Missing Self-Induced Microvascular Rejection of Kidney Allografts: A Population-Based Study.

BACKGROUND: Circulating anti-HLA donor-specific antibodies (HLA-DSA) are often absent in kidney transplant recipients with microvascular inflammation (MVI). Missing self, the inability of donor endothelial cells to provide HLA I-mediated signals to inhibitory killer cell Ig-like receptors (KIRs) on recipient natural killer cells, can cause endothelial damage in vitro, and has been associated with HLA-DSA-negative MVI. However, missing self's clinical importance as a nonhumoral trigger of allograft rejection remains unclear. METHODS: In a population-based study of 924 consecutive kidney transplantations between March 2004 and February 2013, we performed high-resolution donor and recipient HLA typing and recipient KIR genotyping. Missing self was defined as the absence of A3/A11, Bw4, C1, or C2 donor genotype, with the presence of the corresponding educated recipient inhibitory KIR gene. RESULTS: We identified missing self in 399 of 924 transplantations. Co-occurrence of missing self types had an additive effect in increasing MVI risk, with a threshold at two concurrent types (hazard ratio [HR], 1.78; 95% confidence interval [95% CI], 1.26 to 2.53), independent of HLA-DSA (HR, 5.65; 95% CI, 4.01 to 7.96). Missing self and lesions of cellular rejection were not associated. No HLA-DSAs were detectable in 146 of 222 recipients with MVI; 28 of the 146 had at least two missing self types. Missing self associated with transplant glomerulopathy after MVI (HR, 2.51; 95% CI, 1.12 to 5.62), although allograft survival was better than with HLA-DSA-associated MVI. CONCLUSION: Missing self specifically and cumulatively increases MVI risk after kidney transplantation, independent of HLA-DSA. Systematic evaluation of missing self improves understanding of HLA-DSA-negative MVI and might be relevant for improved diagnostic classification and patient risk stratification.

Oral Lichen Planus - Related Connection with HLA-System Antigens.

AIM: To determine whether there is an immunogenic connection and antigen difference between the HLA antigens in the erosive (EOLP) and reticular (ROLP) oral lichen planus. MATERIALS AND METHOD: 73 patients with ROLP and EOLP have been tested. Typing of the HLA antigens has been made for locus A and B. The typing of the HLA was conducted with the use of microlymphocyto toxic test by Terasaki. The reading of the findings has been conducted with an inverse microscope. When a reaction has 4 points it is considered to be positive. RESULTS: The most frequently typified antigens in ROLP from locus A are HLA А2 (57.57%) and А3 (33.33)%, and for locus B 21.21%. In EOLP it is А9 (8888%). In locus B a connection has been found with HLA B8 (77.77%). The statistical analysis with the ×2 test has shown that the carriers of HLA A9 display a relative risk (RR) of 3.65 and ×2=20.72. Consequently, there is high static importance for locus A p<0,001. For locus B, In EOLP for HLA B8, RR=6. 7 ×2=37.64 and p<0,001. ROLP has shown association with HLA A3, where RR=2. 31 and ×2 =9.14 and p<0.05. CONCLUSIONS: In ROLP A3 antigen and in EOLP A9 and A8 may be considered as carriers with proneness to OLP.

The HLA A03 Supertype and Several Pan Species Major Histocompatibility Complex Class I A Allotypes Share a Preference for Binding Positively Charged Residues in the F Pocket: Implications for Controlling Retroviral Infections.

