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. 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.

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. Differential chemokine reliance for interstitial migration was linked to STAT6 transcription-factor-dependent programming of integrin αVß3 expression: Th2 cell differentiation led to high αVß3 expression relative to Th1 cells. Th1 and Th2 cell modes of motility could be switched simply by manipulating the amount of αVß3 on the cell surface. Deviating motility modes from those established during differentiation impaired effector function. Thus, programmed expression of αVß3 tunes effector T cell reliance on environmental cues for optimal exploration of inflamed tissues.

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. 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.

Inflammation-induced interstitial migration of effector CD4⁺ T cells is dependent on integrin αV.

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 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. 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.

Pivotal role for αV 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. 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.

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. 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.

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). 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.

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. 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.

Leishmania induces survival, proliferation and elevated cellular dNTP levels in human monocytes promoting acceleration of HIV co-infection.

Leishmaniasis is a parasitic disease that is widely prevalent in many tropical and sub-tropical regions of the world. Infection with Leishmania has been recognized to induce a striking acceleration of Human Immunodeficiency Virus Type 1 (HIV-1) infection in coinfected individuals through as yet incompletely understood mechanisms. Cells of the monocyte/macrophage lineage are the predominant cell types coinfected by both pathogens. Monocytes and macrophages contain extremely low levels of deoxynucleoside triphosphates (dNTPs) due to their lack of cell cycling and S phase, where dNTP biosynthesis is specifically activated. Lentiviruses, such as HIV-1, are unique among retroviruses in their ability to replicate in these non-dividing cells due, at least in part, to their highly efficient reverse transcriptase (RT). Nonetheless, viral replication progresses more efficiently in the setting of higher intracellular dNTP concentrations related to enhanced enzyme kinetics of the viral RT. In the present study, in vitro infection of CD14+ peripheral blood-derived human monocytes with Leishmania major was found to induce differentiation, marked elevation of cellular p53R2 ribonucleotide reductase subunit and R2 subunit expression. The R2 subunit is restricted to the S phase of the cell cycle. Our dNTP assay demonstrated significant elevation of intracellular monocyte-derived macrophages (MDMs) dNTP concentrations in Leishmania-infected cell populations as compared to control cells. Infection of Leishmania-maturated MDMs with a pseudotyped GFP expressing HIV-1 resulted in increased numbers of GFP+ cells in the Leishmania-maturated MDMs as compared to control cells. Interestingly, a sub-population of Leishmania-maturated MDMs was found to have re-entered the cell cycle, as demonstrated by BrdU labeling. In conclusion, Leishmania infection of primary human monocytes promotes the induction of an S phase environment and elevated dNTP levels with notable elevation of HIV-1 expression in the setting of coinfection.

Cutting edge: Regulatory T cells selectively attenuate, not terminate, T cell signaling by disrupting NF-κB nuclear accumulation in CD4 T cells.

A key consequence of regulatory T cell (Treg) suppression of CD4 T cells is the inhibition of IL-2 production, yet how Tregs attenuate IL-2 has not been defined. Current models predict a termination of TCR signaling, by disrupting T-APC contacts, or TCR signal modification, through mechanisms such as cAMP. To directly define Treg effects on TCR signaling in CD4 T cell targets, we visualized changes in nuclear accumulation of transcription factors at time points when IL-2 was actively suppressed. Nuclear accumulation of NFAT was highly dependent on sustained TCR signaling in the targets. However, in the presence of Tregs, NFAT and AP-1 signals were sustained in the target cells. In contrast, NF-κB p65 was selectively attenuated. Thus, Tregs do not generally terminate TCR signals. Rather, Tregs selectively modulate TCR signals within hours of contact with CD4 targets, independent of APCs, resulting in the specific loss of NF-κB p65 signals.

Regulatory T cells inhibit acute IFN-γ synthesis without blocking T-helper cell type 1 (Th1) differentiation via a compartmentalized requirement for IL-10.

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-ß 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.

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. This migration is mediated through small and dynamic focal adhesions that are composed of the same proteins associated with canonical mesenchymal cell focal adhesions, such as integrins, talin, and vinculin. These focal adhesions, furthermore, localize to sites of contractile traction stresses, enabling T cells to pull themselves through confined spaces. Finally, we show that Th1 T cells preferentially follow tracks of other T cells, suggesting that these adhesions modify the extracellular matrix to provide additional environmental guidance cues. These results demonstrate not only that the boundaries between amoeboid and mesenchymal migration modes are ambiguous, but that integrin-mediated focal adhesions play a key role in T cell motility.

