Perforin-independent regulation of dendritic cell homeostasis by CD8(+) T cells in vivo: implications for adaptive immunotherapy.

We investigated here the effects of perforin on CTL responses during interaction of dendritic cells (DC) with cytotoxic T lymphocytes in vivo. Using MHC class I tetramers complexed with the immunodominant CTL epitope of the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP33), we followed the kinetics of DC-induced CTL responses. GP33-presenting DC induced rapid primary expansion of both perforin-competent and -deficient CTL with similar kinetics. Secondary CTL responses in perforin-deficient and normal control mice after DC-booster immunization were more rapid than the primary responses, but never reached the high initial levels, suggesting that reactivated memory CTL eliminated the antigen-presenting DC and thereby limited the booster effect. Whereas killingof DC in vitro was strictly dependent on perforin, elimination of GP33-presenting DC by CTL in vivo was largely independent of perforin and Fas. Taken together, these results suggest that control of DC homeostasis by CTL, i. e. elimination of DC by the effector cells they had elicited, is controlled via multiple and probably redundant signals and represents an important fail-safe mechanism to avoid exaggerated CTL responses.

A MAGE-A1 HLA-A A*0201 epitope identified by mass spectrometry.

Peptides presented by HLA-A*0201 molecules on the surface of the human breast carcinoma cell line KS24.22 after IFN-gamma induction were analyzed by the "Predict-Calibrate-Detect" approach, which combines epitope prediction and high-performance liquid chromatography mass spectrometry. One of the predicted epitopes, MAGE-A1(278-286) (KVLEYVIKV), was found to be presented by HLA-A*0201, with an estimated copy number of 18 molecules/cell. HLA-A*0201 transgenic mice (HHD mice) were used to generate CTL lines that stained positive with an HLA-A*0201 tetramer folded around the KVLEYVIKV peptide and killed peptide-loaded mouse target cells expressing HLA-A*0201. IFN-gamma-treated or -nontreated HLA-A*0201 expressing HeLa cells transiently transfected with a plasmid expressing the MAGE-A1 gene stimulated in vitro cytokine production by the CTL lines. Moreover, IFN-gamma-treated KS24.22 cells, but not IFN-gamma-treated HLA-A*0201(+) MAGE-A1(-) cells or IFN-gamma-treated HLA-A*0201(-) MAGE-A1(+) cells, were killed by these CTLS: Thus, the combination of HLA epitope prediction, peptide analysis, and immunological methods is a powerful approach for the identification of tumor-associated epitopes.

Rapid peptide turnover and inefficient presentation of exogenous antigen critically limit the activation of self-reactive CTL by dendritic cells.

This study evaluated to what extent presentation of exogenously acquired self-Ags via MHC class I molecules on DC might contribute to the activation of self-reactive CTL and subsequent development of autoimmune disease. We show here by using the rat insulin promotor lymphocytic choriomeningitis virus glycoprotein model of autoimmune diabetes that the activation of self-reactive CTL by DC after uptake of exogenous Ag is very limited, first by the short half-life of MHC class I-associated peptides on DC in vitro and in vivo, and second by the rather inefficient MHC class I presentation of cell-associated self-Ags by DC. These two mechanisms are probably crucial in establishing high thresholds for the induction of self-reactive CTL that prevent autoimmune sequelae after release of sequestered and previously immunologically ignored tissue Ags.

A naturally processed rat major histocompatibility complex class I-associated viral peptide as target structure of borna disease virus-specific CD8+ T cells.

The first naturally processed peptide synthesized by a virus and recognized by classical CD8(+) T cells in association with the RT1.A(l) major histocompatibility complex class I molecule of the Lewis rat is reported. Borna disease virus-specific CD8(+) T cells recognize syngeneic target cells pulsed with peptides extracted from Borna disease virus-infected cells. The predicted peptide sequence ASYAQMTTY from the viral p40 protein coeluted with the cytotoxic T-lymphocyte-reactive fraction was identified among natural ligands by tandem mass spectrometry. Numerous naturally processed peptides derived from intracellular bacteria, viruses, or tumors and recognized by CD8(+) T cells of man and mice are known, leading to a better understanding of cellular immune mechanisms against pathogens in these two species. In contrast, for the rat little information exists with regard to the function and role of CD8(+) T cells as part of their cellular immune defense system. This first naturally processed viral epitope in the rat contributes to the understanding of the rat cellular immune response and might trigger the identification of more cytotoxic T-lymphocyte epitopes in this animal.

Hypercholesterolemia exacerbates virus-induced immunopathologic liver disease via suppression of antiviral cytotoxic T cell responses.

