Biomedical subjects

Gomathinayagam Sinnathamby

Publications and source records attributed to Gomathinayagam Sinnathamby.

7 recordsLinked to original sources

PubMed2006

Epitopes derived by incidental translational frameshifting give rise to a protective CTL response.

Aberrant gene expression can be caused by several different mechanisms at the transcriptional, RNA processing, and translational level. Although most of the resulting proteins may have no significant biological function, they can be meaningful for the immune system, which is sensitive to extremely low levels of Ag. We have tested this possibility by investigating the ability of CD8+ T cells (TCD8+) to respond to an epitope whose expression results from incidental ribosomal frameshifting at a sequence element within the HSV thymidine kinase gene. This element, with no apparent functional significance, has been identified due to its ability to facilitate escape from the antiviral compound acyclovir. Using a recombinant vaccinia virus expression system, we find that in vitro and in vivo TCD8+ responses to the frameshift-dependent epitope are easily discernible. Furthermore, the in vivo response is at a sufficient level to mediate protection from a tumor challenge. Thus, the targets of immune responses to infectious agents can extend beyond the products of conventional open reading frames. On a per-cell basis, responses to such minimally expressed epitopes may be exceedingly effective due to the selective expansion of high avidity TCD8+.

Animals↗

PubMed2006

Re-evaluating the generation of a "proteasome-independent" MHC class I-restricted CD8 T cell epitope.

The proteasome is primarily responsible for the generation of MHC class I-restricted CTL epitopes. However, some epitopes, such as NP(147-155) of the influenza nucleoprotein (NP), are presented efficiently in the presence of proteasome inhibitors. The pathways used to generate such apparently "proteasome-independent" epitopes remain poorly defined. We have examined the generation of NP(147-155) and a second proteasome-dependent NP epitope, NP(50-57), using cells adapted to growth in the presence of proteasome inhibitors and also through protease overexpression. We observed that: 1) Ag processing and presentation proceeds in proteasome-inhibitor adapted cells but may become more dependent, at least in part, on nonproteasomal protease(s), 2) tripeptidyl peptidase II does not substitute for the proteasome in the generation of NP(147-155), 3) overexpression of leucine aminopeptidase, thymet oligopeptidase, puromycin-sensitive aminopeptidase, and bleomycin hydrolase, has little impact on the processing and presentation of NP(50-57) or NP(147-155), and 4) proteasome-inhibitor treatment altered the specificity of substrate cleavage by the proteasome using cell-free digests favoring NP(147-155) epitope preservation. Based on these results, we propose a central role for the proteasome in epitope generation even in the presence of proteasome inhibitors, although such inhibitors will likely alter cleavage patterns and may increase the dependence of the processing pathway on postproteasomal enzymes.

Aminopeptidases↗

PubMed2005

Enforced expression of Spi-B reverses T lineage commitment and blocks beta-selection.

The molecular changes that restrict multipotent murine thymocytes to the T cell lineage and render them responsive to Ag receptor signals remain poorly understood. In this study, we report our analysis of the role of the Ets transcription factor, Spi-B, in this process. Spi-B expression is acutely induced coincident with T cell lineage commitment at the CD4(-)CD8(-)CD44(-)CD25(+) (DN3) stage of thymocyte development and is then down-regulated as thymocytes respond to pre-TCR signals and develop beyond the beta-selection checkpoint to the CD4(-)CD8(-)CD44(-)CD25(-) (DN4) stage. We found that dysregulation of Spi-B expression in DN3 thymocytes resulted in a dose-dependent perturbation of thymocyte development. Indeed, DN3 thymocytes expressing approximately five times the endogenous level of Spi-B were arrested at the beta-selection checkpoint, due to impaired induction of Egr proteins, which are important molecular effectors of the beta-selection checkpoint. T lineage-committed DN3 thymocytes expressing even higher levels of Spi-B were diverted to the dendritic cell lineage. Thus, we demonstrate that the prescribed modulation of Spi-B expression is important for T lineage commitment and differentiation beyond the beta-selection checkpoint; and we provide insight into the mechanism underlying perturbation of development when that expression pattern is disrupted.

Animals↗

PubMed2005

A cytosolic pathway for MHC class II-restricted antigen processing that is proteasome and TAP dependent.

