A Conserved E7-derived Cytotoxic T Lymphocyte Epitope Expressed on Human Papillomavirus 16-transformed HLA-A2+ Epithelial Cancers

Human Papillomavirus 16 (HPV-16) has been identified as the causative agent of 50% of cervical cancers and many other HPV-associated tumors. The transforming potential/tumor maintenance capacity of this high risk HPV is mediated by two viral oncoproteins, E6 and E7, making them attractive targets for therapeutic vaccines. Of 21 E6 and E7 peptides computed to bind HLA-A*0201, 10 were confirmed through TAP-deficient T2 cell HLA stabilization assay. Those scoring positive were investigated to ascertain which were naturally processed and presented by surface HLA molecules for CTL recognition. Because IFNγ ELISpot frequencies from healthy HPV-exposed blood donors against HLA-A*0201-binding peptides were unable to identify specificities for tumor targeting, their physical presence among peptides eluted from HPV-16-transformed epithelial tumor HLA-A*0201 immunoprecipitates was analyzed by MS³ Poisson detection mass spectrometry. Only one epitope (E7_11–19) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E7_11–20), previously used as a vaccine candidate, was neither detected by MS³ on HPV-transformed tumor cells nor effectively recognized by 9-mer specific CTL. These data underscore the importance of precisely defining CTL epitopes on tumor cells and offer a paradigm for T cell-based vaccine design.     

The diagnostic potential of MPT63‐derived HLA‐A*0201‐restricted CD8+T‐cell epitopes for active pulmonary tuberculosis

MPT63 protein is found only in Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis. Detection of MPT63‐specific IFN‐γ‐secreting T cells could be useful for the diagnosis of tuberculosis (TB) diseases. In the present study, the HLA‐A*0201 restriction of ten predicted MPT63‐derived CD8 ⁺ T‐cell epitopes was assessed on the basis of T2 cell line and HLA‐A*0201 transgenic mice. The diagnostic potential of immunogenic peptides in active pulmonary TB patients was evaluated using an IFN‐γ enzyme‐linked immunospot assay. It was found that five peptides bound to HLA‐A*0201 with high affinity, whereas the remaining peptides exhibited low affinity for HLA‐A*0201. Five immunogenic peptides (MPT63_18–26, MPT63_29–37, MPT63_20–28, MPT63_5–14 and MPT63_10–19) elicited large numbers of cytotoxic IFN‐γ‐secreting T cells in HLA‐A*0201 transgenic mice. Each of the five immunogenic peptides was recognized by peripheral blood mononuclear cells from 45% to 73% of 40 HLA‐A*0201 positive TB patients. The total diagnostic sensitivity of the five immunogenic peptides was higher than that of a T‐SPOT.TB assay (based on ESAT‐6 and CFP‐10) (93% versus 90%). It is noticeable that the diagnostic sensitivity of the combination of five immunogenic peptides and T‐SPOT.TB assay reached 100%. These MPT63‐derived HLA‐A*0201‐restricted CD8 ⁺ T‐cell epitopes would likely contribute to the immunological diagnosis of M. tuberculosis infection and may provide the components for designing an effective TB vaccine.     

Exploring skyline for both MSE‐based label‐free proteomics and HRMS quantitation of small molecules

The MS^E (where MS^E is low energy (MS) and elevated energy (E) mode of acquisition) acquisition method commercialized by Waters on its Q‐TOF instruments is regarded as a unique data‐independent fragmentation approach that improves the accuracy and dynamic range of label‐free proteomic quantitation. Due to its special format, MS^E acquisition files cannot be independently analyzed with most widely used open‐source proteomic software specialized for processing data‐dependent acquisition files. In this study, we established a workflow integrating Skyline, a popular and versatile peptide‐centric quantitation program, and a statistical tool DiffProt to fulfill MS^E‐based proteomic quantitation. Comparison with the vendor software package for analyzing targeted phosphopeptides and global proteomic datasets reveals distinct advantages of Skyline in MS^E data mining, including sensitive peak detection, flexible peptide filtering, and transparent step‐by‐step workflow. Moreover, we developed a new procedure such that Skyline MS1 filtering was extended to small molecule quantitation for the first time. This new utility of Skyline was examined in a protein–ligand interaction experiment to identify multiple chemical compounds specifically bound to NDM‐1 (where NDM is New Delhi metallo‐β‐lactamase 1), an antibiotics‐resistance target. Further improvement of the current weaknesses in Skyline MS1 filtering is expected to enhance the reliability of this powerful program in full scan‐based quantitation of both peptides and small molecules.     

