Trefoil Factor 2 Promotes Type 2 Immunity and Lung Repair through Intrinsic Roles in Hematopoietic and Nonhematopoietic Cells.

Trefoil factors (TFFs) are small secreted proteins that regulate tissue integrity and repair at mucosal surfaces, particularly in the gastrointestinal tract. However, their relative contribution(s) to controlling baseline lung function or the extent of infection-induced lung injury are unknown issues. With the use of irradiation bone marrow chimeras, we found that TFF2 produced from both hematopoietic- and nonhematopoietic-derived cells is essential for host protection, proliferation of alveolar type 2 cells, and restoration of pulmonary gas exchange after infection with the hookworm parasite Nippostrongylus brasiliensis. In the absence of TFF2, lung epithelia were unable to proliferate and expressed reduced lung mRNA transcript levels for type 2 response-inducing IL-25 and IL-33 after infectious injury. Strikingly, even in the absence of infection or irradiation, TFF2 deficiency compromised lung structure and function, as characterized by distended alveoli and reduced blood oxygen levels relative to wild-type control mice. Taken together, we show a previously unappreciated role for TFF2, produced by either hematopoietic or nonhematopoietic sources, as a pro-proliferative factor for lung epithelial cells under steady-state and infectious injury conditions.

Immune responses and long-term disease recurrence status after telomerase-based dendritic cell immunotherapy in patients with acute myeloid leukemia.

BACKGROUND: Telomerase activity in leukemic blasts frequently is increased among patients with high-risk acute myeloid leukemia (AML). In the current study, the authors evaluated the feasibility, safety, immunogenicity, and therapeutic potential of human telomerase reverse transcriptase (hTERT)-expressing autologous dendritic cells (hTERT-DCs) in adult patients with AML. METHODS: hTERT-DCs were produced from patient-specific leukapheresis, electroporated with an mRNA-encoding hTERT and a lysosomal-targeting sequence, and cryopreserved. A total of 22 patients with a median age of 58 years (range, 30-75 years) with intermediate-risk or high-risk AML in first or second complete remission (CR) were enrolled. hTERT-DCs were generated for 24 patients (73%). A median of 17 intradermal vaccinations (range, 6-32 intradermal vaccinations) containing 1×107 cells were administered as 6 weekly injections followed by 6 biweekly injections. A total of 21 patients (16 in first CR, 3 in second CR, and 2 with early disease recurrence) received hTERT-DCs. RESULTS: hTERT-DCs were well tolerated with no severe toxicities reported, with the exception of 1 patient who developed idiopathic thrombocytopenic purpura. Of the 19 patients receiving hTERT-DCs in CR, 11 patients (58%) developed hTERT-specific T-cell responses that primarily were targeted toward hTERT peptides with predicted low human leukocyte antigen (HLA)-binding affinities. With a median follow-up of 52 months, 58% of patients in CR (11 of 19 patients) were free of disease recurrence at the time of their last follow-up visit; 57% of the patients who were aged ≥60 years (4 of 7 patients) also were found to be free of disease recurrence at a median follow-up of 54 months. CONCLUSIONS: The generation of hTERT-DCs is feasible and vaccination with hTERT-DCs appears to be safe and may be associated with favorable recurrence-free survival. Cancer 2017;123:3061-72. © 2017 American Cancer Society.

A critical role for dendritic cells in the evolution of IL-1ß-mediated murine airway disease.

Chronic airway inflammation and fibrosis, known as airway remodeling, are defining features of chronic obstructive pulmonary disease and are refractory to current treatments. How and whether chronic inflammation contributes to airway fibrosis remain controversial. In this study, we use a model of chronic obstructive pulmonary disease airway disease utilizing adenoviral delivery of IL-1ß to determine that adaptive T cell immunity is required for airway remodeling because mice deficient in α/ß T cells (tcra(-/-)) are protected. Dendritic cells (DCs) accumulate around chronic obstructive pulmonary disease airways and are critical to prime adaptive immunity, but they have not been shown to directly influence airway remodeling. We show that DC depletion or deficiency in the crucial DC chemokine receptor ccr6 both protect from adenoviral IL-1ß-induced airway adaptive T cell immune responses and fibrosis in mice. These results provide evidence that chronic airway inflammation, mediated by accumulation of α/ß T cells and driven by DCs, is critical to airway fibrosis.

TGF-ß-Dependent Dendritic Cell Chemokinesis in Murine Models of Airway Disease.

