A BAFF ligand-based CAR-T cell targeting three receptors and multiple B cell cancers.

B cell-activating factor (BAFF) binds the three receptors BAFF-R, BCMA, and TACI, predominantly expressed on mature B cells. Almost all B cell cancers are reported to express at least one of these receptors. Here we develop a BAFF ligand-based chimeric antigen receptor (CAR) and generate BAFF CAR-T cells using a non-viral gene delivery method. We show that BAFF CAR-T cells bind specifically to each of the three BAFF receptors and are effective at killing multiple B cell cancers, including mantle cell lymphoma (MCL), multiple myeloma (MM), and acute lymphoblastic leukemia (ALL), in vitro and in vivo using different xenograft models. Co-culture of BAFF CAR-T cells with these tumor cells results in induction of activation marker CD69, degranulation marker CD107a, and multiple proinflammatory cytokines. In summary, we report a ligand-based BAFF CAR-T capable of binding three different receptors, minimizing the potential for antigen escape in the treatment of B cell cancers.

Engineered Hydroxyapatite Nanoadjuvants with Controlled Shape and Aspect Ratios Reveal Their Immunomodulatory Potentials.

Hydroxyapatite (HAP) has been formulated as adjuvants in vaccines for human use. However, the optimal properties required for HAP nanoparticles to elicit adjuvanticity and the underlying immunopotentiation mechanisms have not been fully elucidated. Herein, a library of HAP nanorods and nanospheres was synthesized to explore the effect of the particle shape and aspect ratio on the immune responses in vitro and adjuvanticity in vivo. It was demonstrated that long aspect ratio HAP nanorods induced a higher degree of cell membrane depolarization and subsequent uptake, and the internalized particles elicited cathepsin B release and mitochondrial reactive oxygen species generation, which further led to pro-inflammatory responses. Furthermore, the physicochemical property-dependent immunostimulation capacities were correlated with their humoral responses in a murine hepatitis B surface antigen immunization model, with long aspect ratio HAP nanorods inducing higher antigen-specific antibody productions. Importantly, HAP nanorods significantly up-regulated the IFN-γ secretion and CD107α expression on CD8+ T cells in immunized mice. Further mechanistic studies demonstrated that HAP nanorods with defined properties exerted immunomodulatory effects by enhanced antigen persistence and immune cell recruitments. Our study provides a rational design strategy for engineered nanomaterial-based vaccine adjuvants.

Regulation of lipid homeostasis by the TBC protein dTBC1D22 via modulation of the small GTPase Rab40 to facilitate lipophagy.

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy.

Circ-LAMP1 contributes to the growth and metastasis of cholangiocarcinoma via miR-556-5p and miR-567 mediated YY1 activation.

Dysregulation of circular RNAs (circRNAs) executes important regulatory roles in carcinogenesis. Nonetheless, few studies focused on the mechanisms of circRNAs in cholangiocarcinoma (CCA). qRT-PCR was applied to verify the dysregulated circRNAs in CCA. Fisher's exact test, Kaplan-Meier analysis and Cox regression model were utilized to investigate the clinical implications of circ-LAMP1 in the patients with CCA. The viability, apoptosis, migration and invasion of CCA cells were detected after silencing/overexpression of circ-LAMP1. Xenograft and lung metastasis assays were performed to verify the in vitro results. The regulatory networks of circ-LAMP1 were unveiled by bioinformatic analysis, RNA immunoprecipitation (RIP), RNA pulldown and luciferase reporter assays. Up-regulation of circ-LAMP1 was found in CCA tissue samples and cell lines. Enhanced level of circ-LAMP1 was linked to clinical severity, high post-operative recurrence and poor prognosis for the patients with CCA. Gain/loss-of-function assays confirmed the oncogenic role of circ-LAMP1 in mediating cell growth, apoptosis, migration and invasion. Nevertheless, the level of circ-LAMP1 had no effect on normal biliary epithelium proliferation and apoptosis. Animal study further verified the in vitro data. Mechanistically, circ-LAMP1 directly sponged miR-556-5p and miR-567, thereby releasing their suppression on YY1 at post-transcriptional level. Rescue assay indicated that the oncogenic role of circ-LAMP1 is partially dependent on its modulation of YY1 in CCA. In summary, this study suggested that circ-LAMP1 might be used as a promising biomarker/therapeutic target for CCA.