By convention, presentation of major histocompatibility complex (MHC) class I-restricted epitopes involves processing by cytosolic proteasomes, whereas MHC class II-restricted epitopes are generated by endosomal proteases. Here, we show that two MHC class II-restricted epitopes within influenza virus were generated by a proteasome- and TAP-dependent pathway that was accessed by exogenous virus in dendritic cells (DCs) but not cell types with less permeable endosomes. Both epitopes were presented by recycling MHC class II molecules. Challenging mice with influenza or vaccinia viruses demonstrated that a substantial portion of the MHC class II-restricted response was directed against proteasome-dependent epitopes. By complementing endosomal activities, this pathway broadens the array of MHC class II-restricted epitopes available for CD4(+) T cell activation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Differential requirements for endosomal reduction in the presentation of two H2-E(d)-restricted epitopes from influenza hemagglutinin.

We examined the role of reduction in the presentation of two H2-E(d)-restricted epitopes (site 1 epitope (S1) and site 3 epitope (S3)) occupying distinct domains of the influenza hemagglutinin major subunit that contains four intrachain disulfide bonds and is connected to the virion by one interchain bond. S3 is situated within the stalk region that unfolds in response to mild acidification, and loads onto recycling H2-E(d) in the early endosome, while S1, located in the structurally constrained globular domain, loads onto nascent H2-E(d) in the late endosome. Predicting dependence upon reduction for either epitope seemed plausible but the results from several approaches were clear: presentation of S1 but not S3 is reduction dependent. Surprisingly, IFN-gamma-inducible lysosomal thiol reductase (GILT), the only reductase thus far known to be involved in MHC class II-restricted processing, is not necessary for the generation of S1. However, GILT is necessary for presentation of either epitope when the virus is pretreated with a reducible cross-linker. The results suggest that unfolding of the Ag, perhaps a prerequisite for proteolytic processing in many cases, proceeds either spontaneously in the early endosome or via reduction in a later endosome. They further imply mechanisms for GILT-independent reduction in the late endosome, with GILT perhaps being reserved for more intractable Ags.

Animals↗

PubMed2004

Vaccinia virus as a tool for immunologic studies.

Studies that involve antigen processing and presentation often require de novo biosynthesis of the antigen both in vitro and in vivo. Additionally, biosynthesis of the antigen or engineered variants within the antigen-presenting cells is usually simpler than providing purified recombinant proteins from bacteria, yeast, or insect cells. For these purposes, recombinant vaccinia virus-based expression has several advantages over other expression systems employed in the field. Insertion of large pieces of recombinant DNA into the vaccinia virus genome, easy recombination and selection of vaccinia viruses, and the ability of these viruses to infect a variety of cells are some key aspects that have made this system popular. Although their efficacy is proven in studies of major histocompatibility complex (MHC) class I-restricted antigen processing and presentation, it is challenging to use them in MHC class II-restricted antigen processing and presentation owing to many reasons specified in this chapter. This chapter aims to describe the commonly used procedures in this field that employ vaccinia virus systems, particularly troubleshooting common problems encountered during experiments.

Animals↗

PubMed2003

Presentation by recycling MHC class II molecules of an influenza hemagglutinin-derived epitope that is revealed in the early endosome by acidification.

We investigated the roles of nascent and recycling MHC class II molecules (MHC II) in the presentation of two well-defined I-E(d)-restricted epitopes that are within distinct regions of the influenza virus hemagglutinin (HA) protein. The site 3 epitope (S3; residues 302-313) lies in the stalk region that unfolds in response to mild acidification, while the site 1 epitope (S1; residues 107-119) is situated in the stable globular domain. In a murine B lymphoma cell line and an I-E(d)-transfected fibroblast cell line, presentation from inactivated virus of S3 is inhibited by primaquine, a compound that prevents recycling of cell surface proteins, including MHC II, while S1 presentation is unaffected. In contrast, brefeldin A, an agent that inhibits exit of proteins from the endoplasmic reticulum, selectively inhibited S1 presentation without affecting S3 presentation, suggesting that S1 presentation requires nascent MHC II. The use of agents that perturb endosomal function revealed a requirement for acidification of internalized viral particles for presentation of both epitopes. Notably, all compounds tested had similar effects on presentation of the two epitopes derived from endogenously synthesized HA. Thus, recycling I-E(d) molecules appear to be crucial for capturing and presenting an epitope that is revealed in mild acidic conditions following the uptake of virions or the synthesis of Ag, while nascent I-E(d) molecules are required for presentation of a second epitope located in a structurally constrained region of the same polypeptide. Viral glycoproteins, such as HA, may have been a major impetus for the evolutionary establishment of this recycling pathway.

Animals↗