Tumor antigens and proteomics from the point of view of the major histocompatibility complex peptides

The major histocompatibility complex (MHC) peptide repertoire of cancer cells serves both as a source for new tumor antigens for development of cancer immunotherapy and as a rich information resource about the protein content of the cancer cells (their proteome). Thousands of different MHC peptides are normally displayed by each cell, where most of them are derived from different proteins and thus represent most of the cellular proteome. However, in contrast to standard proteomics, which surveys the cellular protein contents, analyses of the MHC peptide repertoire correspond more to the rapidly degrading proteins in the cells (i.e. the transient proteome). MHC peptides can be efficiently purified by affinity chromatography from membranal MHC molecules, or preferably following transfection of vectors for expression of recombinant soluble MHC molecules. The purified peptides are resolved and analyzed by capillary high-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry, and the data are deciphered with new software tools enabling the creation of large databanks of MHC peptides displayed by different cell types and by different MHC haplotypes. These lists of identified MHC peptides can now be used for searching new tumor antigens, and for identification of proteins whose rapid degradation is significant to cancer progression and metastasis. These lists can also be used for identification of new proteins of yet unknown function that are not detected by standard proteomics approaches. This review focuses on the presentation, identification and analysis of MHC peptides significant for cancer immunotherapy. It is also concerned with the aspects of human proteomics observed through large-scale analyses of MHC peptides.     

Kinetics of registry selection of chimeric peptides binding to MHC II

Major histocompatability complex type II proteins (MHC II) are alphabeta-heterodimeric glycoproteins that present peptides to the T cell receptor (TCR) of CD4(+) T-cells. This presentation may result in activation of these T-cells, depending on the nature of the peptide. Peptides interact specifically with MHC II with nine peptide amino acid positions, and the corresponding MHC II pocket positions are usually labeled P1-P9. However, the length of peptides binding to MHC II may be greater than nine amino acids, and therefore these peptides may potentially bind to the MHC II in more than one registry. To investigate the mechanism by which a long peptide binds to I-E(k), a murine MHC II, a chimeric peptide with two nonoverlapping registries, f-IAYLKQATKQLRMATPLLMR was designed. The IAYLKQATK peptide segment is based on moth cytochrome c 95-103 (MCC 95-103), and the QLRMATPLLMR segment is based on murine Ii CLIP 89-99 M90L (Ii CLIP 89-99 M90L). This chimeric peptide forms two isomeric complexes. The MCC and Ii CLIP registries dissociate from I-E(k) with t(1/2) values of >800 and 4.94 h, respectively. The registry composition of this MHC II/chimeric peptide complex was found to change as a function of time in approaching thermodynamic equilibrium: the results are consistent with a kinetic model that involves no intramolecular isomer interconversion. The model depicts uncorrelated binding to the MHC II determined by relative association rates to the two registries. This is followed by dissociation and subsequent rebinding, leading ultimately to a preponderance of the most stable complex. Similar results were obtained at pH 5.3. The behavior of this chimeric peptide approximates the binding of a 1:1 solution mixture of two peptides to MHC II, where the more stable complex is selected over time. We have also found that a chimeric peptide and a human MHC II, HLA-DR40401, form isomers with relative association rates to DR0401 at pH 5.3 of 15% for one isomer and 85% for the second isomer.     

Degenerate T-cell recognition of peptides on MHC molecules creates large holes in the T-cell repertoire

The cellular immune system screens peptides presented by host cells on MHC molecules to assess if the cells are infected. In this study we examined whether the presented peptides contain enough information for a proper self/nonself assessment by comparing the presented human (self) and bacterial or viral (nonself) peptides on a large number of MHC molecules. For all MHC molecules tested, only a small fraction of the presented nonself peptides from 174 species of bacteria and 1000 viral proteomes ([Formula: see text]0.2%) is shown to be identical to a presented self peptide. Next, we use available data on T-cell receptor-peptide-MHC interactions to estimate how well T-cells distinguish between similar peptides. The recognition of a peptide-MHC by the T-cell receptor is flexible, and as a result, about one-third of the presented nonself peptides is expected to be indistinguishable (by T-cells) from presented self peptides. This suggests that T-cells are expected to remain tolerant for a large fraction of the presented nonself peptides, which provides an explanation for the "holes in the T-cell repertoire" that are found for a large fraction of foreign epitopes. Additionally, this overlap with self increases the need for efficient self tolerance, as many self-similar nonself peptides could initiate an autoimmune response. Degenerate recognition of peptide-MHC-I complexes by T-cells thus creates large and potentially dangerous overlaps between self and nonself.     