Lysosomal protein surface expression discriminates fat- from bone-forming human mesenchymal precursor cells.

Tissue resident mesenchymal stem/stromal cells (MSCs) occupy perivascular spaces. Profiling human adipose perivascular mesenchyme with antibody arrays identified 16 novel surface antigens, including endolysosomal protein CD107a. Surface CD107a expression segregates MSCs into functionally distinct subsets. In culture, CD107alow cells demonstrate high colony formation, osteoprogenitor cell frequency, and osteogenic potential. Conversely, CD107ahigh cells include almost exclusively adipocyte progenitor cells. Accordingly, human CD107alow cells drove dramatic bone formation after intramuscular transplantation in mice, and induced spine fusion in rats, whereas CD107ahigh cells did not. CD107a protein trafficking to the cell surface is associated with exocytosis during early adipogenic differentiation. RNA sequencing also suggested that CD107alow cells are precursors of CD107ahigh cells. These results document the molecular and functional diversity of perivascular regenerative cells, and show that relocation to cell surface of a lysosomal protein marks the transition from osteo- to adipogenic potential in native human MSCs, a population of substantial therapeutic interest.

The Oncometabolite 5'-Deoxy-5'-Methylthioadenosine Blocks Multiple Signaling Pathways of NK Cell Activation.

Tumor cells develop various mechanisms to escape immune surveillance. In this context, oncometabolites secreted by tumor cells due to deregulated metabolic pathways, have been in the spotlight of researchers during the last years. 5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase (MTAP) deficiency in tumors results in the accumulation of MTA within the tumor microenvironment and thereby negatively influencing immune functions of various immune cells, including T and NK cells. The influence of MTA on T cell activation has been recently described in more detail, while its impact on NK cells is still largely unknown. Therefore, we aimed to illuminate the molecular mechanism of MTA-induced NK cell dysfunction. NK cell cytotoxicity against target cells was reduced in the presence of MTA in a dose-dependent manner, while NK cell viability remained unaffected. Furthermore, we revealed that MTA blocks NK cell degranulation and cytokine production upon target cell engagement as well as upon antibody stimulation. Interestingly, the immune-suppressive effect of MTA was less pronounced in healthy donors harboring an expansion of NKG2C+ NK cells. Finally, we demonstrated that MTA interferes with various signaling pathways downstream of the CD16 receptor upon NK cell activation, including the PI3K/AKT/S6, MAPK/ERK, and NF-κB pathways. In summary, we revealed that MTA blocks NK cell functions like cytotoxicity and cytokine production by interfering with the signaling cascade of activating NK cell receptors. Specific targeting of MTA metabolism in MTAP-deficient tumors therefore could offer a promising new strategy to reverse immune dysfunction of NK cells within the tumor microenvironment.

Evaluation of HIV-specific T-cell responses in HIV-infected older patients with controlled viremia on long-term antiretroviral therapy.