Small airway chronic inflammation is a major pathologic feature of chronic obstructive pulmonary disease (COPD) and is refractory to current treatments. Dendritic cells (DCs) accumulate around small airways in COPD. DCs are critical mediators of Ag surveillance and Ag presentation and amplify adaptive immune responses. How DCs accumulate around airways remains largely unknown. We use 2-photon DC imaging of living murine lung sections to directly visualize the dynamic movement of living DCs around airways in response to either soluble mediators (IL-1ß) or environmental stimuli (cigarette smoke or TLR3 ligands) implicated in COPD pathogenesis. We find that DCs accumulate around murine airways primarily by increasing velocity (chemokinesis) rather than directional migration (chemotaxis) in response to all three stimuli. DC accumulation maximally occurs in a specific zone located 26-50 µm from small airways, which overlaps with zones of maximal DC velocity. Our data suggest that increased accumulation of DCs around airways results from increased numbers of highly chemokinetic DCs entering the lung from the circulation with balanced rates of immigration and emigration. Increases in DC accumulation and chemokinesis are partially dependent on ccr6, a crucial DC chemokine receptor, and fibroblast expression of the integrin αvß8, a critical activator of TGF-ß. αvß8-Mediated TGF-ß activation is known to enhance IL-1ß-dependent fibroblast expression of the only known endogenous ccr6 chemokine ligand, ccl20. Taken together, these data suggest a mechanism by which αvß8, ccl20, and ccr6 interact to lead to DC accumulation around airways in response to COPD-relevant stimuli.

Stabilized imaging of immune surveillance in the mouse lung.

Real-time imaging of cellular and subcellular dynamics in vascularized organs requires image resolution and image registration to be simultaneously optimized without perturbing normal physiology. This problem is particularly pronounced in the lung, in which cells may transit at speeds >1 mm s(-1) and in which normal respiration results in large-scale tissue movements that prevent image registration. Here we report video-rate, two-photon imaging of a physiologically intact preparation of the mouse lung that is stabilizing and nondisruptive. Using our method, we obtained evidence for differential trapping of T cells and neutrophils in mouse pulmonary capillaries, and observed neutrophil mobilization and dynamic vascular leak in response to stretch and inflammatory models of lung injury in mice. The system permits physiological measurement of motility rates of >1 mm s(-1), observation of detailed cellular morphology and could be applied in the future to other organs and tissues while maintaining intact physiology.

Selective and site-specific mobilization of dermal dendritic cells and Langerhans cells by Th1- and Th2-polarizing adjuvants.

Dendritic cells (DCs) initiate and polarize adaptive immune responses toward varying functional outcomes. By means of intravital two-photon microscopy, we report that dermal dendritic cells (DDCs) and Langerhans cells (LCs) are differentially mobilized during contact sensitization and by adjuvants such as unmethylated CpG oligonucleotide (CpG) and LPS that induce T helper type 1 (Th1) responses, or papain that induces T helper type 2 (Th2) responses. In ear pinna, contact sensitization, CpG, LPS, and papain all mobilized DDCs in three distinct phases: increased motility and dendritic probing, directed migration, and entry into lymphatic vessels. During the same treatments, the adjacent LCs in ear pinna remained immotile over a 48-hr period of observation. In contrast, footpads lacked DDCs and Th1-polarizing adjuvants selectively induced a delayed mobilization of LCs after 48 hr. Th1 polarization of CD4(+) T cells was independent of the immunization site, whereas ear immunization favored Th2 polarization, correlating with site-specific DC distribution and dynamics. Our results provide an initial description of peripheral DC dynamics in response to adjuvants and imply that LC mobilization enhances a Th1 response and is not sufficient to trigger a Th2 response, whereas mobilization of DDCs alone is sufficient to trigger T-cell proliferation and to polarize initial T-cell activation toward a Th2 response.

Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation.

For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai in Drosophila cells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca2+ concentration near the interface, indicating Ca2+ influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca2+ signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca2+ influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca2+ signaling during subsequent antigen encounter.

Macrophages promote epithelial proliferation following infectious and non-infectious lung injury through a Trefoil factor 2-dependent mechanism.

