Tilman Dumrese

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.

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.

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 thanthe 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 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 cyto toxic 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.

Efficient T cell repertoire selection in tetraparental chimeric mice independent of thymic epithelial MHC

Nonthymic epithelial cells were compared with thymic epithelial cells for their role in T cell repertoire selection. Tetraparental aggregation chimeras were generated from T and B cell-deficient mice (H-2d SCID or H-2b Rag−/−) and thymus-deficient nude mice (H-2b or H-2d). These tetraparental mice showed primary protective CD8+ T cell responses, after lymphocytic choriomeningitis virus infection, that were peptide-specifically restricted to either thymic or nonthymic epithelial MHC at comparable levels. These chimeras also mounted neutralizing IgG responses dependent on cognate CD4+ T helper cell activity restricted to nonthymic epithelial MHC. Therefore, in contrast to earlier results with irradiation or thymus chimeras, these relatively undisturbed tetraparental mice reveal that the MHC of nonthymic epithelial cells efficiently selects a functional T cell repertoire.

Identification of Chlamydia pneumoniae-Derived Mouse CD8 Epitopes

ABSTRACT Chlamydia pneumoniae is a common intracellular human pathogen that has been associated with several severe pathological conditions, including coronary heart disease and atherosclerosis. There is no vaccine against C. pneumoniae infection, but CD8+ T cells have been shown to be crucial for protection during experimental infection. However, the effector functions and epitope specificity of the protective CD8+ T cell remain unknown. The aim of this study was to identify C. pneumoniae-derived mouse CD8 epitopes by using a recent epitope prediction method. Of four C. pneumoniae proteins (the major outer membrane protein, outer membrane protein 2, polymorphic outer membrane protein 5, and heat shock protein 60), 53 potential CD8+ T-cell epitopes were predicted by H-2 class I binding algorithms. Nineteen of the 53 peptides were identified as CD8 epitopes by testing for induction of a cytotoxic response after immunization. To test whether the predicted epitopes are naturally processed and presented by C. pneumoniae-infected cells, we generated a panel of seven peptide-specific cytotoxic T lymphocyte lines that were subsequently tested for recognition of C. pneumoniae-infected target cells. By using this strategy, we were able to identify three C. pneumoniae CD8 epitopes that were, indeed, processed and presented on infected cells. Identification of these natural CD8 epitopes provides tools for characterization of CD8+ T-cell function in vivo and generation of epitope-specific prevention strategies.

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.Al 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.

The HLA-B* 1516 motif demonstrates HLA-B-specific P2 pocket characteristics.

HLA polymorphism is a phenomenon which has to be contended with for the design of peptide-based vaccines. Considering the number of different HLA alleles present in an outbred population, as wide a range of peptide motifs as possible has to be known in order to foster our knowledge of peptide-MHC interaction and for use in the prediction of T-cell epitopes. To determine the peptide motif of HLAB*1516 as expressed by a TAP-deficient patient (de la Salle et al. 1997), we performed sequence analysis of HLAB*1516-extracted peptide pools and individual ligands. The results provided an unexpected insight into general features of peptide-MHC interaction. A cDNA encoding HLA-B*1516 was obtained by reverse transcription -polymerase chain reaction (de la Salle et al. 1997) and cloned into the NT expression vector (a gift from C. Bonnerot, Institute Curie, Paris). After transfecting the HMy2.C1R cell line (ATCC CRL 1993) with HLAB*1516, we expanded the cells up to 35±50 l. MHC class I molecules and bound peptides were isolated according to standard protocols (Falk et al. 1991) with the monoclonal antibody (mAb)W6/32 (Barnstable et al. 1978). After the elution of peptides by acid treatment (0.1% trifluoroacetic acid), ultrafiltration, and HPLC separation, fractions were pooled (dominant peaks were sequenced individually) and sequenced by Edman degradation on the sequencer model 494 A (Applied Biosystems, Weiterstadt, Germany). Evaluation of pool sequencing data was carried out as described (Falk et al. 1991; Stevanovic 1997). Raw pool sequencing data are not shown, but summaries of several experiments are given in Table 1. Peptides eluted from B*1516 molecules are nonamers or occasionally octamers. Pool sequencing revealed P2 anchor amino acids by clear signals at position 2 for serine and threonine, whereby other amino acids were detected only at background levels. The carboxyl terminus is occupied mainly by small hydrophobic amino acids such as isoleucine and valine, but large aromatic amino acids are also present in two ligands. Thus, P9 is a hydrophobic anchor with fairly broad specificity which is often observed in human MHC I molecules. Proline showed strong signals at position 4; this is a phenomenon often seen in class I ligand pools and is possibly either the result of unknown processing events or is necessary for forcing peptides into a kink conformation which could be advantageous for tight binding to the MHC. In the case of HLA-B*1516, the proline signal is much stronger in pool sequencing compared with other class I pools, and five of eleven natural ligands carry proline in P4. Thus, we consider proline at position 4 to be advantageous for binding to B*1516 and classify it as an auxiliary anchor. In other positions, there are preferred residues similar to those present in many class I motifs: hydrophilic amino acids are likely to be present at positions 4 and 8, whereas position 6 is often occupied by hydrophobic amino acids. Very similar results were obtained by pool sequencing of peptides from W6/32-precipitated HLA molecules from the TAP-negative line ST-EMO, followed by subtraction of the A3 motif (data not shown). From C1RB*1516, ten natural ligands that all confirmed the motif derived from pool sequencing were identified. Nine were 9mers and one was an 8mer. From ST-EMO cells, one 8mer ligand fitting into the B*1516 motif but not into the A3 motif was identified (Table 1). Thus, this peptide (not derived from a signal sequence) must be loaded to the MHC in a TAP-independent way. To analyze the fine specificity of the B*1516 motif in greater detail, peptide binding assays using the ST-EMO cell line were performed. The TAP2cell line ST-EMO (de la Salle et al.1997) that was used for binding studies is an HLA homozygous Epstein-Barr virus-transformed B-cell line expressing low level HLA-A*0301, HLA-B*1516, and HLA-Cw*1402 on plasma membrane due to a stop mutaF.H. Seeger ? D. Arnold ? T. Dumrese ? H. Schild ? H.-G. Rammensee ? S. Stevanovic ( ) Institute of Cell Biology, Department of Immunology, Auf der Morgenstelle 15 (VerfuÈgungsgebaÈude), D-72076 TuÈbingen, Germany