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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. Initial studies revealed a surprising randomness to the pattern of T cell migration through inflamed tissues, behavior thought to facilitate chance encounters with rare antigen-bearing cells. Subsequent tissue-wide visualization has uncovered a high degree of spatial preference when it comes to T cell activation. Here, we discuss the basic tenants of a successful effector T cell activation niche, taking cues from the dynamics of Tfh positioning in the lymph node germinal center. In peripheral tissues, steady-state microanatomical organization may direct the location of "pop-up" de novo activation niches, often observed as perivascular clusters, that support early effector T cell activation. These perivascular activation niches appear to be regulated by site-specific chemokines that coordinate the recruitment of dendritic cells and other innate cells for local T cell activation, survival, and optimized effector function.

Summary: 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. Dermal inflammation induces tissue-wide de novo generation of discrete perivascular CXCL10+ cell clusters, enriched for CD11c+MHC-II+ monocyte-derived dendritic cells. These chemokine clusters are “hotspots” for both Th1 extravasation and activation in the inflamed skin. CXCR3-dependent Th1 localization to the cluster micro-environment prolongs T-APC interactions and boosts function. Both the frequency and range of these clusters are enhanced via a T helper 1 (Th1)-intrinsic, interferon-gamma (IFNγ)-dependent positive-feedback loop. Thus, the perivascular CXCL10+ clusters act as initial peripheral activation niches, optimizing controlled activation broadly throughout the tissue by coupling Th1 tissue entry with enhanced opportunities for Th1-APC encounter.

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. Tfh entry into the GC is highly dynamic and the signals that maintain Tfh within the GC for support of late LLPC production are poorly understood. The GC is marked by inflammation-induced presentation of specific ECM components. To determine if T cell recognition of these ECM components played a role in Tfh support of the GC, we immunized mice with a T cell-restricted deletion of the ECM-binding integrin αV (αV-CD4 cKO). T cell integrin αV deletion led to a striking defect in the number and size of the GCs following immunization with OVA protein in complete Freund's adjuvant. The GC defect was not due to integrin αV deficiency impeding Tfh generation or follicle entry or the ability of αV-CD4 cKO Tfh to contact and support B cell activation. Instead, integrin αV was essential for T cell-intrinsic accumulation within the GC. Altered Tfh positioning resulted in lower-affinity antibodies and a dramatic loss of LLPCs. Influenza A infection revealed that αV integrin was not required for Tfh support of Bmem but was essential for Tfh support of LLPCs. We highlight an αV integrin-ECM-guided mechanism of Tfh GC accumulation that selectively impacts GC output of LLPCs but not Bmem.

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. Here, we demonstrate a hyperspectral multiphoton microscope that utilizes three different wavelength excitation sources together with multiplexed fluorescence emission detection using angle-tuned bandpass filters. This microscope maintains scattering insensitivity, while providing high enough spectral resolution on the emitted fluorescence and capitalizing on the wavelength-dependent nonlinear excitation of fluorescent dyes to enable clean separation of multiple, spectrally overlapping labels, in vivo. We demonstrated the utility of this instrument for spectral separation of closely-overlapped fluorophores in samples containing ten different colors of fluorescent beads, live cells expressing up to seven different fluorescent protein fusion constructs, and in multiple in vivo preparations in mouse cortex and inflamed skin with up to eight different cell types or tissue structures distinguished.

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. This process is shaped by an array of ‘stop’ and ‘go’ guidance signals including target antigens, chemokines, integrin ligands and the mechanical cues of the inflamed microenvironment. Effector T cells must sense and interpret these competing signals to correctly position themselves to mediate their effector functions for complete and durable responses in infectious disease and malignancy. Tuning T cell migration therapeutically will require a new understanding of this complex decision-making process. In this Review, Deborah Fowell and Minsoo Kim highlight the complexity of the biochemical and mechanical cues that facilitate T cell migration. They explain how effector T cells are able to use these cues to navigate through complex tissue environments to respond to pathogens and other immunological challenges.

T follicular helper cells (Tfh) play a critical role in supplying help to B cells for the selection of high-affinity B cells in germinal centers (GC), that give rise to memory B cells (Bmem) and long-lived plasma cells (LLPCs). The products of the GC reaction appear to be temporally distinct, with Bmem cells produced early and LLPCs late. The temporal requirements for Tfh help are not well understood and we still do not know when and for how long this help is critical. In the total absence of help supplied by Tfh cells, both GCs and LLPCs are greatly diminished. Our previous work (Schrock, Leddon et al 2019, PNAS) suggested that sustained GC Tfh were critical for LLPC generation, but interestingly not for Bmem generation. We hypothesize that a temporal requirement for GC Tfh regulates LLPC versus Bmem GC output. To test this, we have established a number of models that allow for deletion of Tfh at different times post OVA/CFA immunization. To specifically target Tfh cells, CD4 iCRE Bcl6fl/fl mice were used for time-dependent deletion of Bcl6 upon tamoxifen administration at different stages of the GC reaction. Tfh and GC output are determined using multicolor Flow Cytometry panels, BM B cell Elispot for LLPCs and a Bmem Elispot. With these systems in place, we aim to pinpoint the temporal requirement for Tfh for both Bmem and LLPCs, speculating that timing of Tfh help will differ for these two arms of the GC response. Understanding this temporal requirement of Tfh help within the GC can not only help us to better design future vaccines that promote LLPCs, but also guide the development of drugs to modulate Tfh longevity in autoimmune disorders, where aberrant Tfh development or maintenance contribute to pathology.

