Alexander K. Nussbaum

Contribution of Proteasomal β-Subunits to the Cleavage of Peptide Substrates Analyzed with Yeast Mutants

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated β-subunits, we investigated the specificities and contributions of the different β-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active β-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with β5/Pre2 cleaving after hydrophobic, β2/Pup1 after basic, and β1/Pre3 after acidic residues, but with the exception that β2/Pup1 and β1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the “branched chain amino acid-preferring” component are associated with β5/Pre2, β1/Pre3, or β2/Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.

Using the World Wide Web for predicting CTL epitopes

Abstract The development of vaccines and also of methods for the monitoring of cytotoxic T lymphocyte (CTL) responses depends on the identification of epitopes from immunologically relevant antigens. Detailed information about the rules that govern the interactions of peptides with MHC class I molecules, together with an increasing knowledge about the cleavage specificities of proteasomes, has paved the way for the development of computer programs that can predict CTL epitopes. The combination of such programs is likely to speed up the identification of CD8+ T cell epitopes relevant for immune responses to infectious diseases and cancer.

Immunoproteasome-Deficient Mice Mount Largely Normal CD8+ T Cell Responses to Lymphocytic Choriomeningitis Virus Infection and DNA Vaccination

During viral infection, constitutive proteasomes are largely replaced by immunoproteasomes, which display distinct cleavage specificities, resulting in different populations of potential CD8+ T cell epitope peptides. Immunoproteasomes are believed to be important for the generation of many viral CD8+ T cell epitopes and have been implicated in shaping the immunodominance hierarchies of CD8+ T cell responses to influenza virus infection. However, it remains unclear whether these conclusions are generally applicable. In this study we investigated the CD8+ T cell responses to lymphocytic choriomeningitis virus infection and DNA immunization in wild-type mice and in mice lacking the immunoproteasome subunits LMP2 or LMP7. Although the total number of virus-specific cells was lower in LMP2 knockout mice, consistent with their having lower numbers of naive cells before infection, the kinetics of virus clearance were similar in all three mouse strains, and LMP-deficient mice mounted strong primary and secondary lymphocytic choriomeningitis virus-specific CD8+ T cell responses. Furthermore, the immunodominance hierarchy of the four investigated epitopes (nuclear protein 396 (NP396) > gp33 > gp276 > NP205) was well maintained. We observed a slight reduction in the NP205-specific response in LMP2-deficient mice, but this had no demonstrable biological consequence. DNA vaccination of LMP2- and LMP7-deficient mice induced CD8+ T cell responses that were slightly lower than, although not significantly different from, those induced in wild-type mice. Taken together, our results challenge the notion that immunoproteasomes are generally needed for effective antiviral CD8+ T cell responses and for the shaping of immunodominance hierarchies. We conclude that the immunoproteasome may affect T cell responses to only a limited number of viral epitopes, and we propose that its main biological function may lie elsewhere.

The function of the proteasome system in MHC class I antigen processing

Abstract A recent meeting**1st Charite Zeuthener See Workshop on the function of the proteasome system in MHC class I antigen processing, 2–4 April 2000. brought together researchers to discuss the latest developments in the origin and processing of cytotoxic T lymphocyte (CTL) epitopes, with special consideration given to proteasome-related events.

The Contraction Phase of Virus-Specific CD8+ T Cells Is Unaffected by a Pan-Caspase Inhibitor

The effectiveness of protection conferred by CD8+ memory T cells is determined by both their quality and their quantity, which suggests that vaccine efficacy might be improved if it were possible to increase the size of the memory pool. Approximately 90% of virus-specific CD8+ T cells die during the contraction phase and, herein, we have attempted to increase the memory pool by reducing CD8+ T cell death. CD8+ T cell contraction has been attributed to apoptosis, or programmed cell death (PCD), which, classically, is dependent on caspases. Caspase-dependent PCD can be prevented by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (zVAD), and here we evaluate the effect of this compound on virus-specific T cell responses in mice. zVAD prevented caspase-dependent PCD of freshly isolated virus-specific T cells in tissue culture, and a fluorescent analog, FITC-VAD, entered CD8+ T cells following in vivo injection. However, despite using 11 different regimens of zVAD administration in vivo, no significant effects on CD8+ or CD4+ memory T cell numbers were observed. Furthermore, the CD8+ memory T cell responses to secondary virus infection were indistinguishable, both qualitatively and quantitatively, in zVAD-treated and normal mice. The absence of effect cannot be attributed to a technical flaw, because identical doses of zVAD were able to rescue mice from hepatocyte apoptosis and lethal intrahepatic hemorrhage, induced by inoculation of anti-Fas Ab. We conclude that the contraction phase of the virus-specific T cell response is unlikely to require caspase-dependent PCD. We propose that contraction can be mediated by an alternative, caspase-independent pathway(s).

A CTL epitope from human cytomegalovirus IE1 defined by combining prediction of HLA binding and proteasomal processing is the target of dominant immune responses in patients after allogeneic stem cell transplantation

OBJECTIVE AND METHODS In an attempt to define HCMV IE1-derived, HLA-A(*)0201-restricted epitopes, an advanced computer-based epitope prediction combining HLA binding and proteasomal cleavages in silico was performed. RESULTS This prediction algorithm clearly confirmed VLEETSVML to be the most likely CTL epitope. By tetramer staining, HCMV pp65 NLVPMVATV-specific CD8(+) T cells were detectable in 18/24 HCMV seropositive HLA-A(*)0201-expressing individuals (median frequency 0.58%; range 0.1%-4.7%), and IE1 VLEETSVML-specific CD8(+) T cells in 5/24 (median frequency 2.1%; range 0.1%-4.3%), respectively (p<0.01). Also in recipients of an allogeneic SCT, VLEETSVML- and NLVPMVATV-specific CD8(+) T cells were detectable in comparable frequencies, but again the number of patients with detectable pp65-specific CD8(+) T cells was higher (p=0.014). In 4/15 individuals, all demonstrating IE1 VLEETSVML-specific CD8(+) T cells prior to peptide stimulation, VLEETSVML-specific T cell lines (purity of 42.6%-98.6% of all CD3(+)/CD8(+) T cells) were successfully generated after 2-4 weeks of culture using the IFN-gamma secretion assay. CONCLUSION In conclusion, this novel prediction strategy efficiently predicted an immunodominant viral T-cell epitope.

Mechanisms of MHC class I-restricted antigen presentation

The vertebrate immune system monitors whether an organism is invaded by pathogens. Therefore, each cell has to prove itself as healthy. This is achieved by presenting fragments of intracellular protein degradation products on the surface, i.e., each cell displays peptides on specialised proteins known as major histocompatibility complex (MHC) class I proteins. A displayed peptide has to pass certain constraints before its presentation: It has to be excised out of a protein, translocated into the endoplasmic reticulum (ER) and fit into the binding groove of a MHC molecule. In theory, alteration of the cellular protein profile by mutation or infection should force pathogen-specific T-cells to take action via recognition of foreign peptide bound to MHC class I molecules on the cell surface. Unfortunately, pathogens and tumours have evolved many ways to affect antigen presentation and to escape from immune response. Understanding the exact mechanisms of antigen presentation, i.e., protein cleavage and peptide binding by MHC molecules, would allow their manipulation by drugs and lead to the re-establishment of the correct antigen presentation pathway. This review will summarise current knowledge of the mechanisms of antigen presentation and discuss putative targets for therapeutic treatment as well as for vaccination strategies.