High-density lipoprotein mediates anti-inflammatory reprogramming of macrophages via the transcriptional regulator ATF3.

High-density lipoprotein (HDL) mediates reverse cholesterol transport and is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional regulator ATF3, as an HDL-inducible target gene in macrophages that downregulates the expression of Toll-like receptor (TLR)-induced proinflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of new HDL-based therapies.

Metabolic fingerprinting by nuclear magnetic resonance of hepatocellular carcinoma cells during p53 reactivation-induced senescence.

Cellular senescence is characterized by stable cell cycle arrest. Senescent cells exhibit a senescence-associated secretory phenotype that can promote tumor progression. The aim of our study was to identify specific nuclear magnetic resonance (NMR) spectroscopy-based markers of cancer cell senescence. For metabolic studies, we employed murine liver carcinoma Harvey Rat Sarcoma Virus (H-Ras) cells, in which reactivation of p53 expression induces senescence. Senescent and nonsenescent cell extracts were subjected to high-resolution proton (1H)-NMR spectroscopy-based metabolomics, and dynamic metabolic changes during senescence were analyzed using a magnetic resonance spectroscopy (MRS)-compatible cell perfusion system. Additionally, the ability of intact senescent cells to degrade the extracellular matrix (ECM) was quantified in the cell perfusion system. Analysis of senescent H-Ras cell extracts revealed elevated sn-glycero-3-phosphocholine, myoinositol, taurine, and creatine levels, with decreases in glycine, o-phosphocholine, threonine, and valine. These metabolic findings were accompanied by a greater degradation index of the ECM in senescent H-Ras cells than in control H-Ras cells. MRS studies with the cell perfusion system revealed elevated creatine levels in senescent cells on Day 4, confirming the 1H-NMR results. These senescence-associated changes in metabolism and ECM degradation strongly impact growth and redox metabolism and reveal potential MRS signals for detecting senescent cancer cells in vivo.

Basic principles of artificial intelligence in dermatology explained using melanoma.

The use of artificial intelligence (AI) continues to establish itself in the most diverse areas of medicine at an increasingly fast pace. Nevertheless, many healthcare professionals lack the basic technical understanding of how this technology works, which severely limits its application in clinical settings and research. Thus, we would like to discuss the functioning and classification of AI using melanoma as an example in this review to build an understanding of the technology behind AI. For this purpose, elaborate illustrations are used that quickly reveal the technology involved. Previous reviews tend to focus on the potential applications of AI, thereby missing the opportunity to develop a deeper understanding of the subject matter that is so important for clinical application. Malignant melanoma has become a significant burden for healthcare systems. If discovered early, a better prognosis can be expected, which is why skin cancer screening has become increasingly popular and is supported by health insurance. The number of experts remains finite, reducing their availability and leading to longer waiting times. Therefore, innovative ideas need to be implemented to provide the necessary care. Thus, machine learning offers the ability to recognize melanomas from images at a level comparable to experienced dermatologists under optimized conditions.

Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors.

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ-/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.

Autoreactive T cells targeting type II pneumocyte antigens in COVID-19 convalescent patients.