The immune system has to be optimally balanced to be highly effective against infections with cytopathic microbial pathogens and must guarantee efficient destruction of cells infected with noncytopathic agents while leaving the integrity of noninfected cells largely unaltered. We describe here the effects of genetically induced hypercholesterolemia on cellular immunity in apolipoprotein E (ApoE(-/-)) and low density lipoprotein receptor-deficient (LDLR(-/-)) mice during infection with the hepatotropic lymphocytic choriomeningitis virus WE strain. In both ApoE(-/-) and LDLR(-/-) mice hypercholesterolemia aggravated virus-induced immunopathologic liver disease. ApoE(-/-) mice exhibited a higher susceptibility to virus-induced immunopathology than LDLR(-/-) mice and usually succumbed to immunopathologic disease when infected with high doses of virus. Initial virus spread was not influenced by the hypercholesterolemia, whereas clearance of the virus from spleen and nonlymphoid organs, including liver, was delayed. Activation of antiviral CTL, measured by ex vivo cytotoxicity and IFN-gamma production, and recruitment of specific CTL into blood and liver were impaired in hypercholesterolemic mice, indicating that hypercholesterolemia had a significant suppressive effect on cellular immunity. Taken together, these data provide evidence that hypercholesterolemia suppresses antiviral immune responses, thereby changing the host-virus balance, and can increase susceptibility to acute or chronic and potentially lethal virus-induced immunopathologic disease. These findings impinge on our understanding of hypercholesterolemia as a disease parameter and may explain aspects of the frequent association of persistent pathogens with hypercholesterolemia-induced diseases, such as atherosclerosis.

Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach.

Specific immunotherapy of cancer utilizes tumor-directed cytotoxic T lymphocytes (CTL) that lyse tumor cells presenting MHC class I-associated peptides derived from tumor-associated proteins. Many tumor-associated gene products are known, but corresponding T cell epitopes are only known for relatively few of these. The most commonly used approaches to identify such antigens require pre-existing CTL lines or clones. By using a CTL-independent high performance liquid chromatography mass spectrometry (HPLC MS)-based approach we identified HLA-A2-presented peptides from carcinoembryonic antigen and wild-type p53 with a copy number as low as eight molecules per cell. Potential epitopes were predicted from the sequences of known tumor antigens and the corresponding synthetic peptides were analyzed by nanocapillary HPLC MS. In parallel, peptides were extracted from fresh, solid tumor tissue or tumor cell lines and analyzed in the same way. Upon co-elution of a natural peptide with a predicted peptide of the same mass, the peptide sequence was confirmed by on-line tandem MS. This approach allows rapid screening of large numbers of tumor-associated gene products for naturally processed peptides presented by different MHC class I molecules as a prerequisite for efficient epitope identification and rapid transfer to therapeutic vaccine trials.

Role of anchor residues in peptide binding to three HLA-A26 molecules.

To investigate the role of anchor residues in HLA-A26 binding peptides, we analyzed the binding of various peptides to three HLA-A26 molecules using the HLA class I stabilization assay. Of twenty nonamer peptides carrying anchors at P2 and P9, 3, 6 and 3 peptides bound to HLA-A*2601, HLA-A*2602 and HLA-A*2603, respectively The peptide EV-IPMFSAL bound most strongly to these three HLA-A26 molecules. Analysis using mutants of this peptide at P1, P2 or P9 showed that acidic amino acids at P1 and five hydrophobic residues (Val, Thr, Ile, Leu and Phe) at P2 are anchor residues for the three HLA-A26 molecules while with exception of positively charged amino acids, a broad range of amino acids function as P9 anchor residues. These anchors were further evaluated using 38 nonamer peptides carrying anchor residues at P1, P2 and P9. Nineteen of these peptides bound to at least one HLA-A26 molecule. The frequency of HLA-A26 binding peptides was higher for peptides carrying all three anchor residues than for peptides carrying only P2 and P9 anchor residues. These results indicate that in addition to P2 and P9 anchors, the P1 anchor plays an important role in peptide binding to three HLA-A26 molecules.

A protective cytotoxic T cell response to a subdominant epitope is influenced by the stability of the MHC class I/peptide complex and the overall spectrum of viral peptides generated within infected cells.