HIV-infected older individuals may have a diminished immune response because of exhaustion/immune aging of T-cells. Therefore, we have investigated HIV-specific CD4 and CD8 T-cell responses in 100 HIV-infected patients (HIV+) who have aged on long-term antiretroviral therapy (ART) and achieved controlled viremia (mostly undetectable viral load; 92 patients with <20 to <40 HIV RNA copies/mL and 8 <60 to <100) and improved CD4 T-cell counts. We show that the median frequencies of HIV-specific CD4+ and CD8+ IFN-γ T-cells were higher in HIV+ than uninfected individuals (HIV-), including increasing levels of IFN-γproduced by CD4+ T-cells and decreasing levels by CD8+ T-cells with increasing CD4 T-cell counts in HIV+. No correlation was found between T-cell responses and varying levels of undetectable viremia. HIV-specific TNF-α made by CD8+ T-cells was higher in HIV+ than HIV-, including decreasing levels with increasing CD4 T-cell counts in HIV+. Furthermore, the CD8+ T-cell mediators, CD107a and Granzyme-B, were higher in HIV+ than HIV-, and decreased with increasing CD4 T-cell counts in HIV+. Remarkably, HIV-specific CD8 T-cells produced decreasing levels of IFN-γwith increasing age of HIV+, including decreased levels of CD107a and Granzyme-B in older HIV+. However, HIV-specific CD8+ T-cells produced increasing levels of TNF-α with increasing age of the HIV+, suggesting continued inflammation. In conclusion, HIV+ with controlled viremia on long-term ART and with higher CD4 T-cell counts showed reduced HIV-specific CD8 T-cell responses as compared to those with lower CD4 T-cell counts, and older HIV+ exhibited decreasing levels of CD8 T-cell responses with increasing age.

The potential markers of NK-92 associated to cytotoxicity against K562 cells.

Markers associated to NK cytolytic activity are in a great need to regulate NK cell immunotherapy products. We assume that biomarkers which response to cytolysis will change their transcription, expression or secretion. To find NK-92 indicator to cytolytic activity, we have evaluated the potential markers by quantifying the expression of well-known cytotoxicity functional molecules (cytokine IFN-γ, Granzyme B, perforin, CD69 and CD107a), and explored candidate markers by a sweeping transcription picture of NK-92 using a direct cytolysis model (incubation with K562). We found that IFN-γ secretion was highly correlated to cytotoxicity of NK-92, neither Granzyme B, perforin secretion, nor CD69, CD107a positive population were upregulated by K562 stimulation. RNAseq revealed 432 genes expression changed during cytolysis, several genes (BIRC3, CSF2, VCAM1 and TNFRSF9) mRNA expression were validated by real time RT-PCR under K562 being killed or protected from being killed conditions. Results suggested IFN-γ secretion, BIRC3 and TNFRSF9 transcription in NK-92 were responsive to K562 cytolysis. In a word, our results confirmed one marker and reveal an array of novel candidate markers associated with NK-92 cytotoxicity. Further studies are greatly needed to determine the roles these new makers play in NK-92 cytolysis process.

PD-1-positive Natural Killer Cells have a weaker antitumor function than that of PD-1-negative Natural Killer Cells in Lung Cancer.

Antibodies targeting the immune checkpoint inhibitor, programmed cell death 1 (PD-1), have provided a breakthrough in the treatment of lung cancer. However, the function of PD-1 in natural killer (NK) cells of cancer patients remains unclear. Herein, we analyzed the expression of PD-1 on the NK cells in the peripheral blood of patients with lung cancer and found that the level of PD-1+ NK cells in patients was significantly higher than that in healthy individuals. Moreover, these PD-1+ NK cells demonstrated a weaker ability to secrete interferon-gamma (INF-γ), granzyme B, and perforin, and exhibited lower CD107a expression. Importantly, in patients with lung cancer, the percentage of PD-1+ NK cells was significantly positively correlated with the concentration of IL-2 in the plasma, which was also higher than that in healthy individuals. In addition, IL-2 could increase the expression of PD-1 on NK cells in vitro, indicating that high IL-2 level in the plasma is largely responsible for the abundance of PD-1+ NK cells in patients with lung cancer. These findings demonstrate intriguing mechanisms for understanding the expression of PD-1 on NK cells and the function of PD-1+ NK cells in lung cancer. This study confirms and extends previous studies demonstrating that PD-1 can negatively regulate the antitumor function of NK cells.

NK cells: A double edge sword against SARS-CoV-2.

