Igor Bacik

A novel influenza A virus mitochondrial protein that induces cell death

While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection.

Expression of a membrane protease enhances presentation of endogenous antigens to MHC class I-restricted T lymphocytes

We find that expression of the membrane dipeptidyl carboxypeptidase angiotensin-converting enzyme (ACE) enhances presentation of certain endogenously synthesized peptides to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes. ACE appears to function only in an intracellular secretory compartment of antigen-presenting cells. ACE-enhanced antigen presentation requires the expression of the putative antigenic peptide transporters, TAP1 and TAP2. These findings demonstrate that a protease can influence the processing of endogenously synthesized antigens and strongly suggest that longer peptides can be transported from the cytosol to a secretory compartment where trimming of antigenic peptides to the lengths preferred by MHC class I molecules can occur if the appropriate protease is present.

Recycling CD1d1 Molecules Present Endogenous Antigens Processed in an Endocytic Compartment to NKT Cells

Mouse CD1d1 molecules present endogenous glycolipids to NKT cells. Although glycolipid presentation requires CD1d1 transport through the endocytic pathway, the processing requirements for such endogenous Ag presentation by CD1d1 molecules are undefined. We examined CD1d1 Ag presentation to NKT cells by disrupting endocytic trafficking and function in cells expressing normal and mutated CD1d1 expressed by recombinant vaccinia viruses. Consistent with previous studies, we found that preventing CD1d1 localization to endosomes by altering its cytoplasmic targeting sequences abrogated recognition by Vα14Jα281+ NKT cells without affecting recognition by Vα14− NKT cells. Increasing the pH of acidic compartments by incubating cells with chloroquine or bafilomycin A1 blocked CD1d1 recognition by Vα14+ (but not Vα14−) NKT cells without reducing levels of cell surface CD1d1. Similar results were obtained with primaquine, which interferes with the recycling of cell surface glycoproteins. These results suggest that the loading of a subset of glycolipid ligands onto CD1d1 molecules entails the delivery of cell surface CD1d1 molecules and an acidic environment in the endocytic pathway.

Multiple Antigen-Specific Processing Pathways for Activating Naive CD8+ T Cells In Vivo

Current knowledge of the processing of viral Ags into MHC class I-associated ligands is based almost completely on in vitro studies using nonprofessional APCs (pAPCs). This is two steps removed from real immune responses to pathogens and vaccines, in which pAPCs activate naive CD8+ T cells in vivo. Rational vaccine design requires answers to numerous questions surrounding the function of pAPCs in vivo, including their abilities to process and present peptides derived from endogenous and exogenous viral Ags. In the present study, we characterize the in vivo dependence of Ag presentation on the expression of TAP by testing the immunogenicity of model Ags synthesized by recombinant vaccinia viruses in TAP1−/− mice. We show that the efficiency of TAP-independent presentation in vitro correlates with TAP-independent activation of naive T cells in vivo and provide the first in vivo evidence for proteolytic processing of antigenic peptides in the secretory pathway. There was, however, a clear exception to this correlation; although the presentation of the minimal SIINFEKL determinant from chicken egg OVA in vitro was strictly TAP dependent, it was presented in a TAP-independent manner in vivo. In vivo presentation of the same peptide from a fusion protein retained its TAP dependence. These results show that determinant-specific processing pathways exist in vivo for the generation of antiviral T cell responses. We present additional findings that point to cross-priming as the likely mechanism for these protein-specific differences.

Generating MHC class I ligands from viral gene products.

Summary: MHC class I molecules function to present peptides comprised of eight to I 1 residues to CD8+ T lymphocytes. Here we review the efforts of our laboratory lo understand bow cells generate such peptides from viral gene products. We particularly focus on the nature of substrates acted on by cytosolic proteases, the contribution of proteasomes and nun‐proteasomal proteases lo peptide generation, the involvement of ubiquitination in peptide generation, the intracellular localization of proteasome generation of antigenic peptides, and the trimming of peptides in the endoplasmic reticulum.

Human cytomegalovirus protein US2 interferes with the expression of human HFE, a nonclassical class I major histocompatibility complex molecule that regulates iron homeostasis.