Pathogen control requires T-cells to locate and make direct contact with antigen-presenting cells (APCs) for peripheral reactivation and delivery of effector molecules. The signals that orchestrate T-cell-APC interactions are poorly understood in inflamed peripheral tissues. Using intravital imaging (IVI), we have found that Th1 cell entry into the inflamed dermis is nucleated by perivascular CXCL10+ cellular clusters enriched for APCs. Th1 cells form more stable interactions with APCs within these clusters compared to outer regions. CXCL10+ clusters are amplified in a IFNγ-dependent manner, where CD4+ T-cell IFNγ release increases APC recruitment and CXCL10 expression. We hypothesize that these chemokine clusters serve as CXCL10 Peripheral Activation (PAC-10) niches, where Th1 cells achieve early peripheral activation away from the pathogen modulated milieu. We seek to determine the relationship between these spatially restricted PAC-10 niches, infection foci, and pathogen clearance. Using a combination of IVI, flow cytometry, and transcriptomics we characterized niche specific Th1 cells and niche cellular composition to determine if PAC-10 niches modulate Th1 cells for pathogen control. Initial studies reveal that niche specific Th1 cells have increased CD69 and IFNγ expression compared to non-niche counterparts. PAC-10 niches appear to have a spatially distinct innate cell population, likely with enhanced ability to support Th1 activation. Future studies will characterize the functional status of niche specific Th1 cells and evaluate the necessity of PAC-10 niches for pathogen control. These studies will inform therapeutic strategies to boost T-cell activation in infection or hinder activation in autoimmunity. Supported by NIH NIAID P01 AI02851 NIH NIAID R01 AI070826

SUMMARYCorrect 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. Dermal inflammation induced tissue-wide de novo generation of discrete perivascular CXCL10+ cell clusters, enriched for CD11c+MHC-II+ monocyte-derived dendritic cells. These chemokine clusters were ‘hot spots’ for both Th1 extravasation and activation in the inflamed skin. CXCR3-dependent Th1 localization to the cluster micro-environment prolonged T-APC interactions and boosted function. Both the frequency and range of these clusters were enhanced via a Th1-intrinsic, IFNγ-dependent positive feedback loop. Thus, the perivascular CXCL10+ clusters act as initial peripheral activation niches, optimizing controlled activation broadly throughout the tissue by coupling Th1 tissue entry with enhanced opportunities for Th1-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. Dermal inflammation induces tissue-wide de novo generation of discrete perivascular CXCL10

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

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. Newly infiltrated inflammatory monocytes become a chief pool of phagocytes and play a key role in the clearance of highly motile apoptotic neutrophils during the resolution phase. Apoptotic neutrophils further release epidermal growth factor and promote the differentiation of monocytes into tissue-resident antigen-presenting cells for activation of antiviral T cell effector functions. Collectively, these results suggest that the presence of in situ neutrophil resolution at the infected tissue is critical for optimal CD8+ T cell–mediated immune protection. Kim and colleagues provide new insights into the function and fate of neutrophils during influenza infection and their roles in antiviral T cell responses.

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. Tfh entry into the GC is highly dynamic and the signals that maintain Tfh within the GC for support of late LLPC production are poorly understood. The GC is marked by inflammation-induced presentation of specific ECM components. To determine if T cell recognition of these ECM components played a role in Tfh support of the GC, we immunized mice with a T cell-restricted deletion of the ECM-binding integrin α(V) (α(V)-CD4 cKO). T cell integrin α(V) deletion led to a striking defect in the number and size of the GCs following immunization with OVA protein in complete Freund’s adjuvant. The GC defect was not due to integrin α(V) deficiency impeding Tfh generation or follicle entry or the ability of α(V)-CD4 cKO Tfh to contact and support B cell activation. Instead, integrin α(V) was essential for T cell-intrinsic accumulation within the GC. Altered Tfh positioning resulted in lower-affinity antibodies and a dramatic loss of LLPCs. Influenza A infection revealed that α(V) integrin was not required for Tfh support of Bmem but was essential for Tfh support of LLPCs. We highlight an α(V) integrin–ECM-guided mechanism of Tfh GC accumulation that selectively impacts GC output of LLPCs but not Bmem.

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. CCL7-blockade or CCL7-deficiency is often marked by decreased inflammation and poor pathogen control. In the context of Leishmania major infection, CCL7 is specifically upregulated in the skin one-2 weeks after infection but its role in L. major control is unclear. To determine CCL7's impact on the response to L. major we infected WT and CCL7−/− C57BL/6 mice. L. major infection of CCL7-deficient mice led to an unexpected increase in inflammation in the infected skin 2 weeks post-infection. A broad increase in immune cell subsets was observed but was dominated by enhanced neutrophilic infiltration. Increased neutrophil recruitment was associated with an enhanced IL-17 gene profile in the infected skin. CCL7 was shown to directly antagonize neutrophil migration in vitro and CCL7 add-back in vivo specifically reduced neutrophil influx into the infected skin revealing an unexpected role for CCL7 in limiting neutrophil recruitment during L. major infection. Enhanced neutrophilic infiltration in CCL7-deficient mice changed the balance of L. major infected host cells with an increase in the ratio of infected neutrophils over monocytes/macrophages. To determine the consequence of CCL7 deficiency on L. major control we analyzed parasite load cutaneously at the site of infection and viscerally in the draining LN and spleen. The CCL7−/− mice supported robust cutaneous parasite control similar to their WT C57BL/6 counterparts. In contrast, CCL7-deficiency led to greater parasite dissemination and poor parasite control in the spleen. Our studies reveal a novel role for CCL7 in negatively regulating cutaneous inflammation, specifically neutrophils, early during L. major infection. We propose that CCL7-mediated dampening of the early immune response in the skin may limit the ability of the parasite to disseminate without compromising cutaneous control.