BACKGROUND: The role of autoreactive T cells on the course of Coronavirus disease-19 (COVID-19) remains elusive. Type II pneumocytes represent the main target cells of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Autoimmune responses against antigens highly expressed in type II pneumocytes may influence the severity of COVID-19 disease. OBJECTIVE: The aim of this study was to investigate autoreactive T cell responses against self-antigens highly expressed in type II pneumocytes in the blood of COVID-19 patients with severe and non-severe disease. METHODS: We collected blood samples of COVID-19 patients with varying degrees of disease severity and of pre-pandemic controls. T cell stimulation assays with peptide pools of type II pneumocyte antigens were performed in two independent cohorts to analyze the autoimmune T cell responses in patients with non-severe and severe COVID-19 disease. Target cell lysis assays were performed with lung cancer cell lines to determine the extent of cell killing by type II PAA-specific T cells. RESULTS: We identified autoreactive T cell responses against four recently described self-antigens highly expressed in type II pneumocytes, known as surfactant protein A, surfactant protein B, surfactant protein C and napsin A, in the blood of COVID-19 patients. These antigens were termed type II pneumocyte-associated antigens (type II PAAs). We found that patients with non-severe COVID-19 disease showed a significantly higher frequency of type II PAA-specific autoreactive T cells in the blood when compared to severely ill patients. The presence of high frequencies of type II PAA-specific T cells in the blood of non-severe COVID-19 patients was independent of their age. We also found that napsin A-specific T cells from convalescent COVID-19 patients could kill lung cancer cells, demonstrating the functional and cytotoxic role of these T cells. CONCLUSIONS: Our data suggest that autoreactive type II PAA-specific T cells have a protective role in SARS-CoV-2 infections and the presence of high frequencies of these autoreactive T cells indicates effective viral control in COVID-19 patients. Type II-PAA-specific T cells may therefore promote the killing of infected type II pneumocytes and viral clearance.

Non invasive in vivo monitoring of dimethyl fumarate treatment in EAE by assessing the glucose metabolism in secondary lymphoid organs.

[18 F]FDG-PET/CT is a high sensitive functional diagnostic imaging modality to monitor tumor but also immune cell activation by determination of the glucose metabolism. Our results show that the anti-inflammatory effects of immunotherapeutics like DMF can be assessed non invasively in vivo during Th1/Th17 cell-mediated encephalomyelitis (EAE) by [18 F]FDG-PET/CT imaging of the draining lymph nodes.

Noninvasive, longitudinal imaging-based analysis of body adipose tissue and water composition in a melanoma mouse model and in immune checkpoint inhibitor-treated metastatic melanoma patients.

BACKGROUND: As cancer cachexia (CC) is associated with cancer progression, early identification would be beneficial. The aim of this study was to establish a workflow for automated MRI-based segmentation of visceral (VAT) and subcutaneous adipose tissue (SCAT) and lean tissue water (LTW) in a B16 melanoma animal model, monitor diseases progression and transfer the protocol to human melanoma patients for therapy assessment. METHODS: For in vivo monitoring of CC B16 melanoma-bearing and healthy mice underwent longitudinal three-point DIXON MRI (days 3, 12, 17 after subcutaneous tumor inoculation). In a prospective clinical study, 18 metastatic melanoma patients underwent MRI before, 2 and 12 weeks after onset of checkpoint inhibitor therapy (CIT; n = 16). We employed an in-house MATLAB script for automated whole-body segmentation for detection of VAT, SCAT and LTW. RESULTS: B16 mice exhibited a CC phenotype and developed a reduced VAT volume compared to baseline (B16 - 249.8 µl, - 25%; controls + 85.3 µl, + 10%, p = 0.003) and to healthy controls. LTW was increased in controls compared to melanoma mice. Five melanoma patients responded to CIT, 7 progressed, and 6 displayed a mixed response. Responding patients exhibited a very limited variability in VAT and SCAT in contrast to others. Interestingly, the LTW was decreased in CIT responding patients (- 3.02% ± 2.67%; p = 0.0034) but increased in patients with progressive disease (+ 1.97% ± 2.19%) and mixed response (+ 4.59% ± 3.71%). CONCLUSION: MRI-based segmentation of fat and water contents adds essential additional information for monitoring the development of CC in mice and metastatic melanoma patients during CIT or other treatment approaches.

The administration route of tumor-antigen-specific T-helper cells differentially modulates the tumor microenvironment and senescence.