The major histocompatibility complex (MHC) class I region of humans, chimpanzees (Pan troglodytes), and bonobos (Pan paniscus) is highly similar, and orthologues of HLA-A, -B, and -C are present in both Pan species. Based on functional characteristics, the different HLA-A allotypes are classified into different supertypes. One of them, the HLA A03 supertype, is widely distributed among different human populations. All contemporary known chimpanzee and bonobo MHC class I A allotypes cluster genetically into one of the six HLA-A families, the HLA-A1/A3/A11/A30 family. We report here that the peptide-binding motif of the Patr-A*05:01 allotype, which is commonly present in a cohort of western African chimpanzees, has a strong preference for binding peptides with basic amino acids at the carboxyl terminus. This phenomenon is shared with the family members of the HLA A03 supertype. Based on the chemical similarities in the peptide-binding pocket, we inferred that the preference for binding peptides with basic amino acids at the carboxyl terminus is widely present among the human, chimpanzee, and bonobo MHC-A allotypes. Subsequent in silico peptide-binding predictions illustrated that these allotypes have the capacity to target conserved parts of the proteome of human immunodeficiency virus type 1 (HIV-1) and the simian immunodeficiency virus SIVcpz.IMPORTANCE Most experimentally infected chimpanzees seem to control an HIV-1 infection and are therefore considered to be relatively resistant to developing AIDS. Contemporary free-ranging chimpanzees may carry SIVcpz, and there is evidence for AIDS-like symptoms in these free-ranging animals, whereas SIV infections in bonobos appear to be absent. In humans, the natural control of an HIV-1 infection is strongly associated with the presence of particular HLA class I allotypes. The ancestor of the contemporary living chimpanzees and bonobos survived a selective sweep targeting the MHC class I repertoire. We have put forward a hypothesis that this may have been caused by an ancestral retroviral infection similar to SIVcpz. Characterization of the relevant MHC allotypes may contribute to understanding the shaping of their immune repertoire. The abundant presence of MHC-A allotypes that prefer peptides with basic amino acids at the C termini suggests that these molecules may contribute to the control of retroviral infections in humans, chimpanzees, and bonobos.

Differential escape of HCV from CD8+ T cell selection pressure between China and Germany depends on the presenting HLA class I molecule.

Adaptation of hepatitis C virus (HCV) to CD8+ T cell selection pressure is well described; however, it is unclear if HCV differentially adapts in different populations. Here, we studied HLA class I-associated viral sequence polymorphisms in HCV 1b isolates in a Chinese population and compared viral substitution patterns between Chinese and German populations. We identified three HLA class I-restricted epitopes in HCV NS3 with statistical support for selection pressure and found evidence for differential escape pathways between isolates from China and Germany depending on the HLA class I molecule. The substitution patterns particularly differed in the epitope VTLTHPITK1635-1643 , which was presented by HLA-A*03 as well as HLA-A*11, two alleles with highly different frequencies in the two populations. In Germany, a substitution in position seven of the epitope was the most frequent substitution in the presence of HLA-A*03, functionally associated with immune escape and nearly absent in Chinese isolates. In contrast, the most frequent substitution in China was located at position two of the epitope and became the predominant consensus residue. Moreover, substitutions in position one of the epitope were significantly enriched in HLA-A*11-positive individuals in China and associated with different patterns of CD8+ T cell reactivity. Our study confirms the differential escape pathways selected by HCV that depended on different HLA class I alleles in Chinese and German populations, indicating that HCV differentially adapts to distinct HLA class I alleles in these populations. This result has important implications for vaccine design against highly variable and globally distributed pathogens, which may require matching antigen sequences to geographic regions for T cell-based vaccine strategies.

Mapping HLA-A2, -A3 and -B7 supertype-restricted T-cell epitopes in the ebolavirus proteome.