We constructed a hyperspectral multiphoton microscope (HMM) that enabled high spectral-resolution imaging deep into scattering samples and used this instrument for in vivo visualization of the behavior of multiple cell types after an injury.

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. In addition, LFA-1 promoted expression of Bcl-6, a transcriptional repressor critical for Tfh cell differentiation, and inhibition of LFA-1 abolished Tfh cell generation and prevented protective humoral immunity to intestinal helminth infection. Furthermore, we demonstrated that expression of Talin-1, an adaptor protein that regulates LFA-1 affinity, dictated Tfh versus Th2 effector cell differentiation. Collectively, our results define unique functions for LFA-1 in the Tfh cell effector program and suggest that integrin activity is important in lineage decision-making events in the adaptive immune system.

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). Polyclonal and Ag-specific Treg accumulation in the skin differed significantly between adjuvants. CFA and Alum led to robust Treg accumulation, with >50% of all skin CD4+ T cells being Foxp3+. In contrast, Tregs accumulated poorly in the Sm Egg–inflamed skin. Surprisingly, we found no evidence of inflammation-specific changes to the Treg gene program between adjuvant-inflamed skin types, suggesting a lack of selective recruitment or adaptation to the inflammatory milieu. Instead, Treg accumulation patterns were linked to differences in CD80/CD86 expression by APC and the regulation of CD25 expression, specifically in the inflamed skin. Inflammatory cues alone, without cognate Ag, differentially supported CD25 upregulation (CFA and Alum > Sm Egg). Only in inflammatory milieus that upregulated CD25 did the provision of Ag enhance local Treg proliferation. Reduced IL-33 in the Sm Egg–inflamed environment was shown to contribute to the failure to upregulate CD25. Thus, the magnitude of the Treg response in inflamed tissues is controlled at two interdependent levels: inflammatory signals that support the upregulation of the important Treg survival factor CD25 and Ag signals that drive local expansion.

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. Here, we demonstrate a hyperspectral multiphoton microscope that utilizes three different wavelength excitation sources together with multiplexed fluorescence emission detection using angle-tuned bandpass filters. This microscope maintains scattering insensitivity, while providing high enough spectral resolution on the emitted fluorescence and capitalizing on the wavelength-dependent nonlinear excitation of fluorescent dyes to enable clean separation of multiple, spectrally overlapping labels, in vivo. We demonstrated the utility of this instrument for spectral separation of closely overlapped fluorophores in samples containing 10 different colors of fluorescent beads, live cells expressing up to seven different fluorescent protein fusion constructs, and in multiple in vivo preparations in mouse cortex and inflamed skin, with up to eight different cell types or tissue structures distinguished.

The inappropriate trafficking and retention of immune subsets in tissue is a hallmark of many diseases prevalent in the developed world. These issues include autoimmunity, chronic inflammation, and unbalanced tissue repair responses which represent a significant disease burden: diabetes, coronary artery disease, inflammatory bowel disease, hepatitis, rheumatoid arthritis, and asthma. Although knowledge about cell trafficking to inflamed sites has grown in the past decade, there is a current gap in knowledge in identifying when and how immune cells decide to stay or leave these tissues. To determine phenotypic and functional differences between those Th1 cells that remain at the inflamed site and those that exit, we employed the photoconvertible Kaede protein system to enable the labelling of Th1 cells in time and space. Kaede+OT-II Th1 T cells were adoptively transferred into WT mice immunized with CFA/OVA. Th1 cells recruited to the inflamed ear skin were photoconverted from Kaede green to Kaede red. Kaede red Th1 cells retained at the inflamed site could be distinguished from newly recruited Kaede green cells, while Kaede red Th1 cells that subsequently exited the inflamed skin were readily detected in the draining lymph node. Using this system, we are able to analyze the phenotypic (multicolor flow cytometry) and transcriptional signatures (RNA-seq) of Th1 cells that are retained at the inflamed site and those Th1 cells that exit (tissue-migrants). Given the temporal component of our model, we are gaining new insight into when and where expression of conventional markers of trafficking and activation are regulated (CD62L, CD44, and CD69) and seek to identify new pathways that dictate T cell retention and egress at inflammatory sites.

Efficient recruitment and correct positioning of T cells within infected peripheral tissues are crucial for pathogen clearance. Using a dual-chemokine reporter (Groom et Al. Immunity, 2012) mouse and intra-vital imaging, we identified a preferred tissue entry and accumulation site for effector Th1 cells, defined by clusters of CXCL9 and CXCL10 expressing hematopoietic cells, enriched with MHC-class-II+ cells, in restricted perivascular spaces within the inflamed dermis. Unbiased computational analysis of motility dynamics of antigen-specific Th1 cells within these clusters, revealed cell confinement characteristics. CXCR3 on Th1 cells and presence of cognate antigen were critical for persistence within clusters and T cell activation. Newly recruited Th1 cells strongly amplified CXCL9 and CXCL10 expression, mainly within skin monocyte-derived dendritic cells, in an antigen- and interferon-gamma-dependent manner, resulting in augmented chemokine clusters. Our data suggest a CXCR3-mediated mechanism of intrinsic amplification loop for optimal Th1 cell recruitment, positioning and activation in restricted antigen presentation sites within the inflamed skin.

