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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., (Copyright © 2021 by the American Society of Nephrology.)

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 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., (Copyright © 2020 American Society for Microbiology.)

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 VTLTHPITK 1635-1643 , 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., (© 2018 The Authors. Journal of Viral Hepatitis Published by John Wiley & Sons Ltd.)

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

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.

HLA-A*03:181 and HLA-A*03:229 differ from HLA-A*03:01:01:01 by one and three nucleotide substitutions, respectively., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Characterization of two new human-leukocyte antigen (HLA)-A alleles, A*02:572 and HLA-A*03:225., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.

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.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

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.

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 β2-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.

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.

Two major obstacles confronting HIV vaccine design have been the extensive viral diversity of HIV-1 globally and viral evolution driven by escape from CD8(+) cytotoxic T-cell lymphocyte (CTL)-mediated immune pressure. Regions of the viral genome that are not able to escape immune response and that are conserved in sequence and across time may represent the "Achilles' heel" of HIV and would be excellent candidates for vaccine development. In this study, T-cell epitopes were selected using immunoinformatics tools, combining HLA-A3 binding predictions with relative sequence conservation in the context of global HIV evolution. Twenty-seven HLA-A3 epitopes were chosen from an analysis performed in 2003 on 10,803 HIV-1 sequences, and additional sequences were selected in 2009 based on an expanded set of 43,822 sequences. These epitopes were tested in vitro for HLA binding and for immunogenicity with PBMCs of HIV-infected donors from Providence, Rhode Island. Validation of these HLA-A3 epitopes conserved across time, clades, and geography supports the hypothesis that epitopes such as these would be candidates for inclusion in our globally relevant GAIA HIV vaccine constructs.

HLA-A*03:57 has two amino acid exchanges in exon 2 at adjacent positions of the peptide-binding groove., (© 2011 John Wiley & Sons A/S.)

Background and Aims: CD8 T cells are central to the control of hepatitis C virus (HCV) although the key features of a successful CD8 T cell response remain to be defined. In a cohort of Irish women infected by a single source, a strong association between viral clearance and the human lecucocyte (HLA)-A*03 allele has been described, and the aim of this study was to define the protective nature of the associated CD8 T cell response., Methods: A sequence-led approach was used to identify HLA-A*03-restricted epitopes. We examine the CD8 T cell response associated with this gene and address the likely mechanism underpinning this protective effect in this special cohort, using viral sequencing, T cell assays and analysis of fitness of viral mutants., Results: A strong 'HLA footprint' in a novel NS3 epitope (TVYHGAGTK) was observed. A lysine (K) to arginine (R) substitution at position 9 (K1088R) was seen in a significant number of A*03-positive patients (9/12) compared with the control group (1/33, p=0.0003). Threonine (T) was also substituted with alanine (A) at position 8 (T1087A) more frequently in A*03-positive patients (6/12) compared with controls (2/33, p=0.01), and the double substitution of TK to AR was also observed predominantly in HLA-A*03-positive patients (p=0.004). Epitope-specific CD8 T cell responses were observed in 60% of patients three decades after exposure and the mutants selected in vivo impacted on recognition in vitro. Using HCV replicons matched to the viral sequences, viral fitness was found to be markedly reduced by the K1088R substitution but restored by the second substitution T1087A., Conclusions: It is proposed that at least part of the protective effect of HLA-A*03 results from targeting of this key epitope in a functional site: the requirement for two mutations to balance fitness and escape provides an initial host advantage. This study highlights the potential protective impact of common HLA-A alleles against persistent viruses, with important implications for HCV vaccine studies.

