Biology subjects

Moraga, I.

Publications and source records attributed to Moraga, I..

7 recordsLinked to original sources

bioRxiv2026

Lysosomal adaptation to extracellular acidity compromises CD8+ T cell cytotoxicity

Extracellular acidosis is a hallmark of aggressive tumors and suppresses cytotoxic-T lymphocyte (CTL)-mediated killing, but how sustained acid exposure reshapes T cell cytotoxicity is poorly understood. Here we show that chronic acidosis drives lysosomal remodeling in human CD8+ T cells, converting the lysosome into a proton sink whose adaptation ultimately impairs killing by depleting the intracellular granzyme B reservoir through constitutive vesicle release. Quantitative proteomics of CD8+ T cells cultured at pH 6.5 identified lysosomes as the dominant target of acid-driven remodeling: CTLs upregulated the V1 sector of the vacuolar H+-ATPase and hyper-acidified the lysosomal lumen. The remodeled compartment accumulated lysosomal membrane proteins (LAMP1, LAMP2, CD63, CD68) post-transcriptionally, acquired higher cathepsin abundance and increased proteolytic activity. Chronic acidity also enlarged endoplasmic reticulum (ER) and lysosomal Ca2+ stores without altering cytosolic Ca2+, and drives constitutive, Ca2+-dependent release of LAMP1+ lysosome-derived vesicles, which drains the intracellular reservoir of granzyme B, contributing to reduced CTL activity. These findings establish lysosomal adaptation as a cost of surviving extracellular acidosis and identify lysosomal hydrolase activity as a possible target for improving CTL function in the acidic tumor environment.

immunology↗

bioRxiv2023

Differential functional coupling in Gp130-JAK complexes expands the plasticity of the interleukin-6 signaling axis

Cytokines dimerize/oligomerize surface receptors to activate signaling. While cytokine receptors preferentially bind only one member of the JAK family, ancestral cytokine receptors, such as Gp130, promiscuously recruit different JAKs to elicit their activities. Here, we have explored how the identity of JAKs in Gp130 signaling complexes can regulate functional outcomes. Using a synthetic biology approach, we show that Gp130 bound to different JAKs propagates distinct STAT activation profiles. While Gp130-JAK1 complexes activated both, STAT1 and STAT3 very potently, Gp130-JAK2 complexes exhibited a clear preference for STAT3 activation. Gp130-TYK2 complexes triggered overall weaker signaling but with diminished STAT specificity. The three JAKs competed for binding to Gp130 and led to differential activation of phospho-Tyr in the Gp130 intracellular domain. JAK1, JAK2 and to a lower extent TYK2 bound with comparable affinities to Gp130, and in response to IL-6 stimulation efficiently drove Gp130 dimerization. However, the three JAKs differentially affected Gp130 surface expression, identifying JAK-dependent receptor trafficking as a critical determinant of signaling plasticity. Our results provide new mechanistic insights into how differential functional coupling in Gp130-JAK complexes translates into unique signaling signatures that likely contribute to its large functional diversity.

cell biology↗

bioRxiv2022

Structural insights into the assembly and activation of IL-27 signalling complex

Interleukin 27 (IL-27) is a heterodimeric cytokine that elicits potent immuno-suppressive responses. Comprised of EBI3 and p28 subunits, IL-27 binds GP130 and IL-27R receptor chains to activate the JAK/STAT signalling cascade. However, how these receptors recognize IL-27 and form a complex capable of phosphorylating JAK proteins remains unclear. Here, we used cryo electron microscopy (cryoEM) to solve the structure of the IL-27 receptor recognition complex. Our data show how IL-27 serves as a bridge connecting IL-27R with GP130 to initiate signalling. While both receptors weakly bind the p28 component of the heterodimeric cytokine, EBI3 stabilizes the complex by binding a positively charged surface of IL-27R. We find that assembly of the IL-27 receptor recognition complex is distinct from both IL-12 and IL-6 cytokine families and provides a mechanistic blueprint for tuning IL-27 pleiotropic actions.

immunology↗

bioRxiv2020

Tuning TPO-R signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL)) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable, both experimentally and clinically. We have developed a series of surrogate protein-ligands for TPO-R, in the form of diabodies, that homodimerize the TPO-R on the cell surface in different geometries, in effect tuning downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism and antagonism, thus decoupling the dual functions of TPO/TPO-R. Using scRNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in Essential Thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R. Significance StatementThe TPO cytokine, which signals through its receptor c-MPL (or TPO-R), is essential for megakaryocyte differentiation and maintenance of hematopoietic stem cells (HSCs). Its signaling is deregulated in Essential Thrombocythemia (ET). Here, we engineered diabodies (DBs) against the TPO-R as surrogate TPO ligands to manipulate TPO-R signaling, from full to partial to antagonism, thus decoupling the dual functions of TPO/TPO-R (i.e, HSC maintenance versus megakaryopoiesis). We subsequently discovered that partial agonistic DBs, by reducing the strength of the TPO-R signal, not only preserved HSCs in culture, but also blocked oncogenic signaling in ET. This finding has the potential to improve HSC cultures for transplants, as well as serve as a unique therapeutic approach for ET.