ABSTRACT HFE is a nonclassical class I major histocompatibility complex (MHC) molecule that is mutated in the autosomal recessive iron overload disease hereditary hemochromatosis. There is evidence linking HFE with reduced iron uptake by the transferrin receptor (TfR). Using a panel of HFE and TfR monoclonal antibodies to examine human HFE (hHFE)-expressing cell lines, we demonstrate the expression of stable and fully glycosylated TfR-free and TfR-associated hHFE/β2m complexes. We show that both the stability and assembly of hHFE complexes can be modified by the human cytomegalovirus (HCMV) viral protein US2, known to interfere with the expression of classical class I MHC molecules. HCMV US2, but not US11, targets HFE molecules for degradation by the proteasome. Whether this interference with the regulation of iron metabolism by a viral protein is a means of potentiating viral replication remains to be determined. The reduced expression of classical class I MHC and HFE complexes provides the virus with an efficient tool for altering cellular metabolism and escaping certain immune responses.

Promiscuous liberation of MHC-class I-binding peptides from the C termini of membrane and soluble proteins in the secretory pathway

TAP can efficiently transport peptides up to twice as long as those bound to MHC class I molecules, suggesting a role for endoplasmic reticulum (ER) proteases in the trimming of TAP‐transported peptides. To better define ER processing of antigenic peptides, we examined the capacity of TAP‐deficient cells to present determinants derived from ER‐targeted proteins encoded by recombinant vaccinia viruses. TAP‐deficient cells failed to present antigenic peptides from internal locations in secreted proteins to MHC class I‐restricted T lymphocytes. The same peptides were liberated from the C termini of a secreted protein and the lumenal domains of two membrane proteins delivered to the ER via different routes. These findings suggest that proteases in the secretory compartment can liberate C‐terminal antigenic peptides from virtually any context. We propose that this activity often participates in the removal of N‐terminal extensions from TAP‐transported peptides, thereby creating optimally sized products for MHC class I binding. We further demonstrate that ER trimming of C termini can occur if we express an appropriate carboxypeptidase in the secretory pathway. The absence of such trimming under normal circumstances suggests that carboxypeptidase activity is generally deficient in the ER, consistent with the concordance between the specificity of TAP and MHC class I molecules for the same types of C‐terminal residues.

TAP-independent delivery of antigenic peptides to the endoplasmic reticulum: therapeutic potential and insights into TAP-dependent antigen processing.

We have taken several approaches to investigate the capacity of the secretory pathway to liberate major histocompatibility complex (MHC) class I-restricted antigenic peptides from precursor porypeptides. Cells lacking the peptide transporter (TAP) are unable to deliver peptides from cytosolic antigens to class I molecules. TAP can be bypassed by targeting peptides directly to the endoplasmic reticulum (ER) using NH2-terminal signal sequences. This results in the generation of enormous numbers of MHC class I complexes (50,000 peptides/cell), and recombinant vaccinia viruses expressing such peptides are highly immunogenic. In contrast to signal sequence-targeted peptides, peptides are liberated very inefficiently from internal locations in ER-targeted full-length proteins, indicating that the secretory pathway has a limited capacity for generating antigenic peptides from most polypeptide contexts. We have, however, identified a location in proteins from which peptides can be liberated in numerous contexts in the secretory pathway. Placing a number of different peptides at the COOH termini of a secreted protein and two proteins with type II membrane anchors resulted in their TAP-independent presentation. These findings demonstrate that the secretory compartment possesses proteases able to liberate COOH-terminal antigenic peptides from virtually any context, entirely consistent with a role for these proteases in the processing of TAP-transported antigenic peptide precursors.

Antigen processing: where tumor-specific T-cell responses begin.

It is well established that tumor-specific CD8+ T cells have the capacity to prevent and cure malignancies in animals under experimental conditions. This has raised expectations that it will prove possible to achieve similar successes with human cancers. CD8+ T cells recognize peptides of 8-10 residues derived from cytosolic proteins that are bound to the class I molecules of the major histocompatibility complex. To most effectively manipulate the T-cell response to tumor cells, it is essential to understand the means by which the peptide-class I complex is created in cells. An overview of this process is provided with an emphasis toward the recent findings made by our laboratory.