Natural killer (NK) cells are pivotal effectors of the innate immunity protecting an individual from microbes. They are the first line of defense against invading viruses, given their substantial ability to directly target infected cells without the need for specific antigen presentation. By establishing cellular networks with a variety of cell types such as dendritic cells, NK cells can also amplify and modulate antiviral adaptive immune responses. In this review, we will examine the role of NK cells in SARS-COV2 infections causing the ongoing COVID19 pandemic, keeping in mind the controversial role of NK cells specifically in viral respiratory infections and in inflammatory-driven lung damage. We discuss lessons learnt from previous coronavirus outbreaks in humans (caused by SARS-CoV-1 and MERS-COV).

Severe Human Lassa Fever Is Characterized by Nonspecific T-Cell Activation and Lymphocyte Homing to Inflamed Tissues.

Lassa fever (LF) is a zoonotic viral hemorrhagic fever caused by Lassa virus (LASV), which is endemic to West African countries. Previous studies have suggested an important role for T-cell-mediated immunopathology in LF pathogenesis, but the mechanisms by which T cells influence disease severity and outcome are not well understood. Here, we present a multiparametric analysis of clinical immunology data collected during the 2017-2018 Lassa fever outbreak in Nigeria. During the acute phase of LF, we observed robust activation of the polyclonal T-cell repertoire, which included LASV-specific and antigenically unrelated T cells. However, severe and fatal LF cases were characterized by poor LASV-specific effector T-cell responses. Severe LF was also characterized by the presence of circulating T cells with homing capacity to inflamed tissues, including the gut mucosa. These findings in LF patients were recapitulated in a mouse model of LASV infection, in which mucosal exposure resulted in remarkably high lethality compared to skin exposure. Taken together, our findings indicate that poor LASV-specific T-cell responses and activation of nonspecific T cells with homing capacity to inflamed tissues are associated with severe LF.IMPORTANCE Lassa fever may cause severe disease in humans, in particular in areas of endemicity like Sierra Leone and Nigeria. Despite its public health importance, the pathophysiology of Lassa fever in humans is poorly understood. Here, we present clinical immunology data obtained in the field during the 2018 Lassa fever outbreak in Nigeria indicating that severe Lassa fever is associated with activation of T cells antigenically unrelated to Lassa virus and poor Lassa virus-specific effector T-cell responses. Mechanistically, we show that these bystander T cells express defined tissue homing signatures that suggest their recruitment to inflamed tissues and a putative role of these T cells in immunopathology. These findings open a window of opportunity to consider T-cell targeting as a potential postexposure therapeutic strategy against severe Lassa fever, a hypothesis that could be tested in relevant animal models, such as nonhuman primates.

Abnormal Lysosomal Positioning and Small Extracellular Vesicle Secretion in Arterial Stiffening and Calcification of Mice Lacking Mucolipin 1 Gene.

Recent studies have shown that arterial medial calcification is mediated by abnormal release of exosomes/small extracellular vesicles from vascular smooth muscle cells (VSMCs) and that small extracellular vesicle (sEV) secretion from cells is associated with lysosome activity. The present study was designed to investigate whether lysosomal expression of mucolipin-1, a product of the mouse Mcoln1 gene, contributes to lysosomal positioning and sEV secretion, thereby leading to arterial medial calcification (AMC) and stiffening. In Mcoln1-/- mice, we found that a high dose of vitamin D (Vit D; 500,000 IU/kg/day) resulted in increased AMC compared to their wild-type littermates, which was accompanied by significant downregulation of SM22-α and upregulation of RUNX2 and osteopontin in the arterial media, indicating a phenotypic switch to osteogenic. It was also shown that significantly decreased co-localization of lysosome marker (Lamp-1) with lysosome coupling marker (Rab 7 and ALG-2) in the aortic wall of Mcoln1-/- mice as compared to their wild-type littermates. Besides, Mcoln1-/- mice showed significant increase in the expression of exosome/ sEV markers, CD63, and annexin-II (AnX2) in the arterial medial wall, accompanied by significantly reduced co-localization of lysosome marker (Lamp-1) with multivesicular body (MVB) marker (VPS16), suggesting a reduction of the lysosome-MVB interactions. In the plasma of Mcoln1-/- mice, the number of sEVs significantly increased as compared to the wild-type littermates. Functionally, pulse wave velocity (PWV), an arterial stiffening indicator, was found significantly increased in Mcoln1-/- mice, and Vit D treatment further enhanced such stiffening. All these data indicate that the Mcoln1 gene deletion in mice leads to abnormal lysosome positioning and increased sEV secretion, which may contribute to the arterial stiffness during the development of AMC.