Human leukocyte antigens (HLA) are initially classified by serotyping but recently can be re-grouped by their peptide-presentation characteristics into supertypes. Both HLA-A*0301 and HLA-A*1101 are grouped into A3 supertype. Although a number of cross-presented T cell epitopes of HLA-A*0301 and HLA-A*1101 have been identified, the molecular mechanisms of cross-presentation remain elusive. Herein, the structures of HLA-A*0301 with two HIV-derived immunodominant T cell epitopes were solved and their characteristics in comparison with HLA-A*1101 presenting the same peptides were analyzed. The comparable structures of HLA-A*0301 and HLA-A*1101 with subtle differences illustrate the common modes of cross-presented peptides and the strict HLA-restriction of T cell receptor (TCR)-recognition., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The Simian immunodeficiency virus (SIV)-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection and AIDS-related research, despite the potential that macaques of Chinese origin is a more relevant model. Ongoing efforts to further characterize the Chinese rhesus macaques' major histocompatibility complex (MHC) for composition and function should facilitate greater utilization of the species. Previous studies have demonstrated that Chinese-origin M. mulatta (Mamu) class I alleles are more polymorphic than their Indian counterparts, perhaps inferring a model more representative of human MHC, human leukocyte antigen (HLA). Furthermore, the Chinese rhesus macaque class I allele Mamu-A1*02201, the most frequent allele thus far identified, has recently been characterized and shown to be an HLA-B7 supertype analog, the most frequent supertype in human populations. In this study, we have characterized two additional alleles expressed with high frequency in Chinese rhesus macaques, Mamu-A1*02601 and Mamu-B*08301. Upon the development of MHC-peptide-binding assays and definition of their associated motifs, we reveal that these Mamu alleles share peptide-binding characteristics with the HLA-A2 and HLA-A3 supertypes, respectively, the next most frequent human supertypes after HLA-B7. These data suggest that Chinese rhesus macaques may indeed be a more representative model of HLA gene diversity and function as compared to the species of Indian origin and therefore a better model for investigating human immune responses.

Anti-HLA antibody (Ab) monitoring is essential for the follow-up of transplant patients. However, it can be affected by drugs, especially Ab infused as a conditioning regimen for transplantation. ATG Fresenius, commonly used in this setting, is a polyclonal rabbit Ab raised against the Jurkat human T cell line (HLA-A3, 32; B7, 35). We report here the de novo detection by CDC and flow cytometry (Luminex) of anti-HLA-A3 Ab in the serum of kidney recipients treated with ATG Fresenius. The Ab, of rabbit origin, was detected in every assessable patient (n = 16), with the exception of the HLA-A3 recipients and/or recipients receiving an HLA-A3 graft, before becoming undetectable, at latest, at day 102 after transplantation. It is of major importance that transplantation monitoring laboratories bear in mind the possibility of therapeutic Ab detection when interpreting anti-HLA Ab results., (Copyright 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

We previously reported peptide vaccine candidates for HLA-A3 supertype (-A3, -A11, -A31, -A33)-positive cancer patients. In the present study, we examined whether those peptides can also induce cytotoxic T lymphocyte (CTL) activity restricted to HLA-A2, HLA-A24, and HLA-A26 alleles. Fourteen peptides were screened for their binding activity to HLA-A*0201, -A*0206, -A*0207, -A*2402, and -A*2601 molecules and then tested for their ability to induce CTL activity in peripheral blood mononuclear cells (PBMCs) from prostate cancer patients. Among these peptides, one from the prostate acid phosphatase protein exhibited binding activity to HLA-A*0201, -A*0206, and -A*2402 molecules. In addition, PBMCs stimulated with this peptide showed that HLA-A2 or HLA-A24 restricted CTL activity. Their cytotoxicity toward cancer cells was ascribed to peptide-specific and CD8+ T cells. These results suggest that this peptide could be widely applicable as a peptide vaccine for HLA-A3 supertype-, HLA-A2-, and -A24-positive cancer patients.

Monoclonal antibody production using the hybridoma technology is one of the attractive areas of research. The aim of this study was to investigate the production of monoclonal antibodies to human HLA-A3 antigen for use in microlymphocytotoxicity technique. HLA-A3 antigen was purified from human leukocytes and injected to several mice. The spleen-derived cells of immunized animal were fused with P3-X63-Ag8 myeloma cells. Obtained hybridoma cells were grown in HAT medium and the supernatant of the cells were screened for antibodies using ELISA and microlymphocytotoxicity technique. Produced monoclonal antibody from the hybridoma clone, B1F10 showed the specificity for human HLA-A3. Furthermore, in this study, we encountered with clones of immortal hybrid cells with completely different features, they were adherent and non-producers for antibodies. This observation could imply the possible role of the antigen (HLA) and its nature in the expansion of cell types other than B cells in the spleen of mouse during immunization.