Coordinated efforts between macrophages and epithelia are considered essential for wound healing, but the macrophage-derived molecules responsible for repair are poorly defined. This work demonstrates that lung macrophages rely upon Trefoil factor 2 to promote epithelial proliferation following damage caused by sterile wounding, Nippostrongylus brasiliensis or Bleomycin sulfate. Unexpectedly, the presence of T, B, or ILC populations was not essential for macrophage-driven repair. Instead, conditional deletion of TFF2 in myeloid-restricted CD11cCre TFF2 flox mice exacerbated lung pathology and reduced the proliferative expansion of CD45- EpCAM+ pro-SPC+ alveolar type 2 cells. TFF2 deficient macrophages had reduced expression of the Wnt genes Wnt4 and Wnt16 and reconstitution of hookworm-infected CD11cCre TFF2flox mice with rWnt4 and rWnt16 restored the proliferative defect in lung epithelia post-injury. These data reveal a previously unrecognized mechanism wherein lung myeloid phagocytes utilize a TFF2/Wnt axis as a mechanism that drives epithelial proliferation following lung injury.

Structure of the 4-1BB/4-1BBL complex and distinct binding and functional properties of utomilumab and urelumab.

4-1BB (CD137, TNFRSF9) is an inducible costimulatory receptor expressed on activated T cells. Clinical trials of two agonist antibodies, utomilumab (PF-05082566) and urelumab (BMS-663513), are ongoing in multiple cancer indications, and both antibodies demonstrate distinct activities in the clinic. To understand these differences, we solved structures of the human 4-1BB/4-1BBL complex, the 4-1BBL trimer alone, and 4-1BB bound to utomilumab or urelumab. The 4-1BB/4-1BBL complex displays a unique interaction between receptor and ligand when compared with other TNF family members. Furthermore, our ligand-only structure differs from previously published data. Utomilumab, a ligand-blocking antibody, binds 4-1BB between CRDs 3 and 4. In contrast, urelumab binds 4-1BB CRD-1, away from the ligand binding site. Finally, cell-based assays demonstrate utomilumab is a milder agonist than urelumab. Collectively, our data provide a deeper understanding of the 4-1BB signaling complex, providing a template for future development of next generation 4-1BB targeted biologics.

In Vivo Molecular Optical Coherence Tomography of Lymphatic Vessel Endothelial Hyaluronan Receptors.

Optical Coherence Tomography (OCT) imaging of living subjects offers increased depth of penetration while maintaining high spatial resolution when compared to other optical microscopy techniques. However, since most protein biomarkers do not exhibit inherent contrast detectable by OCT, exogenous contrast agents must be employed for imaging specific cellular biomarkers of interest. While a number of OCT contrast agents have been previously studied, demonstrations of molecular targeting with such agents in live animals have been historically challenging and notably limited in success. Here we demonstrate for the first time that microbeads (µBs) can be used as contrast agents to target cellular biomarkers in lymphatic vessels and can be detected by OCT using a phase variance algorithm. This molecular OCT method enables in vivo imaging of the expression profiles of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a biomarker that plays crucial roles in inflammation and tumor metastasis. In vivo OCT imaging of LVYE-1 showed that the biomarker was significantly down-regulated during inflammation induced by acute contact hypersensitivity (CHS). Our work demonstrated a powerful molecular imaging tool that can be used for high resolution studies of lymphatic function and dynamics in models of inflammation, tumor development, and other lymphatic diseases.

Erratum: Speckle-modulating optical coherence tomography in living mice and humans.

This corrects the article DOI: 10.1038/ncomms15845.

Speckle-modulating optical coherence tomography in living mice and humans.

Optical coherence tomography (OCT) is a powerful biomedical imaging technology that relies on the coherent detection of backscattered light to image tissue morphology in vivo. As a consequence, OCT is susceptible to coherent noise (speckle noise), which imposes significant limitations on its diagnostic capabilities. Here we show speckle-modulating OCT (SM-OCT), a method based purely on light manipulation that virtually eliminates speckle noise originating from a sample. SM-OCT accomplishes this by creating and averaging an unlimited number of scans with uncorrelated speckle patterns without compromising spatial resolution. Using SM-OCT, we reveal small structures in the tissues of living animals, such as the inner stromal structure of a live mouse cornea, the fine structures inside the mouse pinna, and sweat ducts and Meissner's corpuscle in the human fingertip skin-features that are otherwise obscured by speckle noise when using conventional OCT or OCT with current state of the art speckle reduction methods.

Malignant Eccrine Poroma of the Vulva: An Intriguing Case of a Rare Tumor at an Unusual Site.