Cancer treatment with adoptively transferred tumor-associated antigen-specific CD4+ T-helper cells is a promising immunotherapeutic approach. In the pancreatic cancer model RIP-Tag2, the intraperitoneal (i.p.) application of Tag-specific TH1 cells exhibited a profound antitumoral efficiency. We investigated, whether an intravenous (i.v.) application of Tag-TH1 cells induces an equivalent therapeutic effect. Adoptively transferred fluorescent Tag-TH1 cells revealed a pronounced homing to the tumors after either i.p. or i.v. transfer, and both routes induced an almost equivalent therapeutic effect as demonstrated by magnetic resonance imaging, blood glucose level course and histology. The i.v. administration of Tag-TH1 cells induced p16INK4-positive/Ki67-negative tumor senescence more efficiently than i.p. administration. Both routes replenish host CD4+ T cells by transferred T cells and recruitment of B and dendritic cells to the tumors while reducing CD8+ T cells and depleting macrophages. Both administration routes efficiently induced a similar antitumoral efficiency despite the pronounced senescence induction after i.v. administration. Thus, a combinatory i.v./i.p. injection of therapeutic cells might overcome limitations of the individual routes and improve therapeutic efficacy in solid tumors.

T-helper-1-cell cytokines drive cancer into senescence.

Cancer control by adaptive immunity involves a number of defined death and clearance mechanisms. However, efficient inhibition of exponential cancer growth by T cells and interferon-γ (IFN-γ) requires additional undefined mechanisms that arrest cancer cell proliferation. Here we show that the combined action of the T-helper-1-cell cytokines IFN-γ and tumour necrosis factor (TNF) directly induces permanent growth arrest in cancers. To safely separate senescence induced by tumour immunity from oncogene-induced senescence, we used a mouse model in which the Simian virus 40 large T antigen (Tag) expressed under the control of the rat insulin promoter creates tumours by attenuating p53- and Rb-mediated cell cycle control. When combined, IFN-γ and TNF drive Tag-expressing cancers into senescence by inducing permanent growth arrest in G1/G0, activation of p16INK4a (also known as CDKN2A), and downstream Rb hypophosphorylation at serine 795. This cytokine-induced senescence strictly requires STAT1 and TNFR1 (also known as TNFRSF1A) signalling in addition to p16INK4a. In vivo, Tag-specific T-helper 1 cells permanently arrest Tag-expressing cancers by inducing IFN-γ- and TNFR1-dependent senescence. Conversely, Tnfr1(-/-)Tag-expressing cancers resist cytokine-induced senescence and grow aggressively, even in TNFR1-expressing hosts. Finally, as IFN-γ and TNF induce senescence in numerous murine and human cancers, this may be a general mechanism for arresting cancer progression.

Evaluation of the therapeutic potential of the selective p38 MAPK inhibitor Skepinone-L and the dual p38/JNK 3 inhibitor LN 950 in experimental K/BxN serum transfer arthritis.

BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling plays an important role in inflammatory diseases such as rheumatoid arthritis (RA).The aim of our study was to elucidate the therapeutic potential of the highly selective p38 MAPK inhibitor Skepinone-L and the dual inhibitor LN 950 (p38 MAPK and JNK 3) in the K/BxN serum transfer model of RA. Additionally, we aimed to monitor MAPK treatment non-invasively in vivo using the hypoxia tracer [18F]fluoromisonidazole ([18F]FMISO) and positron emission tomography (PET). METHODS: To induce experimental arthritis, we injected glucose-6-phosphate isomerase autoantibody-containing serum in BALB/c mice. MAPK inhibitor or Sham treatment was administered per os once daily. On days 3 and 6 after arthritis induction, we conducted PET imaging with [18F]FMISO. At the end of the experiment, ankles were harvested for histopathological analysis. RESULTS: Skepinone-L and LN 950 were applicable to suppress the severity of experimental arthritis confirmed by reduced ankle swelling and histopathological analysis. Skepinone-L (3.18 ± 0.19 mm) and LN 950 (3.40 ± 0.13 mm) treatment yielded a significantly reduced ankle thickness compared to Sham-treated mice (3.62 ± 0.11 mm) on day 5 after autoantibody transfer, a time-point characterized by severe arthritis. Hypoxia imaging with [18F]FMISO revealed non-conclusive results and might not be an appropriate tool to monitor MAPK therapy in experimental RA. CONCLUSION: Both the selective p38 MAPK inhibitor Skepinone-L and the dual (p38 MAPK and JNK 3) inhibitor LN 950 exhibited significant therapeutic effects during experimental arthritis. Thus, our study contributes to the ongoing discussion on the use of p38 MAPK as a potential target in RA.

