Biology subjects

Garcia, K. C.

Publications and source records attributed to Garcia, K. C..

3 recordsLinked to original sources

bioRxiv2018

Differential induction of interferon stimulated genes between type I and type III interferons is independent of interferon receptor abundance

It is currently believed that type I and III interferons (IFNs) have redundant functions. However, the preferential distribution of type III IFN receptor on epithelial cells suggests functional differences at epithelial surfaces. Here, using human intestinal epithelial cells we could show that although both type I and type III IFNs confer an antiviral state to the cells, they do so with distinct kinetics. Type I IFN signaling is characterized by an acute strong induction of interferon stimulated genes (ISGs) and confers fast antiviral protection. On the contrary, the slow acting type III IFN mediated antiviral protection is characterized by a weaker induction of ISGs in a delayed manner compared to type I IFN. Moreover, while transcript profiling revealed that both IFNs induced a similar set of ISGs, their temporal expression strictly depended on the IFNs, thereby leading to unique antiviral environments. Using a combination of data-driven mathematical modeling and experimental validation, we addressed the molecular reason for this differential kinetic of ISG expression. We could demonstrate that these kinetic differences are intrinsic to each signaling pathway and not due to different expression levels of the corresponding IFN receptors. We report that type III IFN is specifically tailored to act in specific cell types not only due to the restriction of its receptor but also by providing target cells with a distinct antiviral environment compared to type I IFN. We propose that this specific environment is key at surfaces that are often challenged with the extracellular environment.\n\nAuthor summaryThe human intestinal tract plays two important roles in the body: first it is responsible for nutrient absorption and second it is the primary barrier which protects the human body from the outside environment. This complex tissue is constantly exposed to commensal bacteria and is often exposed to both bacterial and viral pathogens. To protect itself, the gut produces, among others, secreted agents called interferons which help to fight against pathogen attacks. There are several varieties (type I, II, and III) of interferons and our work aims at understanding how type I and III interferon act to protect human intestinal epithelial cells (hIECs) during viral infection. In this study, we confirmed that both interferons can protect hIECs against viral infection but with different kinetics. We determined that type I confer an antiviral state to hIECs faster than type III interferons. We uncovered that these differences were intrinsic to each pathway and not the result of differential abundance of the respective interferon receptors. The results of this study suggest that type III interferon may provide a different antiviral environment to the epithelium target cells which is likely critical for maintaining gut homeostasis. Our findings will also help us to design therapies to aid in controlling and eliminating viral infections of the gut.

immunology↗

bioRxiv2017

A Polymorphic Residue That Attenuates Interferon Lambda 4 Activity in Hominid Lineages

As antimicrobial signalling molecules, type III or lambda interferons (IFN{lambda}s) are critical for defence against infection by diverse pathogens. Counter-intuitively, expression of one member of the family, IFN{lambda}4, is associated with decreased clearance of hepatitis C virus (HCV) in the human population; by contrast, a natural in-frame nucleotide insertion that abrogates IFN{lambda}4 production improves viral clearance. To further understand how genetic variation between and within species affects IFN{lambda}4 function, we screened a panel of extant coding variants of human IFN{lambda}4 and identified three variants that substantially affect antiviral activity (P70S, L79F and K154E). The most notable variant was K154E, which enhanced in vitro activity in a range of antiviral and interferon stimulated gene (ISG) assays. This more active E154 variant of IFN{lambda}4 was found only in African Congo rainforest Pygmy hunter-gatherers. Remarkably, E154 was highly conserved as the ancestral residue in mammalian IFN{lambda}4s yet K154 is the dominant variant throughout evolution of the hominid genus Homo. Compared to chimpanzee IFN{lambda}4, the human orthologue had reduced activity due to amino acid substitution of glutamic acid with lysine at position 154. Meta-analysis of published gene expression data from humans and chimpanzees showed that this difference in activity between K154 and E154 in IFN{lambda}4 is consistent with differences in antiviral gene expression in vivo during HCV infection. Mechanistically, our data suggest that human-specific K154 likely affects IFN{lambda}4 activity by reducing secretion and potency. We postulate that evolution of an IFN{lambda}4 with attenuated activity in humans (K154) likely contributes to distinct host-specific responses to and outcomes of infection, such as HCV.

immunology↗

bioRxiv2016

In vitro reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase

T cell signaling initiates upon binding of peptide-major histocompatibility complex (pMHC) on an antigen-presenting cell (APC) to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR-pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase-phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR-pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor-ligand pairs using purified proteins on model membranes. Using a model receptor-ligand pair (FRB-FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR-pMHC interaction causes partial exclusion of CD45. Comparing two developmentally-regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (~50%) than the smaller R0 isoform (~20%), suggesting that CD45 isotypes could regulate signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell signaling, TCR-pMHC interactions with Kds of [≤]15 M were needed to exclude CD45. We further show that the co-receptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor-ligand pairs on the T cell are sufficient to create spatial organization at membrane-membrane interfaces.\n\nSIGNIFICANCE STATEMENTThe interface between a T cell and an antigen-presenting cell (APC) results in the formation of biochemically distinct plasma membrane domains that initiate signaling cascades. Here, using biochemical reconstitution and microscopy, we show that the binding energies of the TCRpMHC and PD-1-PD-L1 complexes are sufficient to create spatial organization at a model membrane-membrane interface. We show that spatial organization depends upon receptor-ligand binding affinity and the relative sizes of the extracellular domains. These biophysical parameters may be used to fine-tune signaling cascades in T cells.

immunology↗