Cells need to adapt to dynamic environments. Yeast that fail to cope with dynamic changes in the abundance of glucose can undergo growth arrest. We show that this failure is caused by imbalanced ...reactions in glycolysis, the essential pathway in energy metabolism in most organisms. The imbalance arises largely from the fundamental design of glycolysis, making this state of glycolysis a generic risk. Cells with unbalanced glycolysis coexisted with vital cells. Spontaneous, nongenetic metabolic variability among individual cells determines which state is reached and, consequently, which cells survive. Transient ATP (adenosine 5'-triphosphate) hydrolysis through futile cycling reduces the probability of reaching the imbalanced state. Our results reveal dynamic behavior of glycolysis and indicate that cell fate can be determined by heterogeneity purely at the metabolic level.
During T cell-dependent (TD) germinal center (GC) responses, naïve B cells are instructed to differentiate towards GC B cells (GCBC), high-affinity long-lived plasma cells (LLPC) or memory B cells ...(Bmem). Alterations in the B cell-fate choice could contribute to immune dysregulation leading to the loss of self-tolerance and the initiation of autoimmune disease. Here we show that mRNA levels of the transcription regulator BOB.1 are increased in the lymph node compartment of patients with rheumatoid arthritis (RA), a prototypical autoimmune disease caused by the loss of immunological tolerance. Investigating to what extent levels of BOB.1 impact B cells during TD immune responses we found that BOB.1 has a crucial role in determining the B cell-fate decision. High BOB.1 levels promote the generation of cells with phenotypic and functional characteristics of Bmem. Mechanistically, overexpression of BOB.1 drives ABF1 and suppresses BCL6, favouring Bmem over LLPC or recycling GCBC. Low levels of BOB.1 are sufficient for LLPC but not for Bmem differentiation. Our findings demonstrate a novel role for BOB.1 in B cells during TD GC responses and suggest that its dysregulation may contribute to the pathogenesis of RA by disturbing the B cell-fate determination.
•The transcription regulator BOB.1 is aberrantly expressed in lymph nodes of patients with rheumatoid arthritis.•BOB.1 a crucial role in determining the B cell-fate decision.•High BOB.1 levels promote the generation of cells with phenotypic and functional characteristics of Bmem.•Enforced overexpression of BOB.1 favours formation of Bmem over long-lived plasma cells or recycling germinal-center B cells.•Low levels of BOB.1 are sufficient for long-lived plasma cells differentiation but not for the differentiation of Bmem.
IntroductionPlasmacytoid dendritic cells (pDCs) are the main producers of type I interferon (IFN) in SLE. pDCs express high secretory carrier membrane protein 5 (SCAMP5). Recent work in transfected ...HEK cells connects SCAMP5 to the type I IFN secretory pathway. To further study the role of SCAMP5 in IFNα secretion by pDCs, we focused on the subcellular distribution of SCAMP5 in human pDCs freshly isolated from peripheral blood.MethodsWe measured SCAMP5 expression by flow cytometry in peripheral blood mononuclear cells of healthy subjects (n=8). Next, we assessed the colocalisation of SCAMP5 with IFNα in pDCs of healthy subjects (n=4) by evaluating bright detail similarity (BDS) scores using ImageStream technology.ResultsWe confirm that SCAMP5 is highly expressed by pDCs derived from peripheral blood. In activated pDCs, we show that SCAMP5 colocalises with IFNα (mean BDS 2.0±0.1; BDS >2.0 in 44% of pDCs).ConclusionSCAMP5 colocalises with IFNα in activated human pDCs, in support of a role of this trafficking protein in the secretion of type I IFN by pDCs.
•Macrophages are important effector cells in mAb therapy of cancer.•ADPh can be studied with (imaging) flow cytometry or (live cell) microscopy.•Neutrophils are potent cytotoxic cells for mAb-therapy ...after targeting FcαRI.
Monoclonal antibodies (mAbs) have become an important addition to chemo- and/or radiotherapy for the treatment of cancer. They have multiple effector functions that can lead to eradication of tumor, including induction of apoptosis, growth inhibition, and initiation of complement-dependent lysis. Furthermore, mAbs can recruit immune effector cells. Traditionally, natural killer cells have been considered as the main effector cell population in mAb-mediated tumor killing. Myeloid cells have potent cytotoxic ability, as well. Monocytes and macrophages have been shown to induce antibody-dependent cytotoxicity and phagocytosis of tumor cells in the presence of IgG anti-tumor mAb. Furthermore, neutrophils are the most abundant population of circulating white blood cells, and as such may constitute a formidable source of effector cells. However, when targeting neutrophils for tumor therapy, antibodies of the IgA subclass may be more effective. This article focuses on enlisting myeloid effector cells for mAb-based immunotherapy of cancer. Additionally, methods to study mAb-dependent phagocytosis of tumor cells by macrophages are compared.