64Cu antibody-targeting of the T-cell receptor and subsequent internalization enables in vivo tracking of lymphocytes by PET.

T cells are key players in inflammation, autoimmune diseases, and immunotherapy. Thus, holistic and noninvasive in vivo characterizations of the temporal distribution and homing dynamics of lymphocytes in mammals are of special interest. Herein, we show that PET-based T-cell labeling facilitates quantitative, highly sensitive, and holistic monitoring of T-cell homing patterns in vivo. We developed a new T-cell receptor (TCR)-specific labeling approach for the intracellular labeling of mouse T cells. We found that continuous TCR plasma membrane turnover and the endocytosis of the specific (64)Cu-monoclonal antibody (mAb)-TCR complex enables a stable labeling of T cells. The TCR-mAb complex was internalized within 24 h, whereas antigen recognition was not impaired. Harmful effects of the label on the viability, DNA-damage and apoptosis-necrosis induction, could be minimized while yielding a high contrast in in vivo PET images. We were able to follow and quantify the specific homing of systemically applied (64)Cu-labeled chicken ovalbumin (cOVA)-TCR transgenic T cells into the pulmonary and perithymic lymph nodes (LNs) of mice with cOVA-induced airway delayed-type hypersensitivity reaction (DTHR) but not into pulmonary and perithymic LNs of naïve control mice or mice diseased from turkey or pheasant OVA-induced DTHR. Our protocol provides consequent advancements in the detection of small accumulations of immune cells in single LNs and specific homing to the sites of inflammation by PET using the internalization of TCR-specific mAbs as a specific label of T cells. Thus, our labeling approach is applicable to other cells with constant membrane receptor turnover.

Necrotic Cell Sensor Clec4e Promotes a Proatherogenic Macrophage Phenotype Through Activation of the Unfolded Protein Response.

BACKGROUND: Atherosclerotic lesion expansion is characterized by the development of a lipid-rich necrotic core known to be associated with the occurrence of complications. Abnormal lipid handling, inflammation, and alteration of cell survival or proliferation contribute to necrotic core formation, but the molecular mechanisms involved in this process are not properly understood. C-type lectin receptor 4e (Clec4e) recognizes the cord factor of Mycobacterium tuberculosis but also senses molecular patterns released by necrotic cells and drives inflammation. METHODS: We hypothesized that activation of Clec4e signaling by necrosis is causally involved in atherogenesis. We addressed the impact of Clec4e activation on macrophage functions in vitro and on the development of atherosclerosis using low-density lipoprotein receptor-deficient (Ldlr-/-) mice in vivo. RESULTS: We show that Clec4e is expressed within human and mouse atherosclerotic lesions and is activated by necrotic lesion extracts. Clec4e signaling in macrophages inhibits cholesterol efflux and induces a Syk-mediated endoplasmic reticulum stress response, leading to the induction of proinflammatory mediators and growth factors. Chop and Ire1a deficiencies significantly limit Clec4e-dependent effects, whereas Atf3 deficiency aggravates Clec4e-mediated inflammation and alteration of cholesterol efflux. Repopulation of Ldlr-/- mice with Clec4e-/- bone marrow reduces lipid accumulation, endoplasmic reticulum stress, and macrophage inflammation and proliferation within the developing arterial lesions and significantly limits atherosclerosis. CONCLUSIONS: Our results identify a nonredundant role for Clec4e in coordinating major biological pathways involved in atherosclerosis and suggest that it may play similar roles in other chronic inflammatory diseases.

In vivo optical imaging of matrix metalloproteinase activity detects acute and chronic contact hypersensitivity reactions and enables monitoring of the antiinflammatory effects of N-acetylcysteine.