This study identifies instability of MHC class I/peptide complexes and intermolecular competition for MHC class I presentation as factors responsible for the subdominance of cytotoxic T lymphocyte (CTL) epitopes. This evidence is based on the characterization of a new CTL epitope derived from the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV). This epitope, peptide GP117-125 (GP117) is presented to T cells by the mouse MHC class I molecule, H-2Db. In short-term experiments induction of GP117-specific CTL by vaccination rendered C57BL/6 mice only partially resistant to infection with wild-type LCMV (LCMV-WE) but completely resistant to challenge with a previously described LCMV variant. The variant virus, LCMV-8.7B23, bears point mutations within both known LCMV-GP, H-2 Db-restricted epitopes GP33-41 (GP33) and GP276-286 (GP276) resulting in a valine to leucine change at position 35 in peptide GP33 (V35L) and an asparagine to serine change at position 280 in peptide GP276 (N280S). Although variant peptide GP33/V35L stimulates a weak CTL response, GP276/N280S does not. Elution of peptide GP117 from both LCMV-WE- and LCMV-8.7B23-infected cells revealed that the difference in the capacity of GP117-specific CTL to protect against LCMV-WE and the virus variant LCMV-8.7B23 was due to differences in the level of GP117 presentation on the surface of both types of cells. Thus, it appears that the protective capacity of CTL specific for the subdominant epitope GP117 is influenced by the extent of presentation of other immunodominant peptide epitopes present within infected cells.

Protective immunity does not correlate with the hierarchy of virus-specific cytotoxic T cell responses to naturally processed peptides.

Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) stimulates major histocompatibility complex class I-restricted cytotoxic T cells (CTLs), which normally resolve the infection. Three peptide epitopes derived from LCMV have been shown to bind the mouse class I molecule H-2 Db and to stimulate CTL responses in LCMV-infected mice. This report describes the identity and abundance of each CTL epitope after their elution from LCMV-infected cells. Based on this information, peptide abundance was found to correlate with the magnitude of each CTL response generated after infection with LCMV. Subsequent experiments, performed to determine the antiviral capacity of each CTL specificity, indicate that the quantitative hierarchy of CTL activity does not correlate with the ability to protect against LCMV infection. This report, therefore, indicates that immunodominant epitopes should be defined, not only by the strength of the CTL response that they stimulate, but also by the ability of the CTLs to protect against infection.

Substrate specificity of cathepsins D and E determined by N-terminal and C-terminal sequencing of peptide pools.

Degradation of protein antigens by cellular proteases is a crucial step in the initiation of a T-cell-mediated immune response. But still little is known about the enzymes responsible for the processing of antigens, including their specificity. In this paper, we show that the combination of automated N-terminal sequencing with a newly developed method for C-terminal sequencing of peptide pools generated by the aspartic proteases cathepsins D and E is a fast and easy method to obtain detailed information of the substrate specificity of these endopeptidases. Using a 15-residue synthetic peptide library and a native protein as substrates, we confirm and extend the knowledge about the cleavage motif of cathepsin E where positions P1 and P1' of the substrate must be occupied exclusively by hydrophobic amino acids with aromatic or aliphatic side chains. However, Val and Ile residues are not allowed at position P1. Position P2' accepts a broad range of amino acids, including charged and polar ones. Additional requirements concerning the substrate positions P3' and P4' were also defined by pool sequencing. Furthermore, pool sequencing analysis of melittin digests with the aspartic proteases cathepsin D and E provided evidence that both enzymes share the same cleavage motif, identical to the one derived from the peptide library and the native protein. Therefore, pool sequencing analysis is a valuable and fast tool to determine the substrate specificity of any endopeptidase.

Evolutionary conserved cathepsin E substrate specificity as defined by N-terminal and C-terminal sequencing of peptide pools.

The substrate specificity of the non-lysosomal aspartic protease cathepsin E from three different species has been studied using the method of automated N-terminal sequencing and a newly developed method for C-terminal sequencing of peptides and peptide pools. The combination of N-terminal and C-terminal sequencing of peptide pools is a fast and easy method to identify and compare the substrate specificity of endopeptidases. Our analysis shows a conserved hydrolytic specificity between human, mouse and bovine cathepsin E, with only small differences in fine specificity. Furthermore, our results confirm and extend the rules governing the interactions of the substrate with the amino acid (aa) side chains of the various pockets within the enzyme's active cleft. We found that the positions flanking the scissile peptide bond P1-P1' are occupied exclusively by hydrophobic aa with both aliphatic or aromatic side chains; Val and Ile, however, are not allowed in the S1 binding site. The S2 and S2' subsites accept hydrophilic aa. Additional requirements concerning the S3' to S5' subsites were also revealed. Finally, the sequences of single peptides generated by cathepsin E from the three different species can be easily aligned to the determined cleavage motif, showing the reliability of our pool sequencing methods.