Chronic inflammatory diseases such as Crohn’s disease and rheumatoid arthritis affect millions of people worldwide. During an inflammatory response, immune cells enter the inflamed tissue to mediate pathogen clearance, however, their activation and overaccumulation in the tissue can lead to chronic inflammation and autoimmunity. Little is known about the signals that regulate immune cell entry into and exit from these inflamed tissues or about the retention cues that play a role in their accumulation. Therefore, a better understanding of these signals would inform the design of therapeutics intended to alleviate chronic inflammation. To study immune cell movement between a site of inflammation and lymphoid organs, our lab has acquired a mouse model that allows for the in vivo labeling of a subset of cells in time and space. The cells of Kaede transgenic mice constitutively express the photoconvertible fluorescent Kaede protein. CFA/OVA-inflamed ears were photoconverted from Kaede green to red by violet light exposure and the trafficking of Kaede red cells out of the skin to the draining lymph node was assessed over the course of 24 hours. Based on preliminary data, we found that, of the subsets assessed, neutrophils and CD4+ T cells made up the largest percentages of immune cells exiting the inflamed tissue on day 5 of inflammation. On the other hand, far fewer conventional dendritic cells and macrophages were seen to enter the draining lymph node from the inflamed tissue. The role of these tissue-emigrant immune cells is not yet clear, but cells such as neutrophils may play a role in shaping adaptive immune responses in the draining lymph node.

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. Global deletion of Axl on Rag1(−/−) background reversed kidney dysfunction and accumulation of myeloid cells in the kidney medulla. Chimeric mice that lack Axl in innate immune cells (in the absence of lymphocytes) significantly improved kidney function and abolished early hypertensive response. The bioinformatics analyses of Axl-related gene-gene interaction networks established tissue-specific variation in regulatory pathways. It was confirmed that complement C3 is important for Axl-mediated interactions between myeloid and vascular cells in hypertensive kidney. In summary, innate immunity is crucial for renal dysfunction in early hypertension, and is highly influenced by the presence of Axl.

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. Differential chemokine reliance for interstitial migration was linked to STAT6 transcription factor-dependent programming of integrin α(V)β(3) expression: Th2 cell differentiation led to high α(V)β(3) expression relative to Th1 cells. Th1 and Th2 cell modes of motility could be switched simply by manipulating the amount of α(V)β(3) on the cell surface. Deviating motility modes from those established during differentiation impaired effector function. Thus, programmed expression of α(V)β(3) tunes effector T cell reliance on environmental cues for optimal exploration of inflamed tissues.

Intracellular pathogen clearance is dependent on successful T cell mediated immunity. Th1 cells must localize to the site of infection to be activated into IFN-γ producing effector T cells. How Th1 cells find and interact with antigen presenting cells (APC) in infected tissues is poorly understood. Preliminary data using intravital imaging and the REX3 mouse, in which CXCL9 and CXCL10 producing cells express red and blue fluorescent proteins (RFP/BFP) respectively, suggest that CXCL9/10-producing cells form dense perivascular clusters that attract and arrest CXCR3+ Th1 cells. We hypothesize that these CXCL10+ cell clusters are enriched for APC that optimize Th1 cell activation and IFN-γ production. To determine the phenotype of the immune cells within the chemokine-producing clusters we crossed the REX3 mouse with a photoactivatable green fluorescent protein (PA-GFP) mouse. This will enable selective labeling of cells within chemokine-rich clusters prior to flow cytometry. Using precise multiphoton delivery of 830 nm light, we have successfully photoactivated the CXCL10+ clustered regions in the inflamed ear and have developed a flow cytometry panel to distinguish between macrophages, cDCs, and monocyte-derived cells that may be positioned within or outside these chemokine-clusters. Additionally, we will determine if these cell clusters enhance Th1 function by sorting photoactivated (in clusters) and non-photoactivated (outside clusters) Th1 cells from the inflamed ear and analyzing IFN-γ expression. Understanding how Th1 cells are positioned and activated at sites of inflammation will allow the field to boost responses to infectious agents and therapeutically attenuate Th1 responses in autoimmunity.

Leukocytes must traverse inflamed tissues to effectively control local infection. Although motility in dense tissues seems to be integrin independent and based on actomyosin-mediated protrusion and contraction, during inflammation, changes to the extracellular matrix (ECM) may necessitate distinct motility requirements. Indeed, we found that the interstitial motility of T cells was critically dependent on Arg-Gly-Asp (RGD)-binding integrins in the inflamed dermis. Inflammation-induced deposition of fibronectin was functionally linked to higher expression of integrin αV on effector CD4⁺ T cells. By intravital multiphoton imaging, we found that the motility of CD4⁺ T cells was dependent on αV expression. Selective blockade or knockdown of αV arrested T helper type 1 (TH1) cells in the inflamed tissue and attenuated local effector function. Our data demonstrate context-dependent specificity of lymphocyte movement in inflamed tissues that is essential for protective immunity.