cell biology↗

bioRxiv2020

CDK8 fine-tunes IL-6 transcriptional activities by limiting STAT3 resident time at the gene loci

Cytokines are highly pleiotropic ligands that critically contribute to a balanced immune response. We have an incomplete understanding of how cytokines elicit their functional pleiotropy, which has limited their therapeutic use. Here, using Interleukin-6 (IL-6) as a model system, we have performed detailed phosphoproteomic and transcriptomic studies in human Th-1 cells to address the molecular bases defining cytokine functional pleiotropy. We have identified CDK8 as a new negative regulator of STAT3 transcriptional activities that contributes to the diversification of IL-6 responses. We found that CDK8 is a major regulator of the IL-6 phosphoproteome and interacts with STAT3 in the nucleus upon IL-6 stimulation. Inhibition of CDK8 activity, using specific small molecules inhibitors, reduced the IL-6-induced phosphoproteome by 23% in Th-1 cells, including STAT3 Ser727 phosphorylation. This, in turn, resulted in retention of tyrosine phosphorylated STAT3 in the nucleus, which increased the binding of STAT3 to target DNA sites in the genome with a concomitant increase in STAT3 mediated transcriptional activity. Importantly, inhibition of CDK8 activity under Th-17 polarizing conditions resulted in an enhancement of Th-17 differentiation. Our results support a model where CDK8 regulates STAT3 transcriptional processivity via modulation of its gene loci resident time, critically contributing to tuning STAT3 mediated responses.

immunology↗

bioRxiv2020

Engineered IL-10 variants elicit potent immuno-modulatory activities at therapeutic low ligand doses

Interleukin-10 is a dimeric cytokine with both immune-suppressive and immune-stimulatory activities. Despite its immuno-modulatory potential, IL-10-based therapies have shown only marginal benefits in the clinic. Here we have explored whether the stability of the IL-10-receptor complex contributes to IL-10 immuno-modulatory potency. For that, we have generated an IL-10 mutant with greatly enhanced affinity for its IL-10R{beta} receptor via yeast surface display. The affinity enhanced IL-10 variants recruited IL-10R{beta} more efficiently into active cell surface signaling complexes than the wild-type cytokine and triggered more potent STAT1 and STAT3 activation in human monocytes and CD8 T cells. This in turn led to more robust induction of IL-10-mediated gene expression programs at a wide range of ligand concentrations in both human cell subsets. IL-10 regulated genes are involved in monocyte energy homeostasis, migration and trafficking, and genes involved in CD8 T cell exhaustion. Interestingly, at non-saturating doses, IL-10 lost key components of its gene-expression program, which may explain its lack of efficacy in clinical settings. Remarkably, our engineered IL-10 variant exhibited a more robust bioactivity profile than IL-10 wt at all the doses tested in monocytes and CD8 T cells. Moreover, CAR-modified T cells expanded with the engineered IL-10 variant displayed superior cytolytic activity than those expanded with IL-10 wt. Our study provides unique insights into how IL-10-receptor complex stability fine-tunes IL-10 biology, and opens new opportunities to revitalize failed IL-10 therapies.

immunology↗

bioRxiv2018

Loss of Adenomatous polyposis coli function renders intestinal epithelial cells resistant to the cytokine IL-22

Interleukin-22 (IL-22) is critical in maintaining homeostasis in the intestine by regulating the balance between pathogenic and commensal bacteria. IL-22 also promotes wound healing and tissue regeneration, which can support the growth of colorectal tumours. Mutations in the tumour suppressor Adenomatous Polyposis Coli gene (Apc) cause intestinal tumorigenesis and are a major driver of familial colorectal cancers. To understand the role of IL-22 in APC-mediated tumorigenesis, we analysed IL-22 signalling in wild-type (WT) and APC-mutant cells in murine small intestinal epithelial organoids and in mice. In WT epithelia, antimicrobial defence, mucus production, and cellular stress response pathways were most strongly upregulated by IL-22. Surprisingly, we found that although IL-22 activated STAT3 in APC-mutant cells, STAT3 target genes were not effectively induced. Our analyses revealed that ApcMin/Min cells were resistant to IL-22 due to reduced expression of the IL-22 receptor, and increased expression of inhibitors of STAT3, including histone deacetylases. We further show that IL-22 induced expression of nitric oxide synthase in WT epithelial cells and corresponding DNA damage. These findings suggest that IL-22 does not promotes tumour formation by driving the proliferation of transformed intestinal epithelial cells. Rather, IL-22 increases genetic instability thereby accelerating transition from heterozygosity (ApcMin/+) to homozygosity (ApcMin/Min) to drive tumour progression.

immunology↗