The cAMP-PKA signalling cascade in budding yeast regulates adaptation to changing environments. We developed yEPAC, a FRET-based biosensor for cAMP measurements in yeast. We used this sensor with ...flow cytometry for high-throughput single cell-level quantification during dynamic changes in response to sudden nutrient transitions. We found that the characteristic cAMP peak differentiates between different carbon source transitions, and is rather homogenous among single-cells, especially for transitions to glucose. The peaks are mediated by a combination of extracellular sensing and intracellular metabolism. Moreover, the cAMP peak follows the Weber-Fechner law; its height scales with the relative, and not the absolute, change in glucose. Lastly, our results suggest that the cAMP peak height conveys information about prospective growth rates. In conclusion, our yEPAC-sensor makes possible new avenues for understanding yeast physiology, signalling and metabolic adaptation. Media: see text.
The impact of nutritional status during fetal life on the overall health of adults has been recognized; however, dietary effects on the developing immune system are largely unknown. Development of ...secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed. Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells. Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.
The DNA array technique allows comprehensive analysis of the genome and transcriptome, but the high throughput array-based
assessment of intracellular signal transduction remains troublesome. The ...goal of this study was to test a new peptide array
technology for studying the activity of all kinases of whole cell lysates, the kinome. Cell lysates from human peripheral
blood mononuclear cells before and after stimulation with lipopolysaccharide were used for in vitro phosphorylation with γ- 33 PATP arrays consisting of 192 peptides (substrates for kinases) spotted on glass. The usefulness of peptide arrays for studying
signal transduction was demonstrated by the generation of the first comprehensive description of the temporal kinetics of
phosphorylation events induced by lipopolysaccharide stimulation. Furthermore analysis of the signals obtained suggested activation
of p21Ras by lipopolysaccharide, and this was confirmed by direct measurement of p21Ras GTP levels in lipopolysaccharide-stimulated
human peripheral blood mononuclear cells, which represents the first direct demonstration of p21Ras activation by stimulation
of a Toll receptor family member. Further confidence in the usefulness of peptide array technology for studying signal transduction
came from Western blot analysis of lipopolysaccharide-stimulated cells, which corroborated the signals obtained using peptide
arrays as well as from the demonstration that kinase inhibitors effected peptide array phosphorylation patterns consistent
with the expected action of these inhibitors. We conclude that this first metabolic array is a useful method to determine
the enzymatic activities of a large group of kinases, offering high throughput analysis of cellular metabolism and signal
transduction.
Priming of CD8⁺ T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to ...human CD169/Siglec-1⁺ antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14⁺ CD169⁺ monocytes and Axl⁺ CD169⁺ DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169⁺ moDCs and Axl⁺ CD169⁺ DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8⁺ T cells. Finally, Axl⁺ CD169⁺ DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169⁺ DCs to drive antitumor T cell responses.
The vitamin A metabolite retinoic acid (RA) plays a crucial role in mucosal immune responses. We demonstrate in this study that RA-producing retinaldehyde dehydrogenase (RALDH) enzymes are ...postnatally induced in mesenteric lymph node (MLN) dendritic cells (DCs) and MLN stromal cells. RALDH enzyme activity in lamina propria-derived CD103(+) MLN-DCs did not depend on TLR signaling. Remarkably, RA itself could directly induce RALDH2 in both DCs and stromal cells in vitro. Furthermore, upon provision of a vitamin A-deficient diet, it was found that RA-mediated signaling was strongly reduced within the small intestines, while RALDH2 mRNA and RALDH enzyme activity in lamina propria DCs and MLN-DCs, as well as RALDH2 mRNA expression in MLN stromal cells, were strongly diminished. Moreover, supply of vitamin A to vitamin A-deficient mice restored RA-mediated signaling in the intestine and RALDH activity in lamina propria-derived CD103(+) MLN-DCs. Our results show that RA-dependent signaling within the intestine is indispensable for RALDH activity in the draining MLN.
Expression of the tumor-associated glycan Tn antigen (αGalNAc-Ser/Thr) has been correlated to poor prognosis and metastasis in multiple cancer types. However, the exact mechanisms exerted by Tn ...antigen to support tumor growth are still lacking. One emerging hallmark of cancer is evasion of immune destruction. Although tumor cells often exploit the glycosylation machinery to interact with the immune system, the contribution of Tn antigen to an immunosuppressive tumor microenvironment has scarcely been studied. Here, we explored how Tn antigen influences the tumor immune cell composition in a colorectal cancer (CRC) mouse model. CRISPR/Cas9-mediated knock out of the
C1galt1c1
gene resulted in elevated Tn antigen levels on the cell surface of the CRC cell line MC38 (MC38-Tn
high
). RNA sequencing and subsequent GO term enrichment analysis of our Tn
high
glycovariant not only revealed differences in MAPK signaling and cell migration, but also in antigen processing and presentation as well as in cytotoxic T cell responses. Indeed, MC38-Tn
high
tumors displayed increased tumor growth
in vivo
, which was correlated with an altered tumor immune cell infiltration, characterized by reduced levels of cytotoxic CD8
+
T cells and enhanced accumulation of myeloid-derived suppressor cells. Interestingly, no systemic differences in T cell subsets were observed. Together, our data demonstrate for the first time that Tn antigen expression in the CRC tumor microenvironment affects the tumor-associated immune cell repertoire.
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