The Gas6/Axl pathway regulates many cell functions and is implicated in hypertension. In this study, we aimed to investigate the role of Axl in immune cells on initiation and progression of salt-dependent hypertension. Deoxycorticosterone acetate (75 mg/60 days release)–salt hypertension was induced for 1 week or 6 weeks in Axl chimeras generated by bone marrow transplant to restrict Axl deficiency to hematopoietic or nonhematopoietic compartments. Depletion of Axl in hematopoietic cells (Axl −/− →Axl +/+ ) reduced (133±2 mm Hg) increase in systolic blood pressure compared with other Axl chimeras (≈150 mm Hg) 1 week after deoxycorticosterone acetate–salt. Urine protein and renal oxidative stress were lowest in Axl −/− →Axl +/+ at 1 week after deoxycorticosterone acetate–salt. Compensatory increase in Gas6 in kidneys of recipient Axl −/− may affect kidney function and blood pressure in early phase of hypertension. Flow cytometry on kidneys from Axl −/− →Axl +/+ showed increase in total leukocytes, B, and dendritic cells and decrease in macrophages compared with Axl +/+ →Axl +/+ . These immune changes were associated with decrease in proinflammatory gene expression, in particular interferon γ. Systolic blood pressure returned to baseline in Axl −/− →Axl +/+ and Axl −/− →Axl −/− but remained increased in Axl +/+ →Axl +/+ and Axl +/+ →Axl −/− chimeras after 6 weeks of deoxycorticosterone acetate–salt. Vascular apoptosis was increased in the global Axl −/− chimeras in the late phase of hypertension. In summary, we found that expression of Axl in hematopoietic cells is critical for kidney pathology in early phase of salt-dependent hypertension. However, Axl in both hematopoietic and nonhematopoietic lineages contributes to the late phase of hypertension.

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 mechanisms of interstitial T cell motility and the environmental cues that may promote or hinder efficient tissue scanning are poorly understood. The extracellular matrix (ECM) appears to play an important scaffolding role in guidance of T cell migration and likely provides a platform for the display of chemotactic factors that may help to direct the positioning of T cells. Here, we discuss how intra-vital imaging has provided insight into the motility patterns and cellular machinery that facilitates T cell interstitial migration and the critical environmental factors that may optimize the efficiency of effector T cell scanning of the inflamed tissue. Specifically, we highlight the local micro-positioning cues T cells encounter as they migrate within inflamed tissues, from surrounding ECM and signaling molecules, as well as a requirement for appropriate long-range macro-positioning within distinct tissue compartments or at discrete foci of infection or tissue damage. The Central Nervous System (CNS) responds to injury and infection by extensively remodeling the extracellular matrix and with the de novo generation of a fibroblastic reticular network that likely influences T cell motility. We examine how inflammation-induced changes to the CNS landscape may regulate T cell tissue exploration and modulate function.

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. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

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. Approach and Results— We report increased apoptosis in peripheral blood from Axl −/− mice because of lower numbers of white blood cells mostly lymphocytes. In vitro studies showed modest reduction in interferon gamma production in Axl −/− type 1 T helper cells. Axl did not affect basic proliferation capacity or production of interleukin 4 in Axl −/− type 2 T helper cells. However, competitive repopulation of Axl −/− bone marrow or adoptive transfer of Axl −/− CD4 + T cells to Rag1 −/− mice showed robust effect of Axl on T lymphocyte expansion in vivo. Adoptive transfer of Axl −/− CD4 + T cells was protective in a later phase of deoxycorticosterone-acetate and salt hypertension. Reduced numbers of CD4 + T cells in circulation and in perivascular adventitia decreased vascular remodeling and increased vascular apoptosis in the late phase of hypertension. Conclusions— These findings suggest that Axl is critical for survival of T lymphocytes, especially during vascular remodeling in hypertension.

Robust pathogen clearance by T cells requires three critical and interdependent steps: the generation of functionally specialized effector T cells in the lymph node (LN); the localization of those effector T cells to, and within, the infected tissue; and the local delivery of signals for T cell activation of effector function. Activation of T cells in the LN leads to the expansion of pathogen-specific T cells and their differentiation into functionally specialized effector cells with the potential for antimicrobial activity. Whether these effector T cells successfully execute their effector function in the infected tissue is contingent on the local balance of signals that support or attenuate function at the infection site. These signals are context-dependent and influenced by the target tissue itself, the type of pathogen, and the type of inflammatory milieu. Signals that enhance effector function facilitate more efficient sensing and elimination of the pathogen, while signals that attenuate function may compromise on the efficiency of pathogen clearance but prevent immune-mediated tissue damage. The outcome of infection is dependent on finely balancing these opposing immune modulators. In this article we outline the mechanisms whereby microenvironmental signals tune effector function at the infection site.