BACKGROUND: Ebolavirus (EBOV) is responsible for one of the most fatal diseases encountered by mankind. Cellular T-cell responses have been implicated to be important in providing protection against the virus. Antigenic variation can result in viral escape from immune recognition. Mapping targets of immune responses among the sequence of viral proteins is, thus, an important first step towards understanding the immune responses to viral variants and can aid in the identification of vaccine targets. Herein, we performed a large-scale, proteome-wide mapping and diversity analyses of putative HLA supertype-restricted T-cell epitopes of Zaire ebolavirus (ZEBOV), the most pathogenic species among the EBOV family. METHODS: All publicly available ZEBOV sequences (14,098) for each of the nine viral proteins were retrieved, removed of irrelevant and duplicate sequences, and aligned. The overall proteome diversity of the non-redundant sequences was studied by use of Shannon's entropy. The sequences were predicted, by use of the NetCTLpan server, for HLA-A2, -A3, and -B7 supertype-restricted epitopes, which are relevant to African and other ethnicities and provide for large (~86%) population coverage. The predicted epitopes were mapped to the alignment of each protein for analyses of antigenic sequence diversity and relevance to structure and function. The putative epitopes were validated by comparison with experimentally confirmed epitopes. RESULTS & DISCUSSION: ZEBOV proteome was generally conserved, with an average entropy of 0.16. The 185 HLA supertype-restricted T-cell epitopes predicted (82 (A2), 37 (A3) and 66 (B7)) mapped to 125 alignment positions and covered ~24% of the proteome length. Many of the epitopes showed a propensity to co-localize at select positions of the alignment. Thirty (30) of the mapped positions were completely conserved and may be attractive for vaccine design. The remaining (95) positions had one or more epitopes, with or without non-epitope variants. A significant number (24) of the putative epitopes matched reported experimentally validated HLA ligands/T-cell epitopes of A2, A3 and/or B7 supertype representative allele restrictions. The epitopes generally corresponded to functional motifs/domains and there was no correlation to localization on the protein 3D structure. These data and the epitope map provide important insights into the interaction between EBOV and the host immune system.

Genetic Analysis with the Immunochip Platform in Behçet Disease. Identification of Residues Associated in the HLA Class I Region and New Susceptibility Loci.

Behcet's disease (BD) is an immuno-mediated vasculitis in which knowledge of its etiology and genetic basis is limited. To improve the current knowledge, a genetic analysis performed with the Immunochip platform was carried out in a population from Spain. A discovery cohort comprising 278 BD cases and 1,517 unaffected controls were genotyped using the Immunochip platform. The validation step was performed on an independent replication cohort composed of 130 BD cases and 600 additional controls. The strongest association signals were observed in the HLA class I region, being HLA-B*51 the highest peak (overall P = 6.82E-32, OR = 3.82). A step-wise conditional logistic regression with classical alleles identified HLA-B*57 and HLA-A*03 as additional independent markers. The amino acid model that best explained the association, includes the position 97 of the HLA-B molecule and the position 66 of the HLA-A. Among the non-HLA loci, the most significant in the discovery analysis were: IL23R (rs10889664: P = 3.81E-12, OR = 2.00), the JRKL/CNTN5 region (rs2848479: P = 5.00E-08, OR = 1.68) and IL12A (rs1874886: P = 6.67E-08, OR = 1.72), which were confirmed in the validation phase (JRKL/CNTN5 rs2848479: P = 3.29E-10, OR = 1.66; IL12A rs1874886: P = 1.62E-08, OR = 1.61). Our results confirm HLA-B*51 as a primary-association marker in predisposition to BD and suggest additional independent signals within the class I region, specifically in the genes HLA-A and HLA-B. Regarding the non-HLA genes, in addition to IL-23R, previously reported in our population; IL12A, described in other populations, was found to be a BD susceptibility factor also in Spaniards; finally, a new associated locus was found in the JRKL/CNTN5 region.

Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance.

T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines.

Identification of two novel HLA-A alleles, HLA-A*03:181 and HLA-A*03:229 in Chinese individuals.

HLA-A*03:181 and HLA-A*03:229 differ from HLA-A*03:01:01:01 by one and three nucleotide substitutions, respectively.

Description of two new HLA-A alleles, HLA-A*02:572 and HLA-A*03:225.

Characterization of two new human-leukocyte antigen (HLA)-A alleles, A*02:572 and HLA-A*03:225.

A Phase 2 Randomized Controlled Trial of Personalized Peptide Vaccine Immunotherapy with Low-dose Dexamethasone Versus Dexamethasone Alone in Chemotherapy-naive Castration-resistant Prostate Cancer.