Background: E75, a HER2/neu immunogenic peptide, is expressed in breast cancer (BCa). We have performed clinical trials of E75 + GM-CSF vaccine in disease-free, node-positive and node-negative BCa patients at high recurrence risk and recurrences were noted in both control and vaccine groups., Methods: Among the 186 BCa patients enrolled, 177 completed the study. Patients were HLA typed; the HLA-A2(+)/A3(+) patients were vaccinated; HLA-A2(-)/A3(-) patients were followed as controls. Standard clinicopathological factors, immunologic response to the vaccine, and recurrences were collected and assessed., Results: The control group recurrence rate was 14.8 and 8.3% in the vaccinated group (P = 0.17). Comparing the 8 vaccinated recurrences (V-R) to the 88 vaccinated nonrecurrent patients (V-NR), the V-R group had higher nodal stage (> or = N2: 75 vs. 5%, P = 0.0001) and higher grade tumors (%grade 3: 88 vs. 31%, P = 0.003). The V-R group did not fail to respond immunologically as noted by equivalent dimer responses and post-DTH responses. Compared to control recurrent patients (C-R), V-R patients trended toward higher-grade tumors and hormone-receptor negativity. C-R patients had 50% bone-only recurrences, compared to V-R patients with no bone-only recurrences (P = 0.05). Lastly, V-R mortality rate was 12.5% compared with 41.7% for the C-R group (P = 0.3)., Conclusions: The vaccinated patients who recurred had more aggressive disease compared to V-NR patients. V-R patients had no difference in immune response to the vaccine either in vitro or in vivo. V-R patients, when compared to C-R patients, trended towards more aggressive disease, decreased recurrence rates, decreased mortality, and no bone-only recurrences.

Most cancer deaths are due to the development of metastases. Increased expression of RhoC is linked to enhanced metastatic potential in multiple cancers. Consequently, the RhoC protein is an attractive target for drug design. The clinical application of immunotherapy against cancer is rapidly moving forward in multiple areas, including the adoptive transfer of anti-tumor-reactive T cells and the use of "therapeutic" vaccines. The over-expression of RhoC in cancer and the fact that immune escape by down regulation or loss of expression of this protein would reduce the morbidity and mortality of cancer makes RhoC a very attractive target for anti-cancer immunotherapy. Herein, we describe an HLA-A3 restricted epitope from RhoC, which is recognized by cytotoxic T cells. Moreover, RhoC-specific T cells show cytotoxic potential against HLA-matched cancer cells of different origin. Thus, RhoC may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies.

Purpose: A prerequisite for the development of vaccination strategies is the identification and characterization of relevant tumor-associated antigen. Using microarray and reverse transcription-PCR analysis, we found matrix metalloproteinase (MMP)-7 to be extensively up-regulated in renal cell carcinomas and expressed in a broad variety of malignant cells. MMP-7 can promote cancer invasion and angiogenesis by proteolytic cleavage of extracellular matrix and basement membrane proteins, thus making it a promising target in the context of immunotherapies., Experimental Design: To analyze the possible use of MMP-7 as a tumor-associated antigen, specific CTLs were induced using monocyte-derived dendritic cells electroporated with MMP-7-mRNA. In addition, to better characterize the fine specificity of these CTLs, MMP-7 MHC class I ligands were isolated and characterized in renal cell carcinoma tissue, which overexpressed MMP-7, by mass spectrometry-based peptide sequencing. Using this approach, we identified a novel HLA-A3-binding antigenic MMP-7 peptide. CTLs generated from healthy donors by in vitro priming with dendritic cells, pulsed with the novel peptide, were used as effectors in (51)Cr-release assays., Results: The induced CTLs elicited an antigen-specific and HLA-restricted cytolytic activity against tumor cells endogenously expressing the MMP-7 protein. Furthermore, we were able to induce MMP-7-specific CTLs using peripheral blood mononuclear cells from a patient with acute lymphoblastic leukemia capable of recognizing the autologous leukemic blasts while sparing nonmalignant cells., Conclusions: Our study describes the identification of a novel broadly expressed T-cell epitope derived from the MMP-7 protein that represents an interesting candidate to be applied in immunotherapies of human malignancies targeting both tumor cells and neovascularization.

Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8(+) T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.

Peptide presentation is critical for immune recognition of pathogen-infected cells by CD8+ T lymphocytes. Although a limited number of immunodominant peptide epitopes are consistently observed in diseases such as HIV-1 infection, the relationship between immunodominance and antigen processing in humans is largely unknown. Here, we have demonstrated that endogenous processing and presentation of a human immunodominant HIV-1 epitope is more efficient than that of a subdominant epitope. Furthermore, we have shown that the regions flanking the immunodominant epitope constitute a portable motif that increases the production and antigenicity of otherwise subdominant epitopes. We used a novel in vitro degradation assay involving cytosolic extracts as well as endogenous intracellular processing assays to examine 2 well-characterized HIV-1 Gag overlapping epitopes presented by the same HLA class I allele, one of which is consistently immunodominant and the other subdominant in infected persons. The kinetics and products of degradation of HIV-1 Gag favored the production of peptides encompassing the immunodominant epitope and destruction of the subdominant one. Notably, cytosolic digestion experiments revealed flanking residues proximal to the immunodominant epitope that increased the production and antigenicity of otherwise subdominant epitopes. Furthermore, specific point mutations in these portable flanking sequences modulated the production and antigenicity of epitopes. Such portable epitope processing determinants provide what we believe is a novel approach to optimizing CTL responses elicited by vaccine vectors.

SART3-derived peptides applicable to prostate cancer patients with HLA-A3 supertype alleles were identified in order to expand the possibility of an anti-cancer vaccine, because the peptide vaccine candidates receiving the most attention thus far have been the HLA-A2 and HLA-A24 alleles. Twenty-nine SART3-derived peptides that were prepared based on the binding motif to the HLA-A3 supertype alleles (HLA-A11, -A31, and -A33) were first screened for their recognizability by immunoglobulin G (IgG) of prostate cancer patients and subsequently for the potential to induce peptide-specific cytotoxic T lymphocytes (CTLs) from HLA-A3 supertype(+) prostate cancer patients. As a result, five SART3 peptides were frequently recognized by IgG, and two of them-SART3 (511-519) and SART3 (734-742)-efficiently induced peptide-specific and cancer-reactive CTLs. Their cytotoxicity toward prostate cancer cells was ascribed to peptide-specific and CD8(+) T cells. These results indicate that these two SART3 peptides could be promising candidates for peptide-based immunotherapy for HLA-A3 supertype(+) prostate cancer patients.

The E3-19K protein from human adenoviruses (Ads) retains class I MHC molecules in the endoplasmic reticulum. As a consequence, the cell surface expression of class I molecules is suppressed, allowing Ads to evade immune surveillance. Using native gel electrophoresis, gel filtration chromatography, and surface plasmon resonance, we show that a soluble form of the Ad type 2 (Ad2) E3-19K protein associates with HLA-A and -B molecules; equilibrium dissociation constants were in the nanomolar range and approximately 2.5-fold higher affinity for HLA-A (-A*0201, -A*0301, -A*1101, -A*3301, and -Aw*6801) relative to HLA-B (-B*0702 and -B*0801) molecules. Among the alleles of the HLA-A locus examined, HLA-A*3101 associated approximately 15-fold less avidly with soluble E3-19K. Soluble E3-19K interacted only very weakly with HLA-Cw*0304, and no interaction with HLA-Cw*0401 could be detected under identical conditions. Site-directed mutagenesis and flow cytometry demonstrated that MHC residue 56 plays a critical role in the association and endoplasmic reticulum retention of HLA-A molecules by E3-19K. This delineates the spatial environment around residue 56 as a putative E3-19K interaction surface on class I molecules. Overall, our data imply that a link may exist between host genetic factors and the susceptibility of individuals to Ad infections.