CD4 + CD25 + Forkhead box P3 (Foxp3) + regulatory T cells (Tregs) control immune responses to self and foreign antigens in secondary lymphoid organs and at tissue sites of inflammation. Tregs can modify the function of many immune cells and have been proposed to block early proliferation, differentiation, and effector function. Acute ablation of Tregs has revealed rapid cytokine production immediately after Treg removal, suggesting that Tregs may regulate effector function acutely rather than regulating the programming for immune function. We developed in vitro and in vivo models that enabled the direct test of Treg regulation of T-helper cell type 1 (Th1) differentiation. CD28 signaling is known to abrogate Treg suppression of IL-2 secretion and proliferation, but our studies show that Treg suppression of IFN-γ during Th1 priming proceeds despite enhanced CD28 signaling. Importantly, during Th1 differentiation, Tregs inhibited early IFN-γ transcription without disrupting expression of Th1-specific T-box transcription factor (Tbet) and Th1 programming. Acute shutoff of effector cytokine production by Tregs was selective for IFN-γ but not TNF-α and was independent of TGF-β and Epstein-Barr virus-induced gene 3. In vivo, Tregs potently controlled CD4 IFN-γ and CD4 effector cell expansion in the lymph node (four- to fivefold reduction) but not Th1 programming, independent of IL-10. Tregs additionally reduced CD4 IFN-γ in the inflamed dermis (twofold reduction) dependent on their production of IL-10. We propose a model for Treg inhibition of effector function based on acute cytokine regulation. Interestingly, Tregs used different regulatory mechanisms to regulate IFN-γ (IL-10–dependent or –independent) subject to the target T-cell stage of activation and its tissue location.

OBJECTIVE The mechanistic basis for the breakdown of T-cell tolerance in type 1 diabetes is unclear and could result from a gain of effector function and/or loss of regulatory function. In humans, the CD4+CD25+Foxp3+ T–cell compartment contains both effector and regulatory T cells, and it is not known how their relative proportions vary in disease states. RESEARCH DESIGN AND METHODS We performed a longitudinal study of CD4+CD25+ T–cell function in children with type 1 diabetes at onset and throughout the 1st year of disease. Function was assessed using single-cell assays of proliferation, cytokine production, and suppression. Type 1 diabetic individuals were compared with age-matched control subjects, and suppression was directly assessed by coculture with control T–cell targets. RESULTS We identify novel functional changes within the type 1 diabetes CD4+CD25+ compartment. Type 1 diabetic CD4+CD25+ cells exhibited a striking increase in proliferative capacity in coculture with CD4 T cells that was present at onset and stable 9–12 months from diagnosis. Elevated type 1 diabetes CD4+CD25+ cell proliferation correlated with increased inflammatory cytokines interleukin 17 and tumor necrosis factor-α but not γ-interferon. Type 1 diabetes CD4+CD25+ cytokine production occurred coincident with suppression of the same cytokines in the control targets. Indeed, enhanced proliferation/cytokines by CD4+CD25+ cells was uncoupled from their suppressive ability. Longitudinally, we observed a transient defect in type 1 diabetes CD4+CD25+ suppression that unexpectedly correlated with measures of improved metabolic function. CONCLUSIONS Type 1 diabetes onset, and its subsequent remission period, is associated with two independent functional changes within the CD4+CD25+ T–cell compartment: a stable increase in effector function and a transient decrease in regulatory T–cell suppression.

Patients with Wiskott-Aldrich syndrome (WAS) have numerous immune cell deficiencies, but it remains unclear how abnormalities in individual cell types contribute to the pathologies of WAS. In T cells, the WAS protein (WASp) regulates actin polymerization and transcription, and plays a role in the dynamics of the immunologic synapse. To examine how these events influence CD4 function, we isolated the WASp deficiency to CD4+ T cells by adoptive transfer into wild-type mice to study T-cell priming and effector function. WAS−/− CD4+ T cells mediated protective T-helper 1 (Th1) responses to Leishmania major in vivo, but were unable to support Th2 immunity to Nippostrongylus brasiliensis or L major. Mechanistically, WASp was not required for Th2 programming but was required for Th2 effector function. WAS−/− CD4+ T cells up-regulated IL-4 and GATA3 mRNA and secreted IL-4 protein during Th2 differentiation. In contrast, cytokine transcription was uncoupled from protein production in WAS−/− Th2-primed effectors. WAS−/− Th2s failed to produce IL-4 protein on restimulation despite elevated IL-4/GATA3 mRNA. Moreover, dominant-negative WASp expression in WT effector T cells blocked IL-4 production, but had no effect on IFNγ. Thus WASp plays a selective, posttranscriptional role in Th2 effector function.

CTLA-4 is constitutively expressed by CD4(+)CD25(+)Foxp3(+) Treg but its precise role in Treg function is not clear. Although blockade of CTLA-4 interferes with Treg function, studies using CTLA-4-deficient Treg have failed to reveal an essential requirement for CTLA-4 in Treg suppression in vivo. Conditional deletion of CTLA-4 in Foxp3(+) T cells disrupts immune homeostasis in vivo but the immune processes disrupted by CTLA-4 deletion have not been determined. We demonstrate that Treg expression of CTLA-4 is essential for Treg control of lymphopenia-induced CD4 T-cell expansion. Despite IL-10 expression, CTLA-4-deficient Treg were unable to control the expansion of CD4(+) target cells in a lymphopenic environment. Moreover, unlike their WT counterparts, CTLA-4-deficient Treg failed to inhibit cytokine production associated with homeostatic expansion and were unable to prevent colitis. Thus, while Treg developing in the absence of CTLA-4 appear to acquire some compensatory suppressive mechanisms in vitro, we identify a non-redundant role for CTLA-4 in Treg function in vivo.