Identification of T- and B-cell epitopes of the E7 protein of human papillomavirus type 16.

There is strong evidence implicating human papillomavirus type 16 (HPV16) in the genesis of human genital cancer. Viral DNA has been identified in invasive carcinoma of the uterine cervix and in cell lines derived from cervical carcinomas. These sequences are actively transcribed, and translation products corresponding to the early (E)-region genes have been identified. The most abundant viral protein is the E7 protein, which has been shown to possess transforming activity for both established and primary cells. In addition, it has been shown to bind to a cellular tumor suppressor, the retinoblastoma gene product (pRb-105). In view of these properties, we have undertaken the immunological analysis of this protein and have identified four T-cell epitopes and three B-cell epitopes by using a series of overlapping peptides spanning the entire HPV16 E7 sequence. Two of the B-cell epitopes were recognized by antisera from mice with three different murine (H-2) haplotypes (k, d, and s) immunized with two different E7 fusion proteins and from Fischer rats seeded with baby rat kidney cells transformed by HPV16 E7 and ras. A third B-cell epitope was recognized by antisera from CBA mice seeded with HPV16 E7-expressing L cells. Two regions of the protein contain common B- and T-cell epitopes, one of which appears to be particularly immunodominant.     

Identification of a WT1 protein‐derived peptide,WT1187, as a HLA‐A*0206‐restricted,WT1‐specific CTL epitope

The Wilms' tumor gene WT1 is overexpressed in various kinds of hematopoietic malignancies as well as solid cancers, and this protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. WT1‐specific CTL epitopes with a restriction of HLA‐A*2402 or HLA‐A*0201 have been already identified. In the present study it has been demonstrated that a 9‐mer WT1‐derived WT1₁₈₇ peptide, which had already been shown to elicit a WT1‐specific CTL response with a restriction of HLA‐A*0201, can also elicit a CTL response with a restriction of HLA‐A*0206. In all three different HLA‐A*0206⁺ healthy donors examined, WT1₁₈₇ peptide‐specific CTL could be generated from peripheral blood mononuclear cells, and the CTL showed cytotoxic activity that depended on dual expression of WT1 and HLA‐A*0206 molecules. The present study describes the first identification of a HLA‐A*0206‐restricted, WT1‐specific CTL epitope. The present results should help to broaden the application of WT1 peptide‐based immunotherapy from only HLA‐A*0201‐positive to HLA‐A*0206‐positive cancer patients as well.     

A WT1 protein‐derived,naturally processed 16‐mer peptide,WT1332, is a promiscuous helper peptide for induction of WT1‐specific Th1‐type CD4+ T cells

The Wilms' tumor gene WT1 is overexpressed in various tumors, and the WT1 protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. A WT1 protein‐derived 16‐mer peptide, WT1₃₃₂ (KRYFKLSHLQMHSRKH), which was naturally generated through processing in cells and could elicit Th1‐type CD4⁺ helper T cell responses with an HLA‐DRB1*0405‐restriction has previously been identified by us. In the present study, it has been demonstrated that WT1₃₃₂ can induce WT1₃₃₂‐specific CD4⁺ T cell responses with the restriction of not only HLA‐DRB1*0405 but also HLA‐DRB1*1501, ‐DRB1*1502, or ‐DPB1*0901. These HLA class II‐restricted WT1₃₃₂‐specific CD4⁺ T cell lines produced IFN‐γ but neither IL‐4 nor IL‐10 with WT1₃₃₂ stimulation, thus showing a Th1‐type cytokine profile. Furthermore, HLA‐DRB1*1501 or ‐DRB1*1502‐restricted WT1₃₃₂‐specific CD4⁺ T cell lines responded to WT1‐expressing transformed cells in an HLA‐DRB1‐restricted manner, which is consistent with our previous finding that WT1₃₃₂ is a naturally processed peptide. These results indicate that the natural peptide, WT1₃₃₂, is a promiscuous WT1‐specific helper epitope. WT1₃₃₂ is expected to apply to cancer patients with various types of HLA class II as a WT1‐specific helper peptide in combination with HLA class I‐restricted WT1 peptides.     