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. This migration is mediated through small and dynamic focal adhesions that are composed of the same proteins associated with canonical mesenchymal focal adhesions, such as integrins, talin, and vinculin. These focal adhesions, furthermore, localize to sites of contractile traction stresses, enabling T cells to pull themselves through confined spaces. Finally, we show that Th1 T cell preferentially follows tracks of other T cells, suggesting that these adhesions are modifying the extracellular matrix to provide additional environmental guidance cues. These results demonstrate not only that the boundaries between amoeboid and mesenchymal migration modes are ambiguous, but that integrin-mediated adhesions play a key role in T cell motility.

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

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. Tumor-associated macrophages often support neoplastic growth and are frequently the most abundant immune cell found in tumors. Here, we used clinically relevant in vivo Braf-mutant melanoma models with fluorescent markers to track the stage-specific changes in macrophages under targeted therapy with Braf/Mek inhibitors and assessed the dynamic evolution of the macrophage population generated by therapy pressure-induced stress. During the onset of a drug-tolerant persister state, Ccr2+ monocyte-derived macrophage infiltration rose, suggesting that macrophage influx at this point could facilitate the onset of stable drug resistance that melanoma cells show after several weeks of treatment. Comparison of melanomas that develop in a Ccr2-proficient or -deficient microenvironment demonstrated that lack of melanoma infiltrating Ccr2+ macrophages delayed onset of resistance and shifted melanoma cell evolution towards unstable resistance. Unstable resistance was characterized by sensitivity to targeted therapy when factors from the microenvironment were lost. Importantly, this phenotype was reversed by coculturing melanoma cells with Ccr2+ macrophages. Overall, this study demonstrates that the development of resistance may be directed by altering the tumor microenvironment to improve treatment timing and the probability of relapse. SIGNIFICANCE: Ccr2+ melanoma macrophages that are active in tumors during the drug-tolerant persister state following targeted therapy-induced regression are key contributors directing melanoma cell reprogramming toward specific therapeutic resistance trajectories.

Critical requirement for the Wiskott-Aldrich syndrome protein in Th2 effector function.

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 IFNgamma. Thus WASp plays a selective, posttranscriptional role in Th2 effector function.

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. Immune and physical barrier disruption was accompanied by progressive skin dysbiosis, highlighting the functional significance of the disrupted cutaneous homeostasis. Interestingly, the dysregulated immunity in the skin preceded the systemic elevation in IgE and lymphocytic infiltration of the colonic lamina propria associated with WASp deficiency. Mechanistically, the enhanced immune cell accumulation in the skin was lymphocyte dependent. Elevated levels of both Type 2 (IL-4, IL-5) and Type 17 (IL-17, IL-22, IL-23) cytokines were present in the skin, as well as the 'itch' factor IL-31. Unexpectedly, the canonical WAS-associated cytokine IL-4 did not play a role in the immune dysfunction. Instead, IL-17 was critical for skin immune infiltration and elevation of both Type 2 and Type 17 cytokines. Our findings reveal a previously unrecognized IL-17-dependent breakdown in immune homeostasis and cutaneous barrier integrity in the absence of WASp, targeting of which may provide new therapeutic possibilities for the treatment of skin pathologies in WAS patients.

Pathogen-imposed skewing of mouse chemokine and cytokine expression at the infected tissue site.

Compartmentalization of immunity ensures tight regulation of T cell activation in the LN and precise effector T cell delivery to inflamed sites. Herein we show that the tissue-specific accumulation of effector T cells can be subverted by a pathogen at the infection site. Using the Leishmania major mouse model of dermal infection, we observed a restricted chemokine profile at the infection site, i.e., the expression of Th2 cell-attracting CCL7 but not of Th1 cell-attracting chemokines. Consistent with these chemokine expression data, recruitment of cytokine-producing T cells to the infection site was also selective. Both IL-4- and IFN-gamma-producing effector T cells homed to inflamed OVA/CFA-immunized dermis, but only IL-4-producing cells homed to L. major-infected dermis. The narrowing of the cytokine repertoire at the site of infection with L. major was driven, in part, by pathogen-induced CCL7. Inflammatory signals failed to disrupt the early restrictive L. major infection site, which suggests that L. major dominantly modifies the local milieu. We have highlighted an emerging principle in pathogen-host interactions: that the cytokine repertoire at the infection site and the LN draining the infection site can be different because of the ability of the pathogen to modify the chemokine profile at the infection site. Thus, pathogens may edit the LN cytokine repertoire through differential recruitment of cytokine-producing cells.