Malignant eccrine poroma is a rare malignancy of the eccrine sweat glands, occurring most frequently on the lower extremities. It affects both sexes equally usually in the 6(th) to 7(th) decade of life. Metastasis to regional lymph nodes may occur in 20% that may be fatal in 60% cases. Its aggressive nature, rarity of occurrence, and unusual presentations make it very important to be evaluated properly by the clinician. We hereby report a case of a 75-year-old female presenting with two exophytic tumors over her vulva with local extension. On histopathological examination, it was diagnosed as malignant eccrine poroma. On magnetic resonance imaging of the pelvic region, metastatic extension in regional lymph nodes was found. She was treated by radical vulvectomy with bilateral inguinal and femoral lymph node dissection followed by radiotherapy.

Contrast-enhanced optical coherence tomography with picomolar sensitivity for functional in vivo imaging.

Optical Coherence Tomography (OCT) enables real-time imaging of living tissues at cell-scale resolution over millimeters in three dimensions. Despite these advantages, functional biological studies with OCT have been limited by a lack of exogenous contrast agents that can be distinguished from tissue. Here we report an approach to functional OCT imaging that implements custom algorithms to spectrally identify unique contrast agents: large gold nanorods (LGNRs). LGNRs exhibit 110-fold greater spectral signal per particle than conventional GNRs, which enables detection of individual LGNRs in water and concentrations as low as 250 pM in the circulation of living mice. This translates to ~40 particles per imaging voxel in vivo. Unlike previous implementations of OCT spectral detection, the methods described herein adaptively compensate for depth and processing artifacts on a per sample basis. Collectively, these methods enable high-quality noninvasive contrast-enhanced imaging of OCT in living subjects, including detection of tumor microvasculature at twice the depth achievable with conventional OCT. Additionally, multiplexed detection of spectrally-distinct LGNRs was demonstrated to observe discrete patterns of lymphatic drainage and identify individual lymphangions and lymphatic valve functional states. These capabilities provide a powerful platform for molecular imaging and characterization of tissue noninvasively at cellular resolution, called MOZART.

Tracking the Spatial and Functional Gradient of Monocyte-To-Macrophage Differentiation in Inflamed Lung.

Myeloid-derived cells such as monocytes, dendritic cells (DCs), and macrophages are at the heart of the immune effector function in an inflammatory response. But because of the lack of an efficient imaging system to trace these cells live during their migration and maturation in their native environment at sub-cellular resolution, our knowledge is limited to data available from specific time-points analyzed by flow cytometry, histology, genomics and other immunological methods. Here, we have developed a ratiometric imaging method for measuring monocyte maturation in inflamed mouse lungs in situ using real-time using 2-photon imaging and complementary methods. We visualized that while undifferentiated monocytes were predominantly found only in the vasculature, a semi-differentiated monocyte/macrophage population could enter the tissue and resembled more mature and differentiated populations by morphology and surface phenotype. As these cells entered and differentiated, they were already selectively localized near inflamed airways and their entry was associated with changes in motility and morphology. We were able to visualize these during the act of differentiation, a process that can be demonstrated in this way to be faster on a per-cell basis under inflammatory conditions. Finally, our in situ analyses demonstrated increases, in the differentiating cells, for both antigen uptake and the ability to mediate interactions with T cells. This work, while largely confirming proposed models for in situ differentiation, provides important in situ data on the coordinated site-specific recruitment and differentiation of these cells and helps elaborate the predominance of immune pathology at the airways. Our novel imaging technology to trace immunogenic cell maturation in situ will complement existing information available on in situ differentiation deduced from other immunological methods, and assist better understanding of the spatio-temporal cellular behavior during an inflammatory response.

High-resolution contrast-enhanced optical coherence tomography in mice retinae.

Optical coherence tomography (OCT) is a noninvasive interferometric imaging modality providing anatomical information at depths of millimeters and a resolution of micrometers. Conventional OCT images limit our knowledge to anatomical structures alone, without any contrast enhancement. Therefore, here we have, for the first time, optimized an OCT-based contrast-enhanced imaging system for imaging single cells and blood vessels in vivo inside the living mouse retina at subnanomolar sensitivity. We used bioconjugated gold nanorods (GNRs) as exogenous OCT contrast agents. Specifically, we used anti-mouse CD45 coated GNRs to label mouse leukocytes and mPEG-coated GNRs to determine sensitivity of GNR detection in vivo inside mice retinae. We corroborated OCT observations with hyperspectral dark-field microscopy of formalin-fixed histological sections. Our results show that mouse leukocytes that otherwise do not produce OCT contrast can be labeled with GNRs leading to significant OCT intensity equivalent to a 0.5 nM GNR solution. Furthermore, GNRs injected intravenously can be detected inside retinal blood vessels at a sensitivity of ∼0.5 nM, and GNR-labeled cells injected intravenously can be detected inside retinal capillaries by enhanced OCT contrast. We envision the unprecedented resolution and sensitivity of functionalized GNRs coupled with OCT to be adopted for longitudinal studies of retinal disorders.

