Scaffold-Based (Matrigel™) 3D Culture Technique of Glioblastoma Recovers a Patient-like Immunosuppressive Phenotype

Conventional 2D cultures are commonly used in cancer research though they come with limitations such as the lack of microenvironment or reduced cell heterogeneity. In this study, we investigated in what respect a scaffold-based (Matrigel™) 3D culture technique can ameliorate the limitations of 2D cultures. NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in around 36% of 3D models, indicating the reoccurrence of an immune suppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. We also investigated the 3D tumor organoids in a co-culture setting with tumor-infiltrating lymphocytes (TILs). Of note, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release, as well as live cell staining and proliferation analysis, pointed toward an elevated resistance of 3D models. In conclusion, we found that the scaffold-based (Matrigel™) 3D culture technique affects the transcriptional profile in a subset of GBM models. Thus, these models allow for depicting clinically relevant aspects of tumor-immune interaction, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system

Diacylglycerol triggers Rim101 pathway dependent necrosis in yeast: a model for lipotoxicity

The loss of lipid homeostasis can lead to lipid overload and is associated with a variety of disease states. However, little is known as to how the disruption of lipid regulation or lipid overload affects cell survival. In this study we investigated how excess diacylglycerol (DG), a cardinal metabolite suspected to mediate lipotoxicity, compromises the survival of yeast cells. We reveal that increased DG achieved by either genetic manipulation or pharmacological administration of 1,2-dioctanoyl-sn-glycerol (DOG) triggers necrotic cell death. The toxic effects of DG are linked to glucose metabolism and require a functional Rim101 signaling cascade involving the Rim21 dependent sensing complex and activation of a calpain-like protease. The Rim101 cascade is an established pathway that triggers a transcriptional response to alkaline or lipid stress. We propose that the Rim101 pathway senses DG-induced lipid perturbation and conducts a signaling response that either facilitates cellular adaptation or triggers lipotoxic cell death. Using established models of lipotoxicity i.e. high fat diet in Drosophila and palmitic acid administration in cultured human endothelial cells, we present evidence that the core mechanism underlying this calpain-dependent lipotoxic cell death pathway is phylogenetically conserved

Induction of autophagy by spermidine promotes longevity.

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MAPK-pathway inhibition mediates inflammatory reprogramming and sensitizes tumors to targeted activation of innate immunity sensor RIG-I.

Kinase inhibitors suppress the growth of oncogene driven cancer but also enforce the selection of treatment resistant cells that are thought to promote tumor relapse in patients. Here, we report transcriptomic and functional genomics analyses of cells and tumors within their microenvironment across different genotypes that persist during kinase inhibitor treatment. We uncover a conserved, MAPK/IRF1-mediated inflammatory response in tumors that undergo stemness- and senescence-associated reprogramming. In these tumor cells, activation of the innate immunity sensor RIG-I via its agonist IVT4, triggers an interferon and a pro-apoptotic response that synergize with concomitant kinase inhibition. In humanized lung cancer xenografts and a syngeneic Egfr-driven lung cancer model these effects translate into reduction of exhausted CD8+ T cells and robust tumor shrinkage. Overall, the mechanistic understanding of MAPK/IRF1-mediated intratumoral reprogramming may ultimately prolong the efficacy of targeted drugs in genetically defined cancer patients

Noncanonical expression of a murine cytomegalovirus early protein CD8 T-cell epitope as an immediate early epitope based on transcription from an upstream gene

Viral CD8 T-cell epitopes, represented by viral peptides bound to major histocompatibility complex class-I (MHC-I) glycoproteins, are often identified by "reverse immunology", a strategy not requiring biochemical and structural knowledge of the actual viral protein from which they are derived by antigen processing. Instead, bioinformatic algorithms predicting the probability of C-terminal cleavage in the proteasome, as well as binding affinity to the presenting MHC-I molecules, are applied to amino acid sequences deduced from predicted open reading frames (ORFs) based on the genomic sequence. If the protein corresponding to an antigenic ORF is known, it is usually inferred that the kinetic class of the protein also defines the phase in the viral replicative cycle during which the respective antigenic peptide is presented for recognition by CD8 T cells. We have previously identified a nonapeptide from the predicted ORFm164 of murine cytomegalovirus that is presented by the MHC-I allomorph H-2 Dd and that is immunodominant in BALB/c (H-2d haplotype) mice. Surprisingly, although the ORFm164 protein gp36.5 is expressed as an Early (E) phase protein, the m164 epitope is presented already during the Immediate Early (IE) phase, based on the expression of an upstream mRNA starting within ORFm167 and encompassing ORFm164

Image_1_Modulation of cytomegalovirus immune evasion identifies direct antigen presentation as the predominant mode of CD8 T-cell priming during immune reconstitution after hematopoietic cell transplantation.tif