BACKGROUND: It is well known that the prognosis of castration-resistant prostate cancer (CRPC) is poor, and several immunotherapeutic strategies have been applied to the clinical trials. Research on immunotherapy has been of special interest for the treatment of CRPC for years. OBJECTIVE: To evaluate the safety of personalized peptide vaccine (PPV) immunotherapy and its clinical outcomes. DESIGN, SETTING, AND PARTICIPANTS: A phase 2 randomized controlled trial of PPV immunotherapy with low-dose dexamethasone versus dexamethasone alone for chemotherapy-naive CRPC began in 2008. Eligible patients (prostate-specific antigen [PSA] <10 ng/ml) were human leukocyte antigen (HLA) A02, A24, or A03 superfamily positive and had asymptomatic or minimally symptomatic CRPC. Patients were allocated (1:1) to PPV plus dexamethasone (1mg/d) or to dexamethasone (1mg/d) alone. A maximum of four HLA-matched peptides (each 3mg) was selected based on the preexisting immunoglobulin G responses against the 24 warehouse peptides and administered every 2 wk. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: PSA, progression-free survival (PFS), time to initiation of chemotherapy, and overall survival (OS) were analyzed using the Kaplan-Meier method, a log-rank test, and proportional hazard analysis. RESULTS AND LIMITATIONS: Overall, 37 patients received peptide vaccinations and 35 received dexamethasone alone. The primary end point was PSA PFS, which was significantly longer in the vaccination group than in the dexamethasone group (22.0 vs 7.0 mo; p=0.0076). Median OS was also significantly longer in the vaccination group (73.9 vs 34.9 mo; p=0.00084). The relatively small number of patients enrolled is the major limitation of the study. CONCLUSIONS: PPV immunotherapy was well tolerated and associated with longer PSA PFS and OS in men with chemotherapy-naive CRPC. A larger phase 3 study is needed to confirm our findings. PATIENT SUMMARY: We compared clinical outcomes of the treatment with personalized peptide vaccine plus dexamethasone versus dexamethasone alone. Our data provide promising evidence of clinical benefit for peptide vaccines. TRIAL REGISTRATION: UMIN-CTR: 000000959.

Multiple structural and epigenetic defects in the human leukocyte antigen class I antigen presentation pathway in a recurrent metastatic melanoma following immunotherapy.

Scant information is available about the molecular basis of multiple HLA class I antigen-processing machinery defects in malignant cells, although this information contributes to our understanding of the molecular immunoescape mechanisms utilized by tumor cells and may suggest strategies to counteract them. In the present study we reveal a combination of IFN-γ-irreversible structural and epigenetic defects in HLA class I antigen-processing machinery in a recurrent melanoma metastasis after immunotherapy. These defects include loss of tapasin and one HLA haplotype as well as selective silencing of HLA-A3 gene responsiveness to IFN-γ. Tapasin loss is caused by a germ-line frameshift mutation in exon 3 (TAPBP(684delA)) along with a somatic loss of the other gene copy. Selective silencing of HLA-A3 gene and its IFN-γ responsiveness is associated with promoter CpG methylation nearby site-α and TATA box, reversible after DNA methyltransferase 1 depletion. This treatment combined with tapasin reconstitution and IFN-γ stimulation restored the highest level of HLA class I expression and its ability to elicit cytotoxic T cell responses. These results represent a novel tumor immune evasion mechanism through impairing multiple components at various levels in the HLA class I antigen presentation pathway. These findings may suggest a rational design of combinatorial cancer immunotherapy harnessing DNA demethylation and IFN-γ response.

Cell surface expression level variation between two common Human Leukocyte Antigen alleles, HLA-A2 and HLA-B8, is dependent on the structure of the C terminal part of the alpha 2 and the alpha 3 domains.

Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses. Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of-B7 (P = 0.002). Transfection experiments with full-length HLA-A2 and -B8 encoding plasmids confirmed this (54,031 molecules per cell vs. 2,466, respectively, P = 0.001) independently of transcript levels suggesting a post-transcriptional regulation. Using chimeric constructs we found that the cytoplasmic tail and the transmembrane region had no impact on the differential cell surface expression. In contrast, ~65% of the difference could be mapped to the six C-terminal amino acids of the alpha 2 domain and the alpha 3 domain (amino acids 176-284), i.e. amino acids not previously shown to be of importance for differential expression levels of HLA class I molecules. We suggest that the differential cell surface expression of two common HLA-A and-B alleles is regulated by a post-translational mechanism that may involve hitherto unrecognized molecules.

Broadening the repertoire of melanoma-associated T-cell epitopes.

Immune therapy has provided a significant breakthrough in the treatment of metastatic melanoma. Despite the remarkable clinical efficacy and established involvement of effector CD8 T cells, the knowledge of the exact peptide-MHC complexes recognized by T cells on the tumor cell surface is limited. Many melanoma-associated T-cell epitopes have been described, but this knowledge remains largely restricted to HLA-A2, and we lack understanding of the T-cell recognition in the context of other HLA molecules. We selected six melanoma-associated antigens (MAGE-A3, NY-ESO-1, gp100, Mart1, tyrosinase and TRP-2) that are frequently recognized in patients with the aim of identifying novel T-cell epitopes restricted to HLA-A1, -A3, -A11 and -B7. Using in silico prediction and in vitro confirmation, we identified 127 MHC ligands and analyzed the T-cell responses against these ligands via the MHC multimer-based enrichment of peripheral blood from 39 melanoma patients and 10 healthy donors. To dissect the T-cell reactivity against this large peptide library, we used combinatorial-encoded MHC multimers and observed the T-cell responses against 17 different peptide-MHC complexes in the patient group and four in the healthy donor group. We confirmed the processing and presentation of HLA-A3-restricted T-cell epitopes from tyrosinase (TQYESGSMDK) and gp100 (LIYRRRLMK) and an HLA-A11-restricted T-cell epitope from gp100 (AVGATKVPR) via the cytolytic T-cell recognition of melanoma cell lines and/or K562 cells expressing the appropriate antigen and HLA molecule. We further found T-cell reactivity against two of the identified sequences among tumor-infiltrating lymphocytes from melanoma patients, suggesting a potential clinical relevance of these sequences.

New polycomb group protein enhancer of zeste homolog (EZH) 2-derived peptide with the potential to induce cancer-reactive cytotoxic T lymphocytes in prostate cancer patients with HLA-A3 supertype alleles.

Analyses on reactivity of anti-cancer cytotoxic T lymphocytes (CTLs) and clinical application of peptide-based anti-cancer vaccine have been mainly focused on patients with HLA-A2 or -A24 alleles. In this study, we identified an enhancer of zeste homolog (EZH) 2-derived peptide applicable for anti-cancer vaccine for prostate cancer patients with HLA-A3 supertype alleles. Five EZH2-derived peptides that were prepared based on the binding motif to the HLA-A3 supertype alleles (HLA-A11, -A31, and -A33) were functionally screened for their potential to induce peptide-specific CTLs from peripheral blood mononuclear cells (PBMCs) of HLA-A3 supertype allele(+) prostate cancer patients. As a result, EZH2733-741 peptide was found to efficiently induce peptide-specific CTLs. The EZH2733-741 peptide-stimulated and purified CD8(+) T cells from PBMCs of HLA-A3 supertype allele(+) prostate cancer patients showed higher cytotoxicity against HLA-A3 supertype allele-expressing LNCaP prostate cancer cells than against parental LNCaP cells. This cytotoxicity against HLA-A3 supertype allele-expressing LNCaP cells was partially but significantly inhibited by the addition of EZH2733-741 peptide-pulsed competitive cells. These results indicate that the EZH2733-741 peptide could be a promising candidate for peptide-based immunotherapy for HLA-A3 supertype allele(+) prostate cancer patients.