The binding of peptides to MHC class I molecules induces MHC/peptide complexes that have specific conformational features. Little is known about the molecular and structural bases required for an optimal MHC/peptide association able to induce a dominant T cell response. We sought to characterize the interaction between purified HLA-A3 molecules and four well known CD8 epitopes from HIV-1 proteins. To define the characteristics of HLA-peptide complex formation and to identify potential structural changes, we used biochemical assays that detect well formed complexes. We tested the amplitude, stability, and kinetic parameters of the interaction between HLA-A3, peptides, and anti-HLA mAbs. Our results show that the four epitopes Nef73-82, Pol325-333, Env37-46, and Gag20-28 bind strongly to HLA-A3 molecules and form very stable complexes that are detected with differential patterns of mAb reactivity. The most striking result is the nonrecognition of the HLA-A3/Gag20-28 complex by the A11.1M mAb specific to HLA-A3/-A11 alleles. To explain this observation, from the data published on HLA-A11 crystallographic structure, we propose molecular models of the HLA-A3 molecule complexed with Nef73-82, Pol325-333, and Gag20-28 epitopes. In the HLA-A3/Gag20-28 complex, we suggest that Arg at position P1 of the peptide may push the alpha2 helix residue Trp-167 of HLA-A3 and affect mAb recognition. Such observations may have great implications for T cell antigen receptor recognition and the immunogenicity of HLA/peptide complexes.

HLA-A3 and -A11 share similar peptide-binding motifs, however, it is unclear if promiscuous epitope presentation by HLA-A3 or HLA-A11 is associated with promiscuous TCR recognition. Here, we show that despite widespread cross-presentation of identical HIV-1 peptides in HIV-1-infected individuals expressing HLA-A3 or HLA-A11, peptides presented by HLA-A3 or HLA-A11 commonly exhibited clear immune distinctiveness with exclusive TCR recognition. Yet, using HLA-A3 and HLA-A11 tetramers for testing T cell cross-recognition of the HIV-1 Nef QK10 epitope, we observed in two study persons that specific CD8+ T cell populations were able to cross-recognize this peptide in the context of both HLA-A3 and HLA-A11. This cross-recognition was mediated by single cross-reactive TCRs, as shown by TCR sequencing in conjunction with TCR Vbeta chain immunostaining. In each cross-reactive cell population, multiple TCR beta chain variants were detected in the presence of only one TCR alpha chain variant. Thus, despite distinct TCR recognition of HLA-A3 or HLA-A11 presented HIV-1 peptides in the vast majority of cases, specific TCRs can cross-recognize their antigen in the context of both HLA-A3 and HLA-A11.

Five HLA-A3 reactive human monoclonal antibodies (mAbs) originating from a parous woman were screened against HLA-typed panels by means of complement-dependent lymphocytotoxicity, high-definition ELISA, and flow cytometry with single antigen beads. Antibody reactivity profiles were compared with triplet amino acid sequence polymorphisms identified by HLAMatchmaker, and a three-dimensional structural modeling program (Cn3D of the National Center for Biotechnology Information) was used to determine the topography of epitopes recognized by each mAb. These mAbs originated from a woman who during pregnancy developed antibodies to the paternal HLA-A3 antigen of her child. Each mAb was specific for one mismatched triplet on HLA-A3, and the reactivity patterns of these IgM-type mAbs were practically the same in lymphocytotoxicity and antigen-binding assays. One mAb was specific for 163dT, a unique triplet present only on A3. The other mAbs reacted with 62Qe, 142mI, or 144tKr; these triplets are present on different groups of HLA-A alleles, some of which, however, did not react. Topographic modeling of triplet-defined epitopes identified clusters of polymorphic surface residues that were shared between reactive alleles. These clusters may serve as primary contact sites for the specificity-determining complementarity-determining region (CDR) loops of antibody. The reactivity with these mAbs required also the presence of self-sequence elsewhere on the HLA molecular surface as a critical secondary contact site for antibody, likely through another CDR loop. For instance, the reactivity of the 62Qe-specific mAb required the presence of a glycine residue in position 56 and the reactivity of the 142mI-specific mAb required the presence of the GTLRG sequence in positions 79-83. Conversely, there were many other amino acid differences between the mAb-reactive alleles and HLA-A3 that did not prevent antibody binding. For instance, the 62Qe-specific mAb-reactive alleles had 35 and the 142mI-reactive alleles had 50 of such "permissive" residue differences. An HLAMatchmaker-based analysis of the reactivity of human mAbs will increase our understanding of the structural definition of HLA epitopes and their reactivity with alloantibodies.