Expression of class I histocompatibility antigens in neuroectodermal tumors is independent of the expression of a transfected neuroblastoma myc gene

We have evaluated the relationship between the neuronal myc gene (NMYC) and class I major histocompatibility complex (MHC) expression in human neuroblastoma (NB) tumor cell lines. Class I MHC surface Ag expression in NB cell lines varied from nearly undetectable to levels nearly as high as in a lymphoblastoid cell line. Class I MHC mRNA levels in NMYC-amplified NB cell lines were lower than levels observed in single copy NMYC NB cell lines. However, considerable variation in class I MHC surface Ag and mRNA expression was evident in NMYC-amplified cell lines. To determine directly whether NMYC might modulate class I MHC expression in NB, we transfected a plasmid containing a recombinant NMYC gene into two tumor cell lines derived from a NB and a related neuroepithelioma tumor. Constitutive overexpression of the recombinant NMYC gene produced no consistent change in class I MHC surface Ag or mRNA levels. To determine whether class I MHC expression might be developmentally regulated in adrenal medullary cells, the precursor cells of adrenal NB tumors, beta 2-microglobulin expression was measured in fetal and adult adrenal glands. beta 2-Microglobulin expression was not evident in the neuroblasts of a 24-wk-old fetal adrenal gland, whereas beta 2-microglobulin expression was present in the adult adrenal medulla. These data suggest that variation in class I MHC expression among NB cells may reflect the developmental stage at which neuroblasts were arrested during tumorigenesis.     

Rapid and sensitive identification of major histocompatibility complex class I-associated tumor peptides by Nano-LC MALDI MS/MS

Identification of major histocompatibility complex (MHC)-associated peptides recognized by T-lymphocytes is a crucial prerequisite for the detection and manipulation of specific immune responses in cancer, viral infections, and autoimmune diseases. Unfortunately immunogenic peptides are less abundant species present in highly complex mixtures of MHC-extracted material. Most peptide identification strategies use microcapillary LC coupled to nano-ESI MS/MS in a challenging on-line approach. Alternatively MALDI PSD analysis has been applied for this purpose. We report here on the first off-line combination of nanoscale (nano) LC and MALDI TOF/TOF MS/MS for the identification of naturally processed MHC peptide ligands. These peptides were acid-eluted from human leukocyte antigen (HLA)-A2, HLA-A3, and HLA-B/-C complexes separately isolated from a renal cell carcinoma cell lysate using HLA allele-specific antibodies. After reversed-phase HPLC, peptides were further fractionated via nano-LC. This additional separation step provided a substantial increase in the number of detectable candidate species within the complex peptide pools. MALDI MS/MS analysis on nano-LC-separated material was then sufficiently sensitive to rapidly identify more than 30 novel HLA-presented peptide ligands. Peptide sequences contained perfect anchor amino acid residues described previously for HLA-A2, HLA-A3, and HLA-B7. The most promising candidate for a T-cell epitope is an HLA-B7-binding nonamer peptide derived from the tumor-associated gene NY-BR-16. To demonstrate the sensitivity of our approach we characterized peptides binding to HLA-C molecules that are usually expressed at the cell surface at approximately only 10% the levels of HLA-A or HLA-B. In fact, multiple renal cell carcinoma peptides were identified that contained anchor amino acid residues of HLA-Cw5 and HLA-Cw7. We conclude that the nano-LC MALDI MS/MS approach is a sensitive tool for the rapid and automated identification of MHC-associated tumor peptides.     

T cell epitope definition by differential mass spectrometry: identification of a novel, immunogenic HLA-B8 ligand directly from renal cancer tissue

In this study, we describe a differential mass spectrometric technique for the immuno-proteomic analysis of the major histocompatibility complex (MHC) peptides of a renal cell carcinoma (RCC) biopsy compared with the healthy kidney tissue of the same patient after nephrectomy. Using a stable isotope labeling approach, we could directly compare and relatively quantify 43 MHC-peptide pairs, most of which were present in similar proportions on both normal kidney and tumor. Significantly, two dominant peptides of monoisotopic masses ([M+H](+)) 973.43 u and 967.59 u, respectively, were found exclusively in the tumor sample. One of these was identified as originating from heme oxygenase-1 (HO-1), a protein involved in induction of apoptosis resistance, immuno-suppression and neoangiogenesis and reported to be up-regulated in various cancer types. Moreover, the corresponding synthetic HO-1-derived peptide was shown to be immunogenic in vitro by generation of CD8+ T cell lines with peptide-specific cytolytic activity. Thus, this peptide is an example of a differentially identified T cell epitope that could be considered as a target for immunotherapy.     