Human herpesvirus 6B immediate-early I protein contains functional HLA-A*02, HLA-A*03, and HLA-B*07 class I restricted CD8(+) T-cell epitopes.

Human herpesvirus 6B (HHV-6B) is a ubiquitous pathogen with frequent reactivation observed in immunocompromised patients such as BM transplant (BMT) recipients. Adoptive immunotherapy is a promising therapeutic avenue for the treatment of opportunistic infections, including herpesviruses. While T-cell immunotherapy can successfully control CMV and EBV reactivations in BMT recipients, such therapy is not available for HHV-6 infections, in part due to a lack of identified protective CD8(+) T-cell epitopes. Our goal was to identify CD8(+) T-cell viral epitopes derived from the HHV-6B immediate-early protein I and presented by common human leukocyte Ag (HLA) class I alleles including HLA-A*02, HLA-A*03, and HLA-B*07. These epitopes were functionally tested for their ability to induce CD8(+) T-cell expansion and kill HHV-6-infected autologous cells. Cross-reactivity of specific HHV-6B-expanded T cells against HHV-6A-infected cells was also confirmed for a conserved epitope presented by HLA-A*02 molecule. Our findings will help push forward the field of adoptive immunotherapy for the treatment and/or the prevention of HHV-6 reactivation in BMT recipients.

Final report of the phase I/II clinical trial of the E75 (nelipepimut-S) vaccine with booster inoculations to prevent disease recurrence in high-risk breast cancer patients.

BACKGROUND: E75 (nelipepimut-S) is a human leukocyte antigen (HLA)-A2/A3-restricted immunogenic peptide derived from the HER2 protein. We have conducted phase I/II clinical trials vaccinating breast cancer patients with nelipepimut-S and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the adjuvant setting to prevent disease recurrence. All patients have completed 60 months follow-up, and here, we report the final analyses. PATIENTS AND METHODS: The studies were conducted as dose escalation/schedule optimization trials enrolling node-positive and high-risk node-negative patients with tumors expressing any degree of HER2 (immunohistochemistry 1-3+). HLA-A2/3+ patients were vaccinated; others were followed prospectively as controls. Local and systemic toxicity was monitored. Clinical recurrences were documented, and disease-free survival (DFS) was analyzed by Kaplan-Meier curves; groups were compared using log-rank tests. RESULTS: Of 195 enrolled patients, 187 were assessable: 108 (57.8%) in the vaccinated group (VG) and 79 (42.2%) in the control group (CG). The groups were well matched for clinicopathologic characteristics. Toxicities were minimal. Five-year DFS was 89.7% in the VG versus 80.2% in the CG (P = 0.08). Due to trial design, 65% of patients received less than the optimal vaccine dose. Five-year DFS was 94.6% in optimally dosed patients (P = 0.05 versus the CG) and 87.1% in suboptimally dosed patients. A voluntary booster program was initiated, and among the 21 patients that were optimally boosted, there was only one recurrence (DFS = 95.2%). CONCLUSION: The E75 vaccine is safe and appears to have clinical efficacy. A phase III trial evaluating the optimal dose and including booster inoculations has been initiated. CLINICAL TRIALS: NCT00841399, NCT00584789.

A spliced antigenic peptide comprising a single spliced amino acid is produced in the proteasome by reverse splicing of a longer peptide fragment followed by trimming.

Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. Peptides produced by splicing can be presented on class I molecules of the MHC and recognized by CTLs. In this study, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two noncontiguous fragments of the melanoma differentiation Ag gp100(PMEL17) spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3-6 aa, the peptide described in this work results from the ultimate association of an 8-aa fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least 3 aa long to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C terminus that needs to be further trimmed to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described in this work.

Identification and characterization of agonist epitopes of the MUC1-C oncoprotein.