Naturally-occurring regulatory T cells (Tregs) are emerging as key regulators of immune responses to self-tissues and infectious agents. Insight has been gained into the cell types and the cellular events that are regulated by Tregs. Indeed, Tregs have been implicated in the control of initial activation events, proliferation, differentiation and effector function. However, the mechanisms by which Tregs disable their cellular targets are not well understood. Here we review recent advances in the identification of distinct mechanisms of Treg action and of signals that enable cellular targets to escape regulation. Roles for inhibitory cytokines, cytotoxic molecules, modulators of cAMP and cytokine competition have all been demonstrated. The growing number of inhibitory mechanisms ascribed to Tregs suggests that Tregs take a multi-pronged approach to immune regulation. It is likely that the relative importance of each inhibitory mechanism is context dependent and modulated by the inflammatory milieu and the magnitude of the immune response. In addition, the target cell may be differentially susceptible or resistant to distinct Treg mechanisms depending on their activation or functional status at the time of the Treg encounter. Understanding when and where each suppressive tool is most effective will help to fine tune therapeutic strategies to promote or constrain specific arms of Treg suppression.

Introduction: Axl, a receptor tyrosine kinase, is required for vascular and immune cell survival. We sought to investigate the effects of Axl on T lymphocyte survival during deoxycorticosterone acetate (DOCA)-salt hypertension in mice. Methods and Results: We found significant reduction in systolic blood pressure (BP) after 5-6 weeks of DOCA-salt in RAG1-/- mice after adoptive transfer of CD4+ T cells from Axl knockout (Axl-/- →RAG1-/-) compared to transferred CD4+ T cells from wild type (Axl+/+ →RAG1-/-) mice. Media area of the mesenteric artery was significantly lower in Axl-/- →RAG1-/- (4.2±0.7x10 3 m 2 ) vs. Axl+/+ →RAG1-/- (6.0±0.9x10 3 m 2 ) or Axl+/+ (6.8±0.6x10 3 m 2 ) mice. There was significant decrease in interferon gamma production by the T cells from Axl-/- (396±23 ng/mL) compared to Axl+/+ (512±42 ng/mL) after T h 1-priming. The number of carboxyfluorescein succinimidyl ester-positive cells in 6 th division was dramatically declined in Axl-/- (~0.3%) vs. Axl+/+ (~1.8%) in culture. Accordingly, we found lower number of lymphocytes in blood from Axl-/- (4.5±0.7x10 9 ) compared to Axl+/+ (7.8±0.7x10 9 ) mice. Blood leukocyte apoptosis was 2.5-fold higher in Axl-/- mice. We next investigated repopulation capacities of the hematopoietic cells from Axl-/- vs. Axl+/+ mice. There was significant decrease in Axl-/- CD3+ T cells (21±3 %) than Axl+/+ (49±3 %) in spleen after 8 weeks of competitive repopulation of bone marrow-derived cells. However, we found even greater reduction of Axl-/- T lymphocytes (15±1 %) vs. Axl+/+ T lymphocytes (52±6 %) in peripheral blood after 8 weeks of competitive repopulation. Finally, percentage of apoptotic cells was the greatest in the media (20±7 %) and adventitia (13±5 %) from Axl-/- →RAG1-/- mice compared to vascular apoptosis (6-14 % in media; and 6-9 % in adventitia) in other groups after 6 weeks of DOCA-salt. Conclusions: Our data suggest that Axl-dependent survival of the T lymphocytes is crucial for the late increase in BP in DOCA-salt hypertension.

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. Study design Cord blood mononuclear cells from infants (GA ⩽32 weeks), with or without placental histological evidence of CA (hChorio), were analyzed by flow cytometry. Clinical information was collected by retrospective chart review. Numbers of putative Treg (CD4+FoxP3+CD25+CD127Dim), CD4+ non-Tregs, and CD4+ T cell intracellular cytokine content following in vitro stimulation were compared with CA status and oxygen requirement at 36 weeks postmenstrual age. Result Absolute Treg numbers were not different in CA and non-CA exposed samples. However, the infants who developed BPD had a significant decrease in Treg and non-regulatory T cell numbers. Greater IL-6 production was observed in hCA group. Conclusion A pro-inflammatory CD4+ T cell status is noted in CA and BPD but the later disease is also associated with decrease in Tregs, suggesting that the development of BPD is marked by distinct inflammatory changes from those of CA exposed infants.

CD73 (5′-ectonucleotidase) is expressed by two distinct mouse CD4 T cell populations: CD25+ (FoxP3+) T regulatory (Treg) cells that suppress T cell proliferation but do not secrete IL-2, and CD25− uncommitted primed precursor Th (Thpp) cells that secrete IL-2 but do not suppress in standard Treg suppressor assays. CD73 on both Treg and Thpp cells converted extracellular 5′-AMP to adenosine. Adenosine suppressed proliferation and cytokine secretion of Th1 and Th2 effector cells, even when target cells were activated by anti-CD3 and anti-CD28. This represents an additional suppressive mechanism of Treg cells and a previously unrecognized suppressive activity of Thpp cells. Infiltration of either Treg or Thpp cells at inflammatory sites could potentially convert 5′-AMP generated by neutrophils or dying cells into the anti-inflammatory mediator adenosine, thus dampening excessive immune reactions.