Identification of HLA‐A*11:01‐restricted Mycobacterium tuberculosis CD8+ T cell epitopes

New vaccines are needed to combat Mycobacterium tuberculosis (MTB) infections. The currently employed Bacillus Calmette‐Guérin vaccine is becoming ineffective, due in part to the emergence of multidrug‐resistant tuberculosis (MDR‐TB) strains and the reduced immune capacity in cases of HIV coinfection. CD8⁺ T cells play an important role in the protective immunity against MTB infections, and the identification of immunogenic CD8⁺ T cell epitopes specific for MTB is essential for the design of peptide‐based vaccines. To identify CD8⁺ T cell epitopes of MTB proteins, we screened a set of 94 MTB antigens for HLA class I A*11:01‐binding motifs. HLA‐A*11:01 is one of the most prevalent HLA molecules in Southeast Asians, and definition of T cell epitopes it can restrict would provide significant coverage for the Asian population. Peptides that bound with high affinity to purified HLA molecules were subsequently evaluated in functional assays to detect interferon‐γ release and CD8⁺ T cell proliferation in active pulmonary TB patients. We identified six novel epitopes, each derived from a unique MTB antigen, which were recognized by CD8⁺ T cells from active pulmonary TB patients. In addition, a significant level of epitope‐specific T cells could be detected ex vivo in peripheral blood mononuclear cells from active TB patients by an HLA‐A*11:01 dextramer carrying the peptide Rv3130c_194‐204 (from the MTB triacylglycerol synthase Tgs1), which was the most frequently recognized epitope in our peptide library. In conclusion, this study identified six dominant CD8⁺ T cell epitopes that may be considered potential targets for subunit vaccines or diagnostic strategies against TB.     

POSTMan (POST‐translational modification analysis), a software application for PTM discovery

Post‐translationally modified peptides present in low concentrations are often not selected for CID, resulting in no sequence information for these peptides. We have developed a software POSTMan (POST‐translational Modification analysis) allowing post‐translationally modified peptides to be targeted for fragmentation. The software aligns LC‐MS runs (MS₁ data) between individual runs or within a single run and isolates pairs of peptides which differ by a user defined mass difference (post‐translationally modified peptides). The method was validated for acetylated peptides and allowed an assessment of even the basal protein phosphorylation of phenylalanine hydroxylase (PHA) in intact cells.     

Analysis of sesterterpenoids from Aspergillus terreus using ESI‐QTOF and ESI‐IT

Introduction – Biosynthesis of terretonin was studied due to the interesting skeleton of this series of sesterterpenoids. Very recently, López‐Gresa reported two new sesterterpenoids (terretonins E and F) which are inhibitors of the mammalian mitochondrial respiratory chain. Mass spectrometry (MS), especially tandem mass spectrometry, has been one of the most important physicochemical methods for the identification of trace natural products due to it rapidity, sensitivity and low levels of sample consumption. The potential application prospect and unique skeleton prompted us to study structural characterisation using MS. Objective – To obtain sufficient information for rapid structural elucidation of this class of compounds using MS. Methodology – The elemental composition of the product ions was confirmed by low‐energy ESI‐CID‐QTOF‐MS/MS analyses. The fragmentation pathways were postulated on the basis of ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ spectra. Common features and major differences between ESI‐QTOF‐MS/MS and IT‐MSⁿ spectra were compared. For ESI‐QTOF‐MS/MS/MS experiments, capillary exit voltage was raised to induce in‐source dissociation. Ammonium acetate or acetic acid were added into solutions to improve the intensity of [M + H]⁺. The collision energy was optimised to achieve sufficient fragmentation. Some fragmentation pathways were unambiguously proposed by the variety of abundance of fragment ions at different collision energies even without MSⁿ spectra. Results – Fragmentation pathways of five representative sesterterpenoids were elucidated using ESI‐QTOF‐MS/MS/MS and ESI‐IT‐MSⁿ in both positive‐ and negative‐ion mode. The key group of characterising fragmentation profiles was ring B, and these fragmentation patterns are helpful to identify different types of sestertepenoids. Conclusion – Complementary information obtained from fragmentation experiments of [M + H]⁺ (or [M + NH₄]⁺) and [M ? H]^? precursor ions is especially valuable for rapid identification of this kind of sesterterpenoid.     

