumor microenvironment. T cells utilize the ECM as a scaffold for interstitial migration, dependent on T cell expression of matrix-binding integrins αβ/αβ and tissue display of the respective RGD-containing ligands.

In situ neutrophil efferocytosis shapes T cell immunity to influenza infection.

Early recruitment of neutrophils from the blood to sites of tissue infection is a hallmark of innate immune responses. However, little is known about the mechanisms by which apoptotic neutrophils are cleared in infected tissues during resolution and the immunological consequences of in situ efferocytosis. Using intravital multiphoton microscopy, we show previously unrecognized motility patterns of interactions between neutrophils and tissue-resident phagocytes within the influenza-infected mouse airway.

Programming of Distinct Chemokine-Dependent and -Independent Search Strategies for Th1 and Th2 Cells Optimizes Function at Inflamed Sites.

T-helper (Th) cell differentiation drives specialized gene programs that dictate effector T cell function at sites of infection. Here, we have shown Th cell differentiation also imposes discrete motility gene programs that shape Th1 and Th2 cell navigation of the inflamed dermis. Th1 cells scanned a smaller tissue area in a G protein-coupled receptor (GPCR) and chemokine-dependent fashion, while Th2 cells scanned a larger tissue area independent of GPCR signals.

Pivotal role for α integrins in sustained Tfh support of the germinal center response for long-lived plasma cell generation.

CD4+ follicular helper T cells (Tfh) are essential for germinal center (GC) reactions in the lymph node that generate high-affinity, long-lived plasma cells (LLPCs). Temporal GC analysis suggests B memory cells (Bmem) are generated early, while LLPCs are generated late in the GC reaction. Distinct roles for Tfh at these temporally different stages are not yet clear.

CCL7 Is a Negative Regulator of Cutaneous Inflammation Following Infection.

The chemokine CCL7 (MCP3) is known to promote the recruitment of many innate immune cell types including monocytes and neutrophils to sites of bacterial and viral infection and eosinophils and basophils to sites of allergic inflammation. CCL7 upregulation has been associated with many inflammatory settings including infection, cardiovascular disease, and the tumor microenvironment. CCL7's pleotropic effects are due in part to its ability to bind numerous chemokine receptors, namely CCR1, CCR2, CCR3, CCR5, and CCR10.

Innate Immune Cells Are Regulated by Axl in Hypertensive Kidney.

The balance between adaptive and innate immunity in kidney damage in salt-dependent hypertension is unclear. We investigated early renal dysfunction and the influence of Axl, a receptor tyrosine kinase, on innate immune response in hypertensive kidney in mice with lymphocyte deficiency (Rag1). The data suggest that increased presence of CD11b myeloid cells in the medulla might explain intensified salt and water retention as well as initial hypertensive response in Rag1 mice.

CCR7 fuels and LFA-1 grips.

Naïve T cells migrate rapidly through the lymph node. A high-resolution look at the chemokine receptor CCR7 and integrin LFA-1 reveals T cells remain highly responsive to their microenvironment by instantaneous tuning of chemokine-regulated actin flow and integrin-regulated adhesion.

T Cell Interstitial Migration: Motility Cues from the Inflamed Tissue for Micro- and Macro-Positioning.

Effector T cells exit the inflamed vasculature into an environment shaped by tissue-specific structural configurations and inflammation-imposed extrinsic modifications. Once within interstitial spaces of non-lymphoid tissues, T cells migrate in an apparent random, non-directional, fashion. Efficient T cell scanning of the tissue environment is essential for successful location of infected target cells or encounter with antigen-presenting cells that activate the T cell's antimicrobial effector functions.

The Integrin LFA-1 Controls T Follicular Helper Cell Generation and Maintenance.

T follicular helper (Tfh) cells are a CD4 T cell subset critical for long-lived humoral immunity. We hypothesized that integrins play a decisive role in Tfh cell biology. Here we show that Tfh cells expressed a highly active form of leukocyte function-associated antigen-1 (LFA-1) that was required for their survival within the germinal center niche.

Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen.

During a primary influenza infection, cytotoxic CD8+ T cells need to infiltrate the infected airways and engage virus-infected epithelial cells. The factors that regulate T cell motility in the infected airway tissue are not well known. To more precisely study T cell infiltration of the airways, we developed an experimental model system using the trachea as a site where live imaging can be performed.

Regulatory T Cell Numbers in Inflamed Skin Are Controlled by Local Inflammatory Cues That Upregulate CD25 and Facilitate Antigen-Driven Local Proliferation.

CD4(+)Foxp3(+) regulatory T cells (Tregs) are key immune suppressors that regulate immunity in diverse tissues. The tissue and/or inflammatory signals that influence the magnitude of the Treg response remain unclear. To define signals that promote Treg accumulation, we developed a simple system of skin inflammation using defined Ags and adjuvants that induce distinct cytokine milieus: OVA protein in CFA, aluminum salts (Alum), and Schistosoma mansoni eggs (Sm Egg).

Role of Axl in T-Lymphocyte Survival in Salt-Dependent Hypertension.

Objective: Survival of immune and nonimmune cells relies on Axl, a receptor tyrosine kinase, which is implicated in hypertension. Activated T lymphocytes are involved in regulation of high blood pressure. The goal of the study was to investigate the role of Axl in T-lymphocyte functions and its contribution to salt-dependent hypertension.

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis.

Preterm cord blood CD4⁺ T cells exhibit increased IL-6 production in chorioamnionitis and decreased CD4⁺ T cells in bronchopulmonary dysplasia.

Background: Chorioamnionitis (CA) is associated with premature delivery and bronchopulmonary dysplasia (BPD). We hypothesize that preterm infants exposed to CA have reduced suppressive regulatory T cells (Treg) and increased non-regulatory T cell pro-inflammatory cytokines, increasing risk for BPD.

Objective: To evaluate cord blood CD4(+) T cell regulatory phenotype and pro-inflammatory cytokine production in CA and BPD groups.

Platelet factor 4 limits Th17 differentiation and cardiac allograft rejection.

Th cells are the major effector cells in transplant rejection and can be divided into Th1, Th2, Th17, and Treg subsets. Th differentiation is controlled by transcription factor expression, which is driven by positive and negative cytokine and chemokine stimuli at the time of T cell activation. Here we discovered that chemokine platelet factor 4 (PF4) is a negative regulator of Th17 differentiation.