Tumor Suppressor Gene-Derived Peptide Antigens

Foreign and self endogenous proteins can be processed and presented as peptides bound to class I and II MHC to CD8 or CD4-positive T cells. In the case of mutant tumor suppressor proteins, proteosomal processing of the mutant protein could occur either in the tumor cell or in an antigen-presenting cell to generate a variety of peptides that can be transported into the endoplasmic reticulum and loaded on the MHC. These peptides may induce tumor suppressor specific T cells in the presence of sufficient T help and costimulation. In human cancer, p53 is frequently found to be both somatically mutant and overexpressed. We and others are currently investigating the potential of peptide-induced cellular immunotherapy to induce cytotoxic T cells to peptides containing point mutant p53, or other oncogene products, thus potentially inducing tumor-specific cellular immunity. There are many potential prerequisites for successful immunotherapeutic targeting of intracellular antigens such as p53, including: (1) the protein must have a sufficient expression level; (2) it should be a candidate for proteolytic degradation and transport into the ER; (3) the tumor-specific epitope must have adequate affinity to the corresponding MHC restriction element; (4) the MHC complex must be expressed at sufficient levels on the cell surface to make the tumor-specific epitope accessible to T cells; and (5) the method of therapeutic immunization must effectively induce oncopeptide-specific cytotoxic T lymphocytes.     

Neuronal cells are deficient in loading peptides onto MHC class I molecules.

Virally infected neurons avoid destruction by cytotoxic T lymphocytes (CTLs) by failing to express major histocompatibility complex (MHC) class I molecules. Like neurons in vivo and in primary culture, the OBL21 neuronal cell line expressed barely detectable levels of MHC class I molecules. This correlated with very low levels of mRNAs for the MHC class I heavy chains (alpha C). OBL21 cells also fail to provide MHC class I molecules with the peptides necessary for their efficient assembly and transport to the cell surface. This function can be restored by treatment with interferon-gamma (IFN-gamma). The mRNA for peptide transporters HAM1 and HAM2 was not detectable in OBL21 neuronal cells, but was induced by IFN-gamma treatment. Hence, the ability of neurons to evade CTL-mediated killing results from expression at low levels of the MHC class I alpha C, the peptide transporters HAM1 and HAM2, and possibly other genes of the peptide-loading machinery.     

Tapasin is a facilitator, not an editor, of class I MHC peptide binding

Tapasin has been proposed to function as a peptide editor to displace lower affinity peptides and/or to favor the binding of high affinity peptides. Consistent with this, cell surface HLA-B8 molecules in tapasin-deficient cells were less stable and the peptide repertoire was substantially altered. However, the binding affinities of peptides expressed in the absence of tapasin were unexpectedly higher, not lower. The peptide repertoire from cells expressing soluble tapasin was similar in both appearance and affinity to that presented in the presence of full-length tapasin, but the HLA-B8 molecules showed altered cell surface stability characteristics. Similarly, the binding affinities of HLA-A*0201-associated peptides from tapasin(+) and tapasin(-) cells were equivalent, although steady state HLA-A*0201 cell surface expression was decreased and the molecules demonstrated reduced cell surface stability on tapasin(-) cells. These data are inconsistent with a role for tapasin as a peptide editor. Instead, we propose that tapasin acts as a peptide facilitator. In this role, it stabilizes the peptide-free conformation of class I MHC molecules in the endoplasmic reticulum and thus increases the number and variety of peptides bound to class I MHC. Full-length tapasin then confers additional stability on class I MHC molecules that are already associated with peptides.     

TCR internalization induced by peptide/MHC ligands requires the transmembrane domains of alphabeta chains of TCR, but not the expression of CD8 and Thy-1 molecules

T-cell receptor (TCR) internalization occurs via TCR recognition of the peptide/MHC molecule complex on antigen presenting cell (APC). In this study, the requirements for inducing the internalization of TCR molecules on Ld major histocompatibility complex (MHC) class I-restricted T-cells were investigated with 2C cytotoxic T-lymphocyte (CTL) clones with defined peptides as the antigen. To evaluate the function of the transmembrane region of TCR alphabeta chains in TCR internalization, we generated T-cell transfectants expressing the wild type and glycosylphosphatidyl inositol (GPI)-linked form of 2C TCR. Among all peptides forming proper ligands to 2C TCR, only the Qp2Ca peptide induced TCR internalization, which was known to have the highest affinity to both Ld MHC class I molecules and TCR in association with Ld molecules. Such TCR internalization was not observed in cells expressing the GPI-linked form of 2C TCR. Furthermore, the expression of CD8 coreceptor and Thy-1 accessory molecules were both not required for Qp2Ca-induced TCR internalization, and these molecules did not accompany TCR internalization. Altogether, these results suggest that TCR internalization on CTL is not a prerequisite for CTL function.     