Purpose: Alveolar rhabdomyosarcoma (ARMS) frequently contains the fusion transcription factor PAX3/FKHR. Therefore, clinical studies have been initiated to utilize the PAX3/FKHR translocation point area as a peptide vaccine against ARMS. Our study was directed at identifying antigenic T-lymphocyte epitopes at the PAX3/FKHR translocation point area., Experimental Design: The peptide sequence surrounding the PAX3/FKHR translocation point was evaluated by MHC binding algorithms for potential T-lymphocyte antigenic epitopes (class I molecules HLA-A1, -A2 and -A3; class II molecules HLA-DR1, -DR4 and -DR7). Using in vitro techniques, dendritic cells loaded with PAX3/FKHR peptides were used to stimulate naive T-lymphocytes. T-lymphocyte activity was then evaluated by 51Cr release and 3H-thymidine uptake assays., Results: Only one HLA-A3-restricted epitope was predicted by the algorithms. The peptide was prepared and tested for its ability to stimulate naive cytotoxic T-lymphocytes (CTLs). Unfortunately, the peptide was unsuccessful at stimulating naive CTL. However, induction of naive helper T-lymphocytes (HTL) to recognize and respond to the PAX3/FKHR translocation peptide was successful. Yet, this HTL peptide activity did not translate into recognition of PAX3/FKHR-containing ARMS tumor cells., Conclusions: It appears that the fusion area of PAX3/FKHR may not be a good source of antigenic anti-tumor peptide epitopes. These results raise serious concerns about the success and applicability of future peptide-based vaccine immunotherapy directed at the PAX3/FKHR translocation point.

Definition of the antibody specificity in the serum of patients waiting for a renal transplant or in need for platelet transfusion is a crucial step for finding adequate donors. Confounding factors are the complexity of the serum antibodies and the expression of several, up to six, different human leukocyte antigens (HLA) on peripheral blood lymphocytes used as target cells in the antibody screening. Single antigen-expressing (SAL) cell lines were generated by transfecting human major histocompatibility complex (MHC) class I sequences into K562, an erythroleukemia-derived cell line lacking MHC class I and II expression. Thirty-seven different SALs have been generated so far. In this study, we present the validation of 16 of those SALs by flow cytometry against a panel of 84 human HLA-specific monoclonal antibodies (30 HLA-A [8 IgG/22 IgM], 45 HLA-B [18 IgG/27 IgM], 6 HLA-A, B [3 IgG/3 IgM], and 3 HLA-C [all IgM]) developed in our laboratory. The SALs proved to be suitable tools to determine acceptable mismatches for highly sensitized patients. This concept of transfecting target sequences in immortalized cell lines opens up new avenues in the definition of serum and cellular reactivity for sensitized patients awaiting a suitable organ or blood component.