Functional characterization of NK cells in Mexican pediatric patients with acute lymphoblastic leukemia: Report from the Mexican Interinstitutional Group for the Identification of the Causes of Childhood Leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children around the globe. Mexico City has one of the highest incidence rates of childhood leukemia worldwide with 49.5 cases per million children under the age of 15 which is similar to that reported for Hispanic populations living in the United States. In addition, it has been noted a dismal prognosis in Mexican and Hispanic ALL pediatric population. Although ALL, like cancer in general, has its origins in endogenous, exogenous, and genetic factors, several studies have shown that the immune system also plays a deterministic role in cancer development. Among various elements of the immune system, T lymphocytes and NK cells seem to dominate the immune response against leukemia. The aim of the present study was to perform a phenotypic and functional characterization of NK cells in ALL Mexican children at the moment of diagnosis and before treatment initiation. A case-control study was conducted by the Mexican Interinstitutional Group for the Identification of the Causes of Childhood Leukemia (MIGICCL). 41 cases were incident ALL children younger than 17 years old and residents of Mexico City. 14 controls were children without leukemia, matched by age and sex with cases. NK cell function was evaluated by degranulation assays towards K562 cells and SLAM-associated protein (SAP) expression was measured by intracellular staining. All assays were performed using peripheral blood mononuclear cells from controls and patients. The results indicate that NK mediated cytotoxicity, measured by CD107a degranulation assays in response to K562 cells, was reduced in ALL patients compared to controls. Interestingly, an impaired NK cell killing of target cells was not equally distributed among ALL patients. In contrast to patients classified as high-risk, standard-risk patients did not display a significant reduction in NK cell-mediated cytotoxicity. Moreover, patients presenting a leukocyte count ≥ 50,000xmm3 displayed a reduction in NK-cell mediated cytotoxicity and a reduction in SAP expression, indicating a positive correlation between a reduced SAP expression and an impaired NK cell-mediated citotoxicity. In the present study it was observed that unlike patients with standard-risk, NK cells from children presenting high-risk ALL, harbor an impaired cytotoxicity towards K562 at diagnosis. In addition, NK cell function was observed to be compromised in patients with a leukocyte count ≥50,000xmm3, where also it was noticed a decreased expression of SAP compared to patients with a leukocyte count <50,000xmm3. These data indicate NK cell-mediated cytotoxicity is not equally affected in ALL patients, nevertheless a positive correlation between low SAP expression and decreased NK cell-mediated cytotoxicity was observed in ALL patients with a leukocyte count ≥50,000xmm3. Finally, an abnormal NK cell-mediated cytotoxicity may represent a prognostic factor for high-risk acute lymphoblastic leukemia.

A Drosophila model of neuronal ceroid lipofuscinosis CLN4 reveals a hypermorphic gain of function mechanism.