Metadherin peptides containing CD4+ and CD8+ T cell epitopes as a therapeutic vaccine candidate against cancer

The concept of peptide‐based vaccines against cancer has made noteworthy progress. Metadherin (MTDH) overexpression and its role in the development of diverse cancers make it an attractive target for cancer immunotherapy. In the current study, six different T cell epitope prediction tools were run to identify MTDH peptides with multiple immunogenic regions. Further, molecular docking was performed to assess HLA‐peptide binding interactions. Nine and eleven peptides fragments containing multiple CD8 ⁺ and CD4 ⁺ T‐cell epitopes, ranging from 9 to 20 amino acids, respectively, were obtained using a consensus immunoinformatics approach. The three peptides that were finally identified as having overlapping CD4 ⁺ and CD8 ⁺ T‐ cell epitopes are ARLREMLSVGLGFLRTELG, FLLGYGWAAACAGAR, YIDDEWSGLNGLSSADP. These peptides were found to not only have multiple T cell epitopes but also to have binding affinity with wide HLA molecules. A molecular docking study revealed that the predicted immunogenic peptides (with single or multiple T cell epitopes) of MTDH have comparable binding energies with naturally bound peptides for both HLA classes I and II. Thus, these peptides have the potential to induce immune responses that could be considered for developing synthetic peptide vaccines against multiple cancers.     

Quantitative analysis of phosphopeptides in search of the disease biomarker from the hepatocellular carcinoma specimen

Reversible phosphorylation of proteins is the most common PTM in cell‐signaling pathways. Despite this, high‐throughput methods for the systematic detection, identification, and quantification of phosphorylated peptides have yet to be developed. In this paper, we describe the establishment of an efficient online titaniuim dioxide (TiO₂)‐based 3‐D LC (strong cationic exchange/TiO₂/C18)‐MS³‐linear ion trap system, which provides fully automatic and highly efficient identification of phosphorylation sites in complex peptide mixtures. Using this system, low‐abundance phosphopeptides were isolated from cell lines, plasma, and tissue of healthy and hepatocellular carcinoma (HCC) patients. Furthermore, the phosphorylation sites were identified and the differences in phosphorylation levels between healthy and HCC patient specimens were quantified by labeling the phosphopeptides with isotopic analogs of amino acids (stable isotope labeling with amino acids in cell culture for HepG2 cells) or water (HO for tissues and plasma). Two examples of potential HCC phospho‐biomarkers including plectin‐1(phopho‐Ser‐4253) and alpha‐HS‐glycoprotein (phospho‐Ser 138 and 312) were identified by this analysis. Our results suggest that this comprehensive TiO₂‐based online‐3‐D LC‐MS³‐linear ion trap system with high‐throughput potential will be useful for the global profiling and quantification of the phosphoproteome and the identification of disease biomarkers.     

Characterization of low‐abundance species in the active pharmaceutical ingredient of CIGB‐300: A clinical‐grade anticancer synthetic peptide

CIGB‐300 is a first‐in‐class synthetic peptide‐based drug of 25 amino acids currently undergoing clinical trials in cancer patients. It contains an amidated disulfide cyclic undecapeptide fused to the TAT cell‐penetrating peptide through a beta‐alanine spacer. CIGB‐300 inhibits the CK2‐mediated phosphorylation leading to apoptosis of tumor cells in vitro, and in vivo in cancer patients. Despite the clinical development of CIGB‐300, the characterization of peptide‐related impurities present in the active pharmaceutical ingredient has not been reported earlier. In the decision tree of ICHQ3A(R2) guidelines, the daily doses intake, the abundance, and the identity of the peptide‐related species are pivotal nodes that define actions to be taken (reporting, identification, and qualification). For this, purity was first assessed by reverse‐phase chromatography (>97%) and low‐abundance impurities (≤0.27%) were collected and identified by mass spectrometry. Most of the impurities were generated during peptide synthesis, the spontaneous air oxidation of the reduced peptide, and the lyophilization step. The most abundant impurity, with no biological activity, was the full‐length peptide containing Met¹⁷ transformed into a sulfoxide residue. Interestingly, parallel and antiparallel dimers of CIGB‐300 linked by 2 intermolecular disulfide bonds exhibited a higher antiproliferative activity than the CIGB‐300 monomer. Likewise, very low abundance trimers and tetramers of CIGB‐300 linked by disulfide bonds (≤0.01%) were also detected. Here we describe for the first time the presence of active dimeric species whose feasibility as novel CIGB‐300 derived entities merits further investigation.     