Immunogenicity of the carcinoembryonic antigen derived peptide 694 in HLA-A2 healthy donors and colorectal carcinoma patients

Carcinoembryonic antigen (CEACAM5) is commonly overexpressed in human colon cancer. Several antigenic peptides recognized by cytolytic CD8+ T-cells have been identified and used in colon cancer phase-I vaccination clinical trials. The HLA-A*0201-binding CEA_694–702 peptide was recently isolated from acid eluted MHC-I associated peptides from a human colon tumor cell line. However, the immunogenicity of this peptide in humans remains unknown. We found that the peptide CEA_694–702 binds weakly to HLA-A*0201 molecules and is ineffective at inducing specific CD8+ T-cell responses in healthy donors. Immunogenic-altered peptide ligands with increased affinity for HLA-A*0201 were identified. Importantly, the elicited cytolytic T lymphocyte (CTL) lines and clones cross-reacted with the wild-type CEA_694–702 peptide. Tumor cells expressing CEA were recognized in a peptide and HLA-A*0201 restricted fashion, but high-CEA expression levels appear to be required for CTL recognition. Finally, CEA-specific T-cell precursors could be readily expanded by in vitro stimulation of peripheral blood mononuclear cell (PBMC) from colon cancer patients with altered CEA peptide. However, the CEA-specific CD8+ T-cell clones derived from cancer patients revealed low-functional avidity and impaired tumor-cell recognition. Together, using T-cells to demonstrate the processing and presentation of the peptide CEA694-702, we were able to corroborate its presentation by tumor cells. However, the low avidity of the specific CTLs generated from cancer patients as well as the high-antigen expression levels required for CTL recognition pose serious concerns for the use of CEA694-702 in cancer immunotherapy.     

Immunomodulatory action of the DNA methyltransferase inhibitor SGI-110 in epithelial ovarian cancer cells and xenografts

We aimed to determine the effect of SGI-110 on methylation and expression of the cancer testis antigens (CTAs) NY-ESO-1 and MAGE-A in epithelial ovarian cancer (EOC) cells in vitro and in vivo and to establish the impact of SGI-110 on expression of major histocompatibility (MHC) class I and Intracellular Adhesion Molecule 1 (ICAM-1) on EOC cells, and on recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. We also tested the impact of combined SGI-110 and NY-ESO-1-specific CD8+ T-cells on tumor growth and/or murine survival in a xenograft setting. EOC cells were treated with SGI-110 in vitro at various concentrations and as tumor xenografts with 3 distinct dose schedules. Effects on global methylation (using LINE-1), NY-ESO-1 and MAGE-A methylation, mRNA, and protein expression were determined and compared to controls. SGI-110 treated EOC cells were evaluated for expression of immune-modulatory genes using flow cytometry, and were co-cultured with NY-ESO-1 specific T-cell clones to determine immune recognition. In vivo administration of SGI-110 and CD8+ T-cells was performed to determine anti-tumor effects on EOC xenografts. SGI-110 treatment induced hypomethylation and CTA gene expression in a dose dependent manner both in vitro and in vivo, at levels generally superior to azacitidine or decitabine. SGI-110 enhanced the expression of MHC I and ICAM-1, and enhanced recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. Sequential SGI-110 and antigen-specific CD8+ cell treatment restricted EOC tumor growth and enhanced survival in a xenograft setting. SGI-110 is an effective hypomethylating agent and immune modulator and, thus, an attractive candidate for combination with CTA-directed vaccines in EOC.     

Unbiased identification of target antigens of CD8+ T cells with combinatorial libraries coding for short peptides

Cytotoxic CD8(+) T cells recognize the antigenic peptides presented by class I major histocompatibility complex (MHC) molecules. These T cells have key roles in infectious diseases, autoimmunity and tumor immunology, but there is currently no unbiased method for the reliable identification of their target antigens. This is because of the low affinities of antigen-specific T cell receptors (TCR) to their target MHC-peptide complexes, the polyspecificity of these TCRs and the requirement that these TCRs recognize protein antigens that have been processed by antigen-presenting cells (APCs). Here we describe a technology for the unbiased identification of the antigenic peptides presented by MHC class I molecules. The technology uses plasmid-encoded combinatorial peptide libraries and a single-cell detection system. We validated this approach using a well-characterized influenza-virus–specific TCR, MHC and peptide combination. Single APCs carrying antigenic peptides can be detected among several million APCs that carry irrelevant peptides. The identified peptide sequences showed a converging pattern of mimotopes that revealed the parent influenza antigen. This technique should be generally applicable to the identification of disease-relevant T cell antigens.     