The aim of this study was to determine whether the severity of contact hypersensitivity reactions (CHSRs) can be observed by noninvasive in vivo optical imaging of matrix metalloproteinase (MMP) activity and whether this is an appropriate tool for monitoring an antiinflammatory effect. Acute and chronic CHSRs were elicited by application of a 1% trinitrochlorobenzene (TNCB) solution for up to five times on the right ear of TNCB-sensitized mice. N-Acetylcysteine (NAC)-treated and sham-treated mice were monitored by measuring ear swelling and optical imaging of MMP activity. In addition, we performed hematoxylin-eosin staining and CD31 immunohistochemistry for histopathologic analysis of the antiinflammatory effects of NAC. The ear thickness and the MMP activity increased in line with the increasing severity of the CHSR. MMP activity was enhanced 2.5- to 2.7-fold during acute CHSR and 3.1- to 4.1-fold during chronic CHSR. NAC suppressed ear swelling and MMP signal intensity in mice with acute and chronic CHSR. During chronic CHSR, the vessel density was significantly reduced in ear sections derived from NAC-treated compared to sham-treated mice. In vivo optical imaging of MMP activity measures acute and chronic CHSR and is useful to monitor antiinflammatory effects.

Augmenting MEK inhibitor efficacy in BRAF wild-type melanoma: synergistic effects of disulfiram combination therapy.

BACKGROUND: MEK inhibitors (MEKi) were shown to be clinically insufficiently effective in patients suffering from BRAF wild-type (BRAF WT) melanoma, even if the MAPK pathway was constitutively activated due to mutations in NRAS or NF-1. Thus, novel combinations are needed to increase the efficacy and duration of response to MEKi in BRAF WT melanoma. Disulfiram and its metabolite diethyldithiocarbamate are known to have antitumor effects related to cellular stress, and induction of endoplasmic reticulum (ER) stress was found to synergize with MEK inhibitors in NRAS-mutated melanoma cells. Therefore, we investigated the combination of both therapeutics to test their effects on BRAF-WT melanoma cells and compared them with monotherapy using the MEKi trametinib. METHODS: The effects of combined therapy with disulfiram or its metabolite diethyldithiocarbamate and the MEKi trametinib were evaluated in a series of BRAF-WT melanoma cell lines by measuring cell viability and apoptosis induction. Cytotoxicity was additionally assessed in 3D spheroids, ex vivo melanoma slice cultures, and in vivo xenograft mouse models. The response of melanoma cells to treatment was studied at the RNA and protein levels to decipher the mode of action. Intracellular and intratumoral copper measurements were performed to investigate the role of copper ions in the antitumor cytotoxicity of disulfiram and its combination with the MEKi. RESULTS: Diethyldithiocarbamate enhanced trametinib-induced cytotoxicity and apoptosis induction in 2D and 3D melanoma culture models. Mechanistically, copper-dependent induction of oxidative stress and ER stress led to Janus kinase (JNK)-mediated apoptosis in melanoma cells. This mechanism was also detectable in patient-derived xenograft melanoma models and resulted in a significantly improved therapeutic effect compared to monotherapy with the MEKi trametinib. CONCLUSIONS: Disulfiram and its metabolite represent an attractive pharmaceutical approach to induce ER stress in melanoma cells that potentiates the antitumor effect of MEK inhibition and may be an interesting candidate for combination therapy of BRAF WT melanoma.

CD19-immunoPET for noninvasive visualization of CD19 expression in B-cell lymphoma patients.