Quantitative peptidomic analysis by a newly developed one-step direct transfer technology without depletion of major blood proteins: its potential utility for monitoring of pathophysiological status in pregnancy-induced hypertension

We have recently developed a new target plate (BLOTCHIP^®) for MALDI‐MS. An advantage of this procedure is that it does not require the lowering of protein concentrations in test samples prior to analysis. Accordingly, this new technology enables the detection of peptides present in blood samples, including those that would otherwise be adsorbed to abundant blood proteins and would thus escape detection. Using this technology, we analyzed the peripheral blood of patients with pregnancy‐induced hypertension (PIH; the most common serious complication of pregnancy) to test a potential utility of the technology for monitoring of the pathophysiological status. In the present study, we found 23 characteristic peptides for PIH in the blood serum of pregnant women. Offline LC‐MALDI MS/MS identified 7 of the 23 peptides as fragments derived from kininogen‐1 (three peptides), fibrinogen‐α, complement component C4‐A/B, α‐2‐HS‐glycoprotein and inter‐α‐trypsin inhibitor heavy chain H4. 2‐D scatter plots with combinations of the peptides found in the present study can be grouped for pregnant women with/without PIH, which would be satisfactory reflected for their status. Additionally, the levels of most of these peptides found were significantly decreased by albumin/IgG depletion prior to BLOTCHIP^® analysis in accordance with conventional proteomics procedures. These results indicated that BLOTCHIP^® analysis can be applied for discovery study of PIH biomarker candidates.     

High Immunogenicity of the Human Leukocyte Antigen Peptidomes of Melanoma Tumor Cells

Human leukocyte antigens (HLA) bind peptides generated by limited proteolysis in cells and present them at the cell surfaces for recognition by T cells. Through this antigen presentation function they control the specificity of T cell responses and thereby adaptive immune responses. Knowledge of HLA-bound peptides is thus key to understanding adaptive immunity and to the development of vaccines and other specific immune intervention strategies. To gain insight into the antigenicity of melanomas, peptides were extracted from HLA isolated from the tumor cells, separated by two-dimensional HPLC, and sequenced by mass spectrometry. The spectra were analyzed by database-dependent MASCOT searches and database-independent de novo sequencing and, where required, confirmed with synthetic peptides, which were also used to determine their immunogenicity. Comparing four different melanoma cell lines, little overlap of the HLA-bound peptides was found, suggesting a high degree of individualization of the HLA peptidomes. This notwithstanding, the peptidomes were highly immunogenic in the patients from whom the tumor cells had been established and in unrelated patients. This broad cross-patient immunogenicity was only exceptionally related to individual peptides. The majority of the identified epitopes were derived from low to medium abundance proteins, mostly involved in sensitive cellular processes such as cell cycle control, DNA replication, control of gene expression, tumor suppressor function, and protein metabolism. The peptidomes thus provide insights into processes potentially related to tumorigenesis. Furthermore, analyses of the peptide sequences yield information on the specificity of peptide selection by HLA applicable to the developing prediction algorithms for T cell epitopes.     

LALF32‐51‐E7, a HPV‐16 therapeutic vaccine candidate,forms protein body‐like structures when expressed in Nicotiana benthamiana leaves

High‐risk human papillomaviruses (HPVs) cause cervical cancer, and while there are good prophylactic vaccines on the market, these are ineffective against established infections, creating a clear need for therapeutic vaccines. The HPV E7 protein is one of the essential oncoproteins for the onset and maintenance of malignancy and is therefore an ideal therapeutic vaccine target. We fused the HPV‐16 E7 protein to the Limulus polyphemus antilipopolysaccharide factor (LALF_32‐51), a small hydrophobic peptide that can penetrate cell membranes and that has immunomodulatory properties. LALF_32‐51‐E7 was transiently expressed in Nicotiana benthamiana, and we previously determined that it accumulated better when targeted to chloroplasts compared to being localized in the cytoplasm. Subsequently, we aimed to prove whether LALF_32‐51‐E7 was indeed associated with the chloroplasts by determining its subcellular localization. The LALF_32‐51‐E7 gene was fused to one encoding enhanced GFP to generate a LG fusion protein, and localization was determined by confocal laser scanning microscopy and transmission electron microscopy (TEM). The fluorescence observed from chloroplast‐targeted LG was distinctively different from that of the cytoplasmic LG. Small spherical structures resembling protein bodies (PBs) were seen that clearly localized with the chloroplasts. Larger but less abundant PB‐like structures were also seen for the cytoplasmic LG. PB‐like structure formation was confirmed for both LG and LALF_32‐51‐E7 by TEM. LALF_32‐51‐E7 was indeed targeted to the chloroplasts by the chloroplast transit peptide used in this study, and it formed aggregated PB‐like structures. This study could open a new avenue for the use of LALF_32‐51 as a PB‐inducing peptide.     