The development of peptide-based vaccines that are useful in the therapeutic treatment of melanoma and other cancers ultimately requires the identification of a sufficient number of antigenic peptides so that most individuals, regardless of their major histocompatibility complex (MHC)-encoded class I molecule phenotype, can develop a cytotoxic T lymphocyte (CTL) response against one or more peptide components of the vaccine. While it is relatively easy to identify antigenic peptides that are presented by the most prevalent MHC class I molecules in the population, it is problematic to identify antigenic peptides that are presented by MHC class I molecules that have less frequent expression in the population. One manner in which this problem can be overcome is by taking advantage of known MHC class I supertypes, which are groupings of MHC class I molecules that bind peptides sharing a common motif. We have developed a mass spectrometric approach which can be used to determine if an antigenic peptide is naturally processed and presented by any given MHC class I molecule. This approach has been applied to the A3 supertype, and the results demonstrate that some, but not all, A3 supertype family-associated peptides can associate with all A3 supertype family members. The approach also demonstrates the shared nature of several newly identified peptide antigens. The use of this technology negates the need to test peptides for their ability to stimulate CTL responses in those cases where the peptide is not naturally processed and bound to the target MHC class I molecule of interest, thus allowing resources to be focused on the most promising vaccine candidates.

Recognition by CD8(+) T lymphocytes (CTL) of epitopes that are derived from conserved gene products, such as Gag and Pol, is well documented and conceptually supports the development of epitope-based vaccines for use against diverse HIV-1 subtypes. However, many CTL epitopes from highly conserved regions within the HIV-1 genome are highly variable, when assessed by comparison of amino acid sequences. The TCR is somewhat promiscuous with respect to peptide binding, and, as such, CTL can often recognize related epitopes. In these studies, we evaluated CTL recognition of five sets of variant HIV-1 epitopes restricted to HLA-A*0201 and HLA-A*1101 using HLA transgenic mice. We found that numerous different amino acid substitutions can be introduced into epitopes without abrogating their recognition by CTL. Based on our findings, we constructed an algorithm to predict those CTL epitopes capable of inducing responses in the HLA transgenic mice to the greatest numbers of variant epitopes. Similarity of CTL specificity for variant epitopes was demonstrated for humans using PBMC from HIV-1-infected individuals and CTL lines produced in vitro using PBMC from HIV-1-uninfected donors. We believe the ability to predict CTL epitope immunogenicity and recognition patterns of variant epitopes can be useful for designing vaccines against multiple subtypes and circulating recombinant forms of HIV-1.

The recognition of MHC class I molecules by killer cell immunoglobulin-like receptors (KIR) is central to the control of NK cell function and can also modulate the CTL activation threshold. Among KIR receptors, KIR3DL2 is thought to interact with HLA-A3 and -A11, although direct evidence has been lacking. In this study, we show that HLA-A3 and -A11 tetramers specifically bind to KIR3DL2*001 transfectants and that this recognition is peptide-specific. Single amino acid substitutions in the nonamer peptide underline a critical role for residue 8 in recognition of KIR3DL2. However, the role of this interaction in vivo still remains to be established.

An effective Severe Acute Respiratory Syndrome (SARS) vaccine is likely to include components that can induce specific cytotoxic T-lymphocyte (CTL) responses. The specificities of such responses are governed by human leukocyte antigen (HLA)-restricted presentation of SARS-derived peptide epitopes. Exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information (Lauemoller et al., Rev Immunogenet 2001: 2: 477-91). The latter was recently established when a causative coronavirus (SARS-CoV) was isolated and full-length sequenced (Marra et al., Science 2003: 300: 1399-404). Here, we have combined advanced bioinformatics and high-throughput immunology to perform an HLA supertype-, genome-wide scan for SARS-specific CTL epitopes. The scan includes all nine human HLA supertypes in total covering >99% of all individuals of all major human populations (Sette & Sidney, Immunogenetics 1999: 50: 201-12). For each HLA supertype, we have selected the 15 top candidates for test in biochemical binding assays. At this time (approximately 6 months after the genome was established), we have tested the majority of the HLA supertypes and identified almost 100 potential vaccine candidates. These should be further validated in SARS survivors and used for vaccine formulation. We suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design.