Cytomegalovirus (CMV) infection is the most critical infectious complication in recipients of hematopoietic cell transplantation (HCT) in the period between a therapeutic hematoablative treatment and the hematopoietic reconstitution of the immune system. Clinical investigation as well as the mouse model of experimental HCT have consistently shown that timely reconstitution of antiviral CD8 T cells is critical for preventing CMV disease in HCT recipients. Reconstitution of cells of the T-cell lineage generates naïve CD8 T cells with random specificities among which CMV-specific cells need to be primed by presentation of viral antigen for antigen-specific clonal expansion and generation of protective antiviral effector CD8 T cells. For CD8 T-cell priming two pathways are discussed: “direct antigen presentation” by infected professional antigen-presenting cells (pAPCs) and “antigen cross-presentation” by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Current view in CMV immunology favors the cross-priming hypothesis with the argument that viral immune evasion proteins, known to interfere with the MHC class-I pathway of direct antigen presentation by infected cells, would inhibit the CD8 T-cell response. While the mode of antigen presentation in the mouse model of CMV infection has been studied in the immunocompetent host under genetic or experimental conditions excluding either pathway of antigen presentation, we are not aware of any study addressing the medically relevant question of how newly generated naïve CD8 T cells become primed in the phase of lympho-hematopoietic reconstitution after HCT. Here we used the well-established mouse model of experimental HCT and infection with murine CMV (mCMV) and pursued the recently described approach of up- or down-modulating direct antigen presentation by using recombinant viruses lacking or overexpressing the central immune evasion protein m152 of mCMV, respectively. Our data reveal that the magnitude of the CD8 T-cell response directly reflects the level of direct antigen presentation.</p

Infection induces a wave of mast cell-derived chemokine CCL5/RANTES <i>in vivo</i>.

<p>Serum levels of chemokine CCL5 were determined in a time course after intravenous infection of MC-sufficient WT C57BL/6 mice compared to congenic MC-deficient <i>Kit^W-sh/W-sh</i> (<i>Kit^W-sh</i>“or “sash”) mutants. Symbols represent data (mean values of duplicate serum aliquots) from individual mice revealing sample sizes and ranges, with the median values indicated. P values are given for comparisons of interest, unless the difference is obvious.</p

Degranulation of mast cells (MC) is triggered by direct infection <i>in vivo</i>.

<p>(A) MC are first-hit target cells of mCMV infection <i>in vivo</i>. Peritoneal exudate cells (PEC) were recovered from the peritoneal cavity at 4 h or 24 h after intraperitoneal infection with fluorescent reporter virus mCMV-GFP. This timing does not allow completion of the viral infection cycle and thus precludes virus spread to secondary target cells. In the cytofluorometric analysis, a gate was set on FcεRI⁺CD117⁺ MC for identification of infected MC expressing the reporter GFP. (B) Selective degranulation of infected, GFP⁺ MC. PEC were pre-gated for expression of FcεRI and analyzed for the expression of CD117 and the degranulation marker CD107a. GFP-expression in FcεRI⁺CD117⁺CD107a⁺ MC identifies degranulating MC as those which are infected. The black-shaded area represents cells within the gate of CD107a⁺ MC, the gray-shaded area represents the “reverse gate” of MC not or no longer expressing CD107a. Shown are the original data from one mouse as an example. Percentages of degranulating CD107a⁺ MC of n = 4 mice tested individually ranged between 9.0% and 24.1% with a mean value of 17.5% and a median value of 18.5%.</p

Activated virus epitope-specific CD8 T cells transmigrate from the vascular compartment into lung interstitium and alveolar epithelium.

<p>(A) Localization of CD3⁺ T cells in intravascular (in) and extravascular (ex) compartments of the lungs. Shown are 2-color immunohistological images of lung tissue sections taken on day 6 after infection of C57BL/6 mice, with endothelial cells being identified by black staining of CD31 and T cells by turquoise-green staining of CD3ε. (v) small vessel; (a) pulmonary alveoli; (b) pulmonary bronchiole lined with bronchiolar epithelium; (s) alveolar septum; (c) capillary in a thickened alveolar septum. The bar marker represents 25 µm. (B) Frequencies of viral epitope-specific, IFNγ-secreting CD8 T cells among CD45⁺ leucocytes in different compartments. CD8 T cells recovered by BAL are those that are only loosely attached to the epithelial lining of the alveoli or bronchioles and are thus definitely located outside of the lung vasculature. Shown are data from ELISpot assays using stimulator cells exogenously-loaded with saturating concentrations of antigenic peptides representing the epitopes indicated. Bars represent most probable numbers determined by intercept-free linear regression analysis and error bars represent the 95% confidence intervals.</p

MC-deficiency is associated with less efficient recruitment of T cells to the infected lungs.

<p>Pulmonary infiltrates were analyzed 6 days after intravenous infection of MC-sufficient WT C57BL/6 and MC-deficient <i>Kit^W-sh</i> mice. (A) Representative immunohistological images show pulmonary T cell (with a negligible contribution of NKT cells, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004100#ppat-1004100-g001" target="_blank">Figure 1B</a>) infiltrates <i>in situ</i> by black staining of CD3ε, comparing lung tissue sections from WT C57BL/6 (left panel) and <i>Kit^W-sh</i> (right panel) mice. Bar markers represent 50 µm. (B) Comparative analysis of absolute quantities of CD3ε⁺ cells per lung, determined by counting of stained cells in tissue sections extrapolated to the whole organ. Symbols (closed circles: WT C57BL/6; open circles: <i>Kit^W-sh</i> mutant) represent data from individual mice revealing sample sizes and ranges, with the median values indicated.</p