Multiantigenic peptide–polymer conjugates as therapeutic vaccines against cervical cancer

Immunotherapy is one of the most promising strategies for the treatment of cancer. Human papillomavirus (HPV) is responsible for virtually all cases of cervical cancer. The main purpose of a therapeutic HPV vaccine is to stimulate CD8⁺ cytotoxic T lymphocytes (CTLs) that can eradicate HPV infected cells. HPV oncoproteins E6 and E7 are continuously expressed and are essential for maintaining the growth of HPV-associated tumor cells. We designed polymer-based multi-antigenic formulations/constructs that were comprised of the E6 and E7 peptide epitopes. We developed an N-terminus-based epitope conjugation to conjugate two unprotected peptides to poly tert-butyl acrylate. This method allowed for the incorporation of the two antigens into a polymeric dendrimer in a strictly equimolar ratio. The most effective formulations eliminated tumors in up to 50% of treated mice. Tumor recurrence was not observed up to 3 months post initial challenge.     

Analysis of novel over‐ and under‐sulfated glycosaminoglycan sequences by enzyme cleavage and multiple stage MS

We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MSⁿ). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di‐ and hexasaccharides were analyzed by ESI MSⁿ. MS² on bisulfated 4,5‐Δ‐HexAGalNAc revealed an additional sulfate ester group at 4,5‐Δ‐HexA. MS² data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5‐Δ‐HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS¹ mode indicated direct correlation between the sulfate distribution and HexA epimerization. MSⁿ performed on ions that, according to mass calculation, correspond to pentasulfated [4,5‐Δ‐HexAGalNAc(GlcAGalNAc)₂], trisulfated [4,5‐Δ‐HexAGalNAc(GlcAGalNAc)₂] with IdoA‐derived 4,5‐Δ‐HexA at the nonreducing end, tetrasulfated [4,5‐Δ‐HexAGalNAc(IdoAGalNAc)₂] and monosulfated [4,5‐Δ‐HexAGalNAc(IdoAGalNAc)₂] with GlcA‐derived 4,5‐Δ‐HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over‐, regular, and under‐sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.     

Use of Interferon-γ Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-γ (IFN-γ) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected¹. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- γ secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-γ secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- γ ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)² in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 10⁵ cells per well typically used in ELISPOT assays^1,3, 1,000 T-cell clone cells in the presence of 1 x 10⁵ autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-γ. This assures successful performance of IFN-γ ELISPOT assay. Similarly, IFN- γ ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- γ ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes⁴.     

Exploration of peptides bound to MHC class I molecules in melanoma

Advancements in high‐resolution HPLC and mass spectrometry have reinvigorated the application of this technology to identify peptides eluted from immunopurified MHC class I molecules. Three melanoma cell lines were assessed using w6/32 isolation, peptide elution and HPLC purification; peptides were identified by mass spectrometry. A total of 13 829 peptides were identified; 83–87% of these were 8–11 mers. Only approximately 15% have been described before. Subcellular locations of the source proteins showed even sampling; mRNA expression and total protein length were predictive of the number of peptides detected from a single protein. HLA‐type binding prediction for 10 078 9/10 mer peptides assigned 88–95% to a patient‐specific HLA subtype, revealing a disparity in strength of predicted binding. HLA‐B*27‐specific isolation successfully identified some peptides not found using w6/32. Sixty peptides were selected for immune screening, based on source protein and predicted HLA binding; no new peptides recognized by antimelanoma T cells were discovered. Additionally, mass spectrometry was unable to identify several epitopes targeted ex vivo by one patient's T cells.