Cell- and antibody-based CD19-directed therapies have demonstrated great potential for treating B-cell non-Hodgkin lymphoma (B-NHL). However, all these approaches suffer from limited response rates and considerable toxicity. Until now, therapy decisions have been routinely based on histopathological CD19 staining of a single lesion at initial diagnosis or relapse, disregarding heterogeneity and temporal alterations in antigen expression. To visualize in vivo CD19 expression noninvasively, we radiolabeled anti-human CD19 monoclonal antibodies with copper-64 (64Cu-αCD19) for positron emission tomography (CD19-immunoPET). 64Cu-αCD19 specifically bound to subcutaneous Daudi xenograft mouse models in vivo. Importantly, 64Cu-αCD19 did not affect the anti-lymphoma cytotoxicity of CD19 CAR-T cells in vitro. Following our preclinical validation, 64Cu-αCD19 was injected into four patients with follicular lymphoma, diffuse large B-cell lymphoma or mantle zone lymphoma. We observed varying 64Cu-αCD19 PET uptake patterns at different lymphoma sites, both within and among patients, correlating with ex vivo immunohistochemical CD19 expression. Moreover, one patient exhibited enhanced uptake in the spleen compared to that in patients with prior B-cell-depleting therapy, indicating that 64Cu-αCD19 is applicable for identifying B-cell-rich organs. In conclusion, we demonstrated the specific targeting and visualization of CD19+ B-NHL in mice and humans by CD19-immunoPET. The intra- and interindividual heterogeneous 64Cu-αCD19 uptake patterns of lymphoma lesions indicate variability in CD19 expression, suggesting the potential of CD19-immunoPET as a novel tool to guide CD19-directed therapies.

Impact of anesthetics on 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) uptake in animal models of cancer and inflammation.

The aim of this study was to evaluate the impact of different anesthetics on 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) uptake in carcinomas and arthritic ankles. To determine the amount of [18F]FLT uptake in subcutaneous CT26 colon carcinomas or arthritic ankles, spontaneously room air/medical air-breathing mice were anesthetized with isoflurane, a combination of medetomidine/midazolam, or ketamine/xylazine. Mice were kept conscious or anesthetized during [18F]FLT uptake before the 10-minute static positron emission tomographic (PET) investigations. [18F]FLT uptake in CT26 colon carcinomas and arthritic ankles was calculated by drawing regions of interest. We detected a significantly reduced (4.4 ± 0.9 %ID/cm3) [18F]FLT uptake in the carcinomas of ketamine/xylazine-anesthetized mice compared to the [18F]FLT-uptake in carcinomas of medetomidine/midazolam- (7.0 ± 1.5 %ID/cm3) or isoflurane-anesthetized mice (6.4 ± 1.5 %ID/cm3), whereas no significant differences were observed in arthritic ankles regardless of whether mice were anesthetized or conscious during tracer uptake. The time-activity curves of carcinomas and arthritic ankles yielded diverse [18F]FLT accumulation related to the used anesthetics. [18F]FLT uptake dynamics are different in arthritic ankles and carcinoma, and the magnitude and pharmacokinetics of [18F]FLT uptake are sensitive to anesthetics. Thus, for preclinical in vivo [18F]FLT PET studies in experimental tumor or inflammation models, we recommend the use of isoflurane anesthesia as it yields a stable tracer uptake and is easy to handle.

Effective T-cell recall responses require the taurine transporter Taut.

T-cell activation and the subsequent transformation of activated T cells into T-cell blasts require profound changes in cell volume. However, the impact of cell volume regulation for T-cell immunology has not been characterized. Here we studied the role of the cell-volume regulating osmolyte transporter Taut for T-cell activation in Taut-deficient mice. T-cell mediated recall responses were severely impaired in taut(-/-) mice as shown with B16 melanoma rejection and hapten-induced contact hypersensitivity. CD4(+) and CD8(+) T cells were unequivocally located within peripheral lymph nodes of unprimed taut(-/-) mice but significantly decreased in taut(-/-) compared with taut(+/+) mice following in vivo activation. Further analysis revealed that Taut is critical for rescuing T cells from activation-induced cell death in vitro and in vivo as shown with TCR, superantigen, and antigen-specific activation. Consequently, reduction of CD4(+) and CD8(+) T cells in taut(-/-) mice upon antigen challenge resulted in impaired in vivo generation of T-cell memory. These findings disclose for the first time that volume regulation in T cells is an element in the regulation of adaptive immune responses and that the osmolyte transporter Taut is crucial for T-cell survival and T-cell mediated immune reactions.

PTEN deficiency in mast cells causes a mastocytosis-like proliferative disease that heightens allergic responses and vascular permeability.