Introduction: Our laboratory has shown that Axl, a receptor tyrosine kinase, is important in both vascular and immune functions during deoxycorticosterone acetate (DOCA)-salt hypertension. We hypothesized that Axl activity specifically in T lymphocytes could explain the dependence of hypertension on Axl. Methods and Results: We did adoptive transfers of either Axl+/+ or Axl-/- CD4+ T cells to RAG1-/- mice that lack mature T cells. Once CD4+ T cell repopulations were confirmed, we induced DOCA-salt hypertension for 6 weeks. Systolic blood pressure (BP, mmHg) increased by 20±5 in Axl+/+RAG-/- mice after DOCA-salt, but Axl-/- RAG-/- mice had increases in BP by only 6+3 after 6 weeks of DOCA-salt. We isolated naïve CD4+ T cells from both Axl+/+ and Axl-/- littermates and primed them under either Th1 or Th2 polarizing conditions in culture. Production of interferon gamma (IFN-γ ng/mL) was significantly decreased (-23%, p9 ) compared to Axl+/+ mice (7.8±0.7x10 9 ). We found a higher population of AnnexinV (marker of early apoptosis)-positive peripheral leukocytes in Axl-/- mice (10±1%) compared to Axl+/+ (4±1%) by flow cytometry; while the percentages of dead cells (~10%) were similar between Axl+/+ and Axl-/- mice. Conclusions: Altogether we show that expression of Axl by T cells drives salt-induced hypertension. The mechanism of Axl-dependent effects on T cells occurs via T-cell-dependent expression of the pro-inflammatory cytokine IFN-γ. In addition, Axl plays a role in inhibiting lymphocyte apoptosis in the circulation. Future work will focus on how Axl expression in T cells affects T cell-dependent vascular remodeling during hypertension.

The extent of the diversity of T-cell responses is much larger than the original Th1/Th2 dichotomy, raising questions about the significance of the original sharply defined Th1 and Th2 phenotypes, the extent of diversity of T-cell cytokine patterns, the information used by the immune system to decide which type of response to induce, the signals that induce differentiation into these and other phenotypes in vivo, and the extent to which differentiation and continued differentiation occur during normal responses. The study of functional heterogeneity within the CD4+ T-cell population with regard to infectious disease was prompted by studies in the early 1970s on the development of humoral and cell-mediated immunity to pathogens and experimental antigens. In addition, IFN-γ stimulates the production of immunoglobulin G antibodies, which mediate opsonization and phagocytosis of particulate organisms. Th1 responses are also associated with viral infections; a direct role for CD4 cells in viral clearance has not been demonstrated for most viruses, but Th1 responses may support the expansion of CD8+ antiviral effectors. The authors expect that T cells expressing non- Th1, non-Th2 patterns may be essential for other diseases and that the crucial differences between the immune responses may be subtle. The requirement for multiple effector phenotypes is almost certainly due to the intensive interplay between the host response and each pathogen, driving the evolution of complex host defense strategies and equally complex evasion and interference tactics by the pathogen.

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. PF4-deficient and platelet-deficient mice had exaggerated immune responses to cardiac transplantation, including increased numbers of infiltrating Th17 cells and increased plasma IL-17. Although PF4 has been described as a platelet-specific molecule, we found that activated T cells also express PF4. Furthermore, bone marrow transplantation experiments revealed that T cell–derived PF4 contributes to a restriction in Th17 differentiation. Taken together, the results of this study demonstrate that PF4 is a key regulator of Th cell development that is necessary to limit Th17 differentiation. These data likely will impact our understanding of platelet-dependent regulation of T cell development, which is important in many diseases, in addition to transplantation.

CD134 (OX40) is a member of the TNF receptor family that is expressed on activated T lymphocytes. T cells from mice that lack expression of CD134 made strong responses to a range of challenges, but they showed impaired proliferation in response to direct stimulation through the TCR with monoclonal anti-CD3ε Ab. CD134-deficient mice controlled infection with Leishmania major, Nippostrongylus brasiliensis, and Theiler’s murine encephalomyelitis virus, and they made overtly normal Ab responses to a variety of antigens. Thus, CD134 is not essential for many T cell responses in vivo, nor is it required for the provision of help to B cells. Nonetheless, a subtle role in the regulation of T cell reactivity is suggested by the effect of CD134 deficiency on in vitro T cell responses.

Two rodent models of autoimmune type 1 diabetes have been used to investigate the role of insulin as an autoantigen in this disease. In lymphopoenia-induced diabetes in the PVG.RT1u rat, neonatal tolerization with insulin B-chain peptides, but not A-chain peptides, conferred significant protection from disease. After rechallenge of adult rats, neonatally B-chain-tolerized animals showed diminished B-chain-specific T-cell proliferation, interleukin (IL)-2 production, and interferon-gamma (IFN-gamma) production, as compared with control animals. The epitope recognized by the PVG.RT1u rat was mapped to residues 1-18 of the B-chain; T-cell lines specific for this epitope were generated, and these conferred diabetes upon adoptive transfer to irradiated syngeneic recipients. In adult nonobese diabetic (NOD) mice, subcutaneous immunization with B-chain peptide 9-23 emulsified in incomplete Freund's adjuvant (IFA) was also potent at preventing onset of diabetes. In contrast to PVG.RT1u rats, NOD mice recognized an epitope within residues 10-29 of the insulin B-chain. The data implicate insulin as a target autoantigen in type 1 diabetes but do not support a role for molecular mimicry to insulin in the pathogenesis of this disease.