The MUC1 tumor-associated antigen is overexpressed in the majority of human carcinomas and several hematologic malignancies. Much attention has been paid to the hypoglycosylated variable number of tandem repeats (VNTR) region of the N-terminus of MUC1 as a vaccine target, and recombinant viral vector vaccines are also being evaluated that express the entire MUC1 transgene. While previous studies have described MUC1 as a tumor-associated tissue differentiation antigen, studies have now determined that the C-terminus of MUC1 (MUC1-C) is an oncoprotein, and its expression is an indication of poor prognosis in numerous tumor types. We report here the identification of nine potential CD8⁺ cytotoxic T lymphocyte epitopes of MUC1, seven in the C-terminus and two in the VNTR region, and have identified enhancer agonist peptides for each of these epitopes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 major histocompatibility complex (MHC) class I alleles, which encompass the majority of the population. The agonist peptides, compared to the native peptides, more efficiently (a) generate T-cell lines from the peripheral blood mononuclear cells of cancer patients, (b) enhance the production of IFN-γ by peptide-activated human T cells, and (c) lyse human tumor cell targets in an MHC-restricted manner. The agonist epitopes described here can be incorporated into various vaccine platforms and for the ex vivo generation of human T cells. These studies provide the rationale for the T-cell-mediated targeting of the oncogenic MUC1-C, which has been shown to be an important factor in both drug resistance and poor prognosis for numerous tumor types.

KIR3DL2 binds to HLA-B27 dimers and free H chains more strongly than other HLA class I and promotes the expansion of T cells in ankylosing spondylitis.

The human leukocyte Ag HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of APC as both classical ß2-microglobulin-associated B27 and B27 free H chain forms (FHC), including disulfide-bonded H chain homodimers (termed B27(2)). B27 FHC forms, but not classical B27, bind to KIR3DL2. HLA-A3, which is not associated with spondyloarthritis (SpA), is also a ligand for KIR3DL2. In this study, we show that B27(2) and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B27(2) tetramers bound KIR3DL2-transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric, and monomeric FHC from HLA-B27-expressing cell lines. Binding to B27(2) and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B27(2) and B27 FHC stimulated KIR3DL2CD3ε-transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFN-γ secretion and promoted greater survival of KIR3DL2(+) CD4 T and NK cells than binding to other HLA-class I. KIR3DL2(+) T cells from B27(+) SpA patients proliferated more in response to Ag presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27(+) SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC.

Cross-allele cytotoxic T lymphocyte responses against 2009 pandemic H1N1 influenza A virus among HLA-A24 and HLA-A3 supertype-positive individuals.

Lack of a universal vaccine against all serotypes of influenza A viruses and recent progress on T cell-related vaccines against influenza A virus illuminate the important role of human leukocyte antigen (HLA)-restricted cytotoxic T lymphocytes (CTLs) in anti-influenza virus immunity. However, the diverse HLA alleles among humans complicate virus-specific cellular immunity research, and elucidation of cross-HLA allele T cell responses to influenza virus specificity requires further detailed work. An ideal CTL epitope-based vaccine would cover a broad spectrum of epitope antigens presented by most, if not all, of the HLAs. Here, we evaluated the 2009 pandemic influenza A (H1N1) virus-specific T cell responses among the HLA-A24(+) population using a rationally designed peptide pool during the 2009 pandemic. Unexpectedly, cross-HLA allele T cell responses against the influenza A virus peptides were detected among both HLA-A11(+) and HLA-A24(+) donors. Furthermore, we found cross-responses in the entire HLA-A3 supertype population (including HLA-A11, -A31, -A33, and -A30). The cross-allele antigenic peptides within the peptide pool were identified and characterized, and the crystal structures of the major histocompatibility complex (MHC)-peptide complexes were determined. The subsequent HLA-A24-defined cross-allele peptides recognized by the HLA-A11(+) population were shown to mildly bind to the HLA-A*1101 molecule. Together with the structural models, these results partially explain the cross-allele responses. Our findings elucidate the promiscuity of the cross-allele T cell responses against influenza A viruses and are beneficial for the development of a T cell epitope-based vaccine applied in a broader population.