Hepatitis C virus (HCV) variation in specific T-cell epitopes may represent a mechanism of viral persistence in chronic infection. We examined the HCV non-structural protein 3 (NS3), including the immunologically relevant epitopes HCV NS3-2 KLVALGINAV (human leukocyte antigen [HLA]-A2-restricted) and HCV NS3-1391 LIFCHSKKK (HLA-A3-restricted), in 22 HLA-A2+ patients with chronic infection. Significant amino acid variation was found in HCV NS3-2 epitope sequences when compared to the HCV-1 prototype virus. Six of the nine different HCV NS3-2 peptide variants were identified in patients with HCV NS3-2-specific CD8+ cells, detected with an HLA-A2 tetramer made with the HCV-1 prototype peptide. Phylogenetic analysis, including HCV reference sequences other than HCV-1, suggested however that most of the variations in the HCV NS3-2 epitope could be related to genetic heterogeneity between HCV reference subtypes. Variation was less common when comparing HCV NS3-2 epitope sequences from the clinical isolates to the most-closely related HCV reference subtype in each case. Some subtype-independent variations were found in epitopic residues probably important for T-cell receptor interaction. In contrast, no significant variation was found in HLA primary anchor sites, flanking regions, or in the contiguous HLA A3-restricted CD8+ T-cell epitope. Ongoing variation was not evident in two selected patients with follow-up. In conclusion, (i) the HCV NS3-2 epitope is not conserved between different HCV strains/subtypes, and (ii) an HLA-A2 tetramer loaded with the HCV-1 prototype NS3-2 peptide may still detect NS3-specific CD8+ cells in some patients with variant viruses. These data may be useful to improve T-cell assays using HCV NS3 peptides, taking into account the genetic diversity of this virus., (Copyright 2004 Wiley-Liss, Inc.)

Maternally induced allotolerance both in clinical and experimental organ transplantation appears to require both in utero and oral exposure to noninherited maternal antigens (NIMA). Soluble major histocompatibility complex (MHC) class I antigens were studied in 18 mother-baby pairs in order to determine the extent of neonatal exposure to NIMA. Enzyme-linked immunoabsorbent assay (ELISA) analysis of cord blood from three genetically HLA-A2 negative babies born to HLA-A2+ mothers and from two HLA-A3 negative babies born to HLA-A3+ mothers revealed significant NIMA HLA-A levels in cord plasma. The level of NIMA-A2 or -A3 in cord blood were approximately 10% of the predicted value for a baby genetically positive for that allele. HLA-A2 or -A3 was undetectable (< 1.0 ng/ml) in cord blood from HLA-A2 or -A3 negative babies whose mothers were also HLA-A2 or -A3 negative. Breast milk from HLA-A2+ mothers contained soluble HLA-A2 (sHLA-A2) at levels averaging 36.2 ng/ml, resulting in milligram quantities of ingested antigen over 3 months of nursing. Western blot analysis of cord plasma confirmed that bands corresponding to NIMA HLA-A protein were present. This study demonstrates that oral and intravenous exposure to NIMA sHLA in the fetus and newborn is much higher than previously thought, and emphasizes the importance of nursing in the overall antigen dose achieved.

Multiple isoforms (TAG-1, TAG-2a, TAG-2b, and TAG-2c) of a novel cancer/testis antigen gene have been identified and are expressed in 84-88% of melanoma cell lines tested. The tumor antigen (TAG) genes are also expressed in K562, a myelogenous leukemia cell line, and they have homology to two chronic myelogenous leukemia-derived clones and a hepatocellular carcinoma clone in the human expressed sequence tags (EST) database, thus indicating that their expression is not restricted to melanomas. In contrast to the fact that many cancer/testis antigens are poorly immunogenic, the TAG-derived peptide, RLSNRLLLR, is recognized by HLA-A3-restricted, melanoma-specific CTLs that were obtained from a melanoma patient with spontaneous reactivity to the peptide. Unlike most cancer/testis antigen genes which are located on the X chromosome, the TAG genes are located on chromosome 5. The genes have the additional unusual features of being coded for in an open reading frame that is initiated by one of three nonstandard initiation codons, and the sequence coding the RLSNRLLLR peptide crosses an exon-exon boundary. The properties of the TAG antigens indicate that they are excellent vaccine candidates for the treatment of melanoma and perhaps other cancers.