The autosomal dominant neuronal ceroid lipofuscinoses (NCL) CLN4 is caused by mutations in the synaptic vesicle (SV) protein CSPα. We developed animal models of CLN4 by expressing CLN4 mutant human CSPα (hCSPα) in Drosophila neurons. Similar to patients, CLN4 mutations induced excessive oligomerization of hCSPα and premature lethality in a dose-dependent manner. Instead of being localized to SVs, most CLN4 mutant hCSPα accumulated abnormally, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructural examination revealed frequent abnormal membrane structures in axons and neuronal somata. The lethality, oligomerization and prelysosomal accumulation induced by CLN4 mutations was attenuated by reducing endogenous wild type (WT) dCSP levels and enhanced by increasing WT levels. Furthermore, reducing the gene dosage of Hsc70 also attenuated CLN4 phenotypes. Taken together, we suggest that CLN4 alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking.

Kinetics of the expression of CD163 and CD107a in the lung and tonsil of pigs after infection with PRRSV-1 strains of different virulence.

The emergence of virulent strains of porcine reproductive and respiratory syndrome virus (PRRSV), causing atypical and severe outbreaks, has been notified worldwide. This study assesses the expression, distribution and kinetics of PRRSV N-protein, CD163 and CD107a in the lung and tonsil from experimentally-infected piglets with three different PRRSV-1 strains: a virulent PRRSV-1 subtype 3 strain (SU1-bel) and two low-virulent subtype 1 strains, Lelystad virus (LV) and 215-06. SU1-bel replicated more efficiently in the lungs and tonsils. The number of CD163+ cells decreased in both tissues from all infected groups at 7 dpi, followed by an increase at the end of the study, highlighting a negative correlation with the number of N-protein+-infected cells. A significant increase in CD107a was observed in all infected groups at 35 dpi but no differences were observed among them. Whereas the initial decrease of CD163+ cells appears to be associated to virus replication and cell death, the later recovery of the CD163+ population may be due to either the induction of CD163 in immature cells, the recruitment of CD163+ cells in the area of infection, or both. These results highlight the ability of macrophage subpopulations in infected animals to recover and restore their potential biological functions at one-month post-infection, with the greatest improvement observed in SU1-bel-infected animals.

The Role of NK Cells in the Control of Viral Infection in HTLV-1 Carriers.

The cytotoxic activities of CD8+ T cells have been considered the main defense mechanism against the human T lymphotropic virus type 1 (HTLV-1). As with CD8+ T cells, NK cells can perform cytotoxic degranulation with production of cytotoxic mediators, such as perforins and granzymes. NK cells are also responsible for antibody-dependent cellular cytotoxicity (ADCC) against infected cells, but few studies have evaluated the role of NK cells in HTLV-1 infection. The aim of this study was to characterize the subsets and measure the frequency of NK cells in HTLV-1 carriers (HC) and in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and correlate these findings with the proviral load and development of HAM/TSP. The diagnosis of HTLV-1 infection was performed with a detection antibody against viral antigens by ELISA and confirmed by Western blot. Phenotypic characterization of NK cells was performed by flow cytometry. The frequencies of CD56+, CD56+CD3-, CD56+CD16+, and CD56dim cells were decreased in HAM/TSP patients. The frequency of CD56+CD3- cells was inversely correlated with proviral load in HC but not in HAM/TSP patients. HAM/TSP patients showed decreased frequency of CD56+ and CD56dim cells expressing CD16, the main receptor for ADCC. These data indicate that NK cells may play a key role in the control of HTLV-1 infection by preventing the progression of HC to HAM/TSP.

miR-20a regulates sensitivity of colorectal cancer cells to NK cells by targeting MICA.

Colorectal cancer (CRC) is one of the leading cancer-related causes of deaths in the world. Recently, microRNAs have been reported to regulate the tumor growth, invasion and the immunosuppression. In the present study, we found that miR-20a was increased in human CRC specimens compared with the healthy normal tissues. However, miR-20a overexpression and knockdown did not impair the CRC cell growth in vitro Our results indicated that CD107a+ NK cells are increased in CRC group. Furthermore, cytotoxicity assays demonstrated that miR-20a knockdown promoted the CRC cells sensitive to NK cells, whereas miR-20a overexpression showed the opposite results. Our results suggest that the regulation of NK cells by miR-20a depends on NKG2D. Luciferase reporter assays revealed that the NKG2D ligand Major Histocompatibility Complex (MHC) class I-related chain genes A (MICA) is the direct target of miR-20a. Flow cytometry showed the MICA protein level is significantly reduced in miR-20a-overexpressing CRC cells and increased in miR-20a knockdown CRC cells. Taken together, our results suggest that miR-20a regulates sensitivity of CRC cells to NK cells by targeting MICA.