A comparative study of unfolding of Erythrina cristagalli Lectin in chemical denaturant and Fluoroalcohols.

The unfolding of dimeric Erythrina cristagalli lectin (ECL) has been investigated and compared under different denaturing conditions in presence of chemical denaturant, guanidine hydrochloride (GdnHCl) and fluoroalcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP). The GdnHCl-induced unfolding exhibits three-state mechanism involving structured intermediate that corresponds to tertiary monomer. The intermediate has been characterized by 8- anilino-1-naphthalenesulfonate (ANS) binding, which shows ~ 30 fold increase in ANS fluorescence and selective chemical modification with N-bromosuccinimide when Trp 45 and Trp 207 are possibly oxidized. The results are supported by red edge excitation shift (10 nm), acrylamide quenching and phosphorescence studies which give a (0,0) band at 412.6 nm. TFE and HFIP show differing roles and characteristics of ECL unfolding. In TFE, but not in HFIP, a molten globulelike monomeric intermediate is formed, being characterized by ANS binding and concentration dependent studies. TFEand HFIP-induced secondary structure changes of ECL, as monitored by far-UV CD, show that conversion of ß-sheet to α-helix occurs at lower HFIP concentration compared to TFE perturbation, helical content reaching to 65 % in 80 % HFIP and 53 % in 90 % TFE. Temperature-dependent studies reveal that induced helix entails reduced thermal stability. FTIR results show partial ß-sheet to α-helix conversion but with quantitative yield. The tryptophan environment of TFE- and HFIP-induced states is dissimilar involving oxidation of four and three tryptophans respectively, and also differs from the fully unfolded state in GdnHCl when all five tryptophans undergo oxidation. The results offer insights into the unfolding problem of ECL.

Spatiotemporally separated antigen uptake by alveolar dendritic cells and airway presentation to T cells in the lung.

Asthma pathogenesis is focused around conducting airways. The reasons for this focus have been unclear because it has not been possible to track the sites and timing of antigen uptake or subsequent antigen presentation to effector T cells. In this study, we use two-photon microscopy of the lung parenchyma and note accumulation of CD11b(+) dendritic cells (DCs) around the airway after allergen challenge but very limited access of these airway-adjacent DCs to the contents of the airspace. In contrast, we observed prevalent transepithelial uptake of particulate antigens by alveolar DCs. These distinct sites are temporally linked, as early antigen uptake in alveoli gives rise to DC and antigen retention in the airway-adjacent region. Antigen-specific T cells also accumulate in the airway-adjacent region after allergen challenge and are activated by the accumulated DCs. Thus, we propose that later airway hyperreactivity results from selective retention of allergen-presenting DCs and antigen-specific T cells in airway-adjacent interaction zones, not from variation in the abilities of individual DCs to survey the lung.

Pea lectin unfolding reveals a unique molten globule fragment chain.

Pea lectin (PSL) is a dimeric protein in which each subunit comprises two intertwined, post-translationally processed polypeptide chains--a long ß-fragment and a short α-fragment. Using guanidine hydrochloride-induced denaturation, we have investigated and characterized the species obtained in the unfolding equilibrium of PSL by steady-state and time-resolved fluorescence, phosphorescence, and selective chemical modification. During unfolding, the fragment chains become separated, and the unfolding pattern reveals a ß-fragment as intermediate that has the molten globule characteristics. As examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, the fragment intermediate shows ~20 fold increase in ANS fluorescence, and a large increase in ANS lifetime (12.8 ns). The tryptophan environment of the molten globule ß-fragment has been probed by selective modification with N-bromosuccinimide (NBS), which shows that two tryptophans, possibly Trp 53 and Trp 152 are oxidized while the other Trp 128 remains resistant to oxidation. The different types of tryptophan environment for the intermediate are supported by phosphorescence studies at 77 K, which gives a (0,0) band at 410 nm. These results seem to indicate that the larger fragment chain of PSL can independently behave as a monomeric or single domain protein that undergoes unfolding through intermediate state(s), and may provide important insight into the folding problem of oligomeric proteins in general and lectins in particular.

Generation of bone marrow derived murine dendritic cells for use in 2-photon imaging.

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and <85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.