Kit regulation of mast cell proliferation and differentiation has been intimately linked to the activation of phosphatidylinositol 3-OH kinase (PI3K). The activating D816V mutation of Kit, seen in the majority of mastocytosis patients, causes a robust activation of PI3K signals. However, whether increased PI3K signaling in mast cells is a key element for their in vivo hyperplasia remains unknown. Here we report that dysregulation of PI3K signaling in mice by deletion of the phosphatase and tensin homolog (Pten) gene (which regulates the levels of the PI3K product, phosphatidylinositol 3,4,5-trisphosphate) caused mast cell hyperplasia and increased numbers in various organs. Selective deletion of Pten in the mast cell compartment revealed that the hyperplasia was intrinsic to the mast cell. Enhanced STAT5 phosphorylation and increased expression of survival factors, such as Bcl-XL, were observed in PTEN-deficient mast cells, and these were further enhanced by stem cell factor stimulation. Mice carrying PTEN-deficient mast cells also showed increased hypersensitivity as well as increased vascular permeability. Thus, Pten deletion in the mast cell compartment results in a mast cell proliferative phenotype in mice, demonstrating that dysregulation of PI3K signals is vital to the observed mast cell hyperplasia.

Preclinical Identification Of Tumor-Draining Lymph Nodes Using a Multimodal Non-invasive In vivo Imaging Approach.

PURPOSE: Resection of the tumor-draining lymph -node (TDLN) represents a standard method to identify metastasis for several malignancies. Interestingly, recent preclinical studies indicate that TDLN resection diminishes the efficacy of immune checkpoint inhibitor-based cancer immunotherapies. Thus, accurate preclinical identification of TDLNs is pivotal to uncovering the underlying immunological mechanisms. Therefore, we validated preclinically, and clinically available non-invasive in vivo imaging approaches for precise TDLN identification. PROCEDURES: For visualization of the lymphatic drainage into the TDLNs by non-invasive in vivo optical imaging, we injected the optical imaging contrast agents Patent Blue V (582.7 g mol-1) and IRDye® 800CW polyethylene glycol (PEG; 25,000-60,000 g mol-1), subcutaneously (s.c.) in close proximity to MC38 adenocarcinomas at the right flank of experimental mice. For determination of the lymphatic drainage and the glucose metabolism in TDLNs by non-invasive in vivo PET/magnetic resonance imaging (PET/MRI), we injected the positron emission tomography (PET) tracer (2-deoxy-2[18F]fluoro-D-glucose (18F-FDG) [181.1 g mol-1]) in a similar manner. For ex vivo cross-correlation, we isolated TDLNs and contralateral nontumor-draining lymph nodes (NTDLNs) and performed optical imaging, biodistribution, and autoradiography analysis. RESULTS: The clinically well-established Patent Blue V was superior for intraoperative macroscopic identification of the TDLNs compared with IRDye® 800CW PEG but was not sensitive enough for non-invasive in vivo detection by optical imaging. Ex vivo Patent Blue V biodistribution analysis clearly identified the right accessory axillary and the proper axillary lymph node (LN) as TDLNs, whereas ex vivo IRDye® 800CW PEG completely failed. In contrast, functional non-invasive in vivo 18F-FDG PET/MRI identified a significantly elevated uptake exclusively within the ipsilateral accessory axillary TDLN of experimental mice and was able to differentiate between the accessory axillary and the proper LN. Ex vivo biodistribution and autoradiography confirmed our in vivo 18F-FDG PET/MRI results. CONCLUSIONS: When taken together, our results demonstrate the feasibility of 18F-FDG-PET/MRI as a valid method for non-invasive in vivo, intraoperative, and ex vivo identification of the lymphatic drainage and glucose metabolism within the TDLNs. In addition, using Patent Blue V provides additive value for the macroscopic localization of the lymphatic drainage both visually and by ex vivo optical imaging analysis. Thus, both methods are valuable, easy to implement, and cost-effective for preclinical identification of the TDLN.