CD8alpha and CD8beta in Atlantic halibut, Hippoglossus hippoglossus: cloning, characterization and gene expression during viral and bacterial infection

CD8 is expressed on cytotoxic T-cells where it functions as a co-receptor for the TCR by binding to MHC class I proteins that present peptides on the cell surface. In this study we describe the cloning and sequencing of full length cDNAs encoding CD8alpha and CD8beta from Atlantic halibut (Hippoglossus hippoglossus L.) and subsequent isolation and characterization of the CD8alpha and CD8beta genes. The predicted halibut CD8alpha and CD8beta proteins are similar to those of mammals and other fish. Real time RT-PCR revealed that the highest levels of CD8 mRNA were found in the thymus, while some expression was also seen in the spleen, the gills, and the anterior and posterior kidney. In situ hybridization confirmed that the halibut thymus contained numerous CD8alpha and CD8beta expressing cells, while the anterior kidney had no CD8alpha positive cells but only a few CD8beta expressing cells. Only moderate changes in CD8 mRNA expression in other organs during either nodavirus or Vibrio anguillarum infection were observed. Both CD8alpha and CD8beta were significantly (P<0.05) down-regulated in spleen at 48h compared to their levels at 12h post-infection with nodavirus and V. anguillarum.     

Identification of Four-Jointed Box 1 (FJX1)-Specific Peptides for Immunotherapy of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1), a tumor antigen, is overexpressed in NPCs, we investigated if short 9–20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients.     

Uneven tissue distribution of minor histocompatibility proteins versus peptides is caused by MHC expression

Naturally processed minor histocompatibility (H) peptides corresponding to H-4b, H-Y, and an unmapped BALB.B minor H gene were quantified in a relative way in 15 different tissues of male BALB.B mice. For one of these minor H antigens, we could also determine the relative content of the respective protein. For each minor H peptide, an individual tissue distribution was found. Tissues expressing little or no MHC (major histocompatibility complex), like brain, contained only small amounts of minor H peptides or none at all, although the same tissues contained minor H protein in substantial quantities. By contrast, Kb-expressing brains from mice transgenic for Kb under control of the glial acidic protein promoter contained both minor H peptide and protein in high amounts. Thus, the expression of minor H peptides in a given tissue is dependent on coexpression of the restricting MHC class I molecules.     

In vitro maximum binding of antigenic peptides to murine MHC class II molecules does not always take place at the acidic pH of the in vivo endosomal compartment.

Presentation of Ag to the T cell requires binding of specific peptide fragments of the Ag to MHC II molecules. The ability of a peptide to bind to MHC class II appears to be pH dependent. Recent reports indicate that the binding of peptide to MHC class II molecules takes place primarily within an endosomal compartment of the cell at around pH 5. In this study, we have explored the in vitro pH dependence of peptide binding to different haplotypes of murine MHC class II molecules. The binding of peptides to MHC II was analyzed and quantitated by silica gel TLC, using radiolabeled peptides. The MBP peptide fragments, MBP(1-14)A4 and MBP(88-101)Y88, bound maximally at pH 8 to IAk and IAs, respectively. The binding of PLP peptide fragment, PLP(138-151)Y138, to IAs was maximal at around neutral pH. The maximum binding of an OVA peptide fragment, OVA(323-340)Y340, to IAd, was found to occur at pH 6. Results presented in this report thus suggest that the in vitro maximum binding of peptide is pH dependent and does not always occur at pH 5. The optimum pH range for maximum binding may depend on the nature and net charge of the peptide and its interaction with MHC class II molecules.     

Comparative analysis of epitope predictions: proposed library of putative vaccine candidates for HIV

Designing a vaccine for a disease is one of the crucial tasks that involve millions and billions of dollars, several decades and yet there is no guarantee of successful results. Several pharmaceutical companies are investing their money and time in such activities. Computational biology could be of great help in these activities by proving a library of plausible candidates that might actually show some positive responses. MHC binding peptide prediction is one such area where the immense power of computers could be used to get a breakthrough. In this direction several databases and servers have been developed by many labs to predict the MHC binding peptides. These short peptides on the antigen surface are recognized by the MHC molecule and are presented to the receptors of T-cells for further immune response. Peptides that bind to a given MHC molecule share sequence similarity. Here we present a comparative study of servers that can predict the MHC binding peptides in a given protein sequence of the antigen. Based on this comparative analysis on HIV data, we are able to propose a library of putative vaccine candidates for the env GP-160 protein of HIV-1.