Impact of autophagic regulation on splenic red pulp macrophages during cerebral malarial infection.

Splenic red pulp macrophages play a critical role infiltration of infected RBC and elimination of pathogens during malarial infection. However, the efficiency of pathogenic processing and the intricate pathway followed by them to boost the downstream immune response has not been studied in details. We checked the status of autophagic regulation within the cells both before and after the infection and also modulated the autophagic flux with either its inducer or inhibitor. We found that the upregulation of autophagic gene and the corresponding pathway is correlated with better parasite clearance and survivability, with an enhanced downstream immune response. It also increases their phagocytic potential with better Lysosomal associated protein I and II synthesis. The autophagolysosome formation increases as well, and more vacuole bound LC3B protein are detected. Chemokine synthesized from Red Pulp macrophage helps in mediating the induction for recruiting neutrophil and CD4 + T cells to the splenic red pulp region. The skewing of M1 macrophage polarity is observed post autophagic induction with a better costimulatory molecule like CD80, CD86 expression and antigen presenting molecule MHC I, MHC II is observed. This study shows the possibility of an alternative or adjuvant therapy regimen for the malarial patient by inducing the autophagic pathway that targets the red pulp macrophages. This might be helpful for better pathogen degradation and processing. The subsequent clearance of parasite will result in a better outcome for the patients.

Abnormal glycogen storage in tuberous sclerosis complex caused by impairment of mTORC1-dependent and -independent signaling pathways.

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome that causes tumor formation in multiple organs. TSC is caused by inactivating mutations in the genes encoding TSC1/2, negative regulators of the mammalian target of rapamycin complex 1 (mTORC1). Diminished TSC function is associated with excess glycogen storage, but the causative mechanism is unknown. By studying human and mouse cells with defective or absent TSC2, we show that complete loss of TSC2 causes an increase in glycogen synthesis through mTORC1 hyperactivation and subsequent inactivation of glycogen synthase kinase 3ß (GSK3ß), a negative regulator of glycogen synthesis. Specific TSC2 pathogenic mutations, however, result in elevated glycogen levels with no changes in mTORC1 or GSK3ß activities. We identify mTORC1-independent lysosomal depletion and impairment of autophagy as the driving causes underlying abnormal glycogen storage in TSC irrespective of the underlying mutation. The defective autophagic degradation of glycogen is associated with abnormal ubiquitination and degradation of essential proteins of the autophagy-lysosome pathway, such as LC3 and lysosomal associated membrane protein 1 and 2 (LAMP1/2) and is restored by the combined use of mTORC1 and Akt pharmacological inhibitors. In complementation to current models that place mTORC1 as the central therapeutic target for TSC pathogenesis, our findings identify mTORC1-independent pathways that are dysregulated in TSC and that should therefore be taken into account in the development of a therapeutic treatment.

Remodeling of secretory lysosomes during education tunes functional potential in NK cells.

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however, the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. Secretory lysosomes are part of the acidic lysosomal compartment that mediates intracellular signalling in several cell types. Here we show that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) accumulate granzyme B in dense-core secretory lysosomes that converge close to the centrosome. This discrete morphological phenotype is independent of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. Meanwhile, interference of signaling from acidic Ca2+ stores in primary NK cells reduces target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis, or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1, leads to increased granzyme B and enhanced functional potential, thereby mimicking the educated state. These results indicate an intrinsic role for lysosomal remodeling in NK cell education.