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Cross reactivities are known for human leukocyte antigen inside HLA-B7 related Cross-Reactive Group (B7CREG). Some CE-IVD flow-cytometry kits use double monoclonal antibodies (mAb) to distinguish HLA-B27 and HLA-B7 but practice reveals more complexes results. This study explores the performances of this test. Analysis of 466 consecutive cases using HLA-B27 IOTest™ kit on a Navios™ cytometer from Beckman-Coulter, partially compared to their genotypes. Expected haplotypes HLA-B27-/HLA-B7- (undoubtedly HLA-B27 negative) and HLA-B27+/HLA-B7- (undoubtedly HLA-B27+) were clearly identified according to the manufacturer's instructions. On the opposite, patients strongly labeled with anti-HLA-B7 showed three different phenotypes regarding anti-HLA-B27 labeling: (1) most of the cases were poorly labeled in accordance with cross reactivity inside B7CREG (HLA-B27-/HLA-B7+ haplotype); (2) rare cases had strong B7 and B27 labeling corresponding to HLA-B27+/HLA-B7+ haplotype; (3) even less cases had strong labeling by anti-HLA-B7 but non for anti-HLA-B27, all expressing HLA-B44 and no B7CREG molecules. Surprisingly, more cases were not labeled with anti-HLA-B7 antibody but partially labeled with anti-HLA-B27 suggesting another cross reactivity out of B7CREG. mAb HLA typing suggests new, cross reactivities of anti-HLA-B27 antibody to more molecules out of B7CREG and of anti-HLA-B7 antibody but not anti-HLA-B27 to HLA-B44 molecule also out of B7CREG. HLA-B27 could surely be excluded in most samples labeled with HLA-B27, below a "grey zone" on intermediate intensity. More comparison is needed in future studies., (© 2024 International Society for Advancement of Cytometry.)

Rapidly appearing SARS-CoV-2 mutations can affect T cell epitopes, which can help the virus to evade either CD8 or CD4 T-cell responses. We developed T-cell COVID-19 Atlas (T-CoV, https://t-cov.hse.ru) - the comprehensive web portal, which allows one to analyze how SARS-CoV-2 mutations alter the presentation of viral peptides by HLA molecules. The data are presented for common virus variants and the most frequent HLA class I and class II alleles. Binding affinities of HLA molecules and viral peptides were assessed with accurate in silico methods. The obtained results highlight the importance of taking HLA alleles diversity into account: mutation-mediated alterations in HLA-peptide interactions were highly dependent on HLA alleles. For example, we found that the essential number of peptides tightly bound to HLA-B*07:02 in the reference Wuhan variant ceased to be tight binders for the Indian (Delta) and the UK (Alpha) variants. In summary, we believe that T-CoV will help researchers and clinicians to predict the susceptibility of individuals with different HLA genotypes to infection with variants of SARS-CoV-2 and/or forecast its severity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

NP 105-113 -B*07:02-specific CD8 + T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP 105-113 -B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP 105-113 -B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP 105-113 -B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP 105-113 -B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design., (© 2021. The Author(s).)

Human metapneumovirus (HMPV) is a leading cause of acute lower respiratory tract illness in children and adults. Repeated infections are common and can be severe in young, elderly, and immunocompromised persons due to short-lived protective humoral immunity. In turn, few protective T cell epitopes have been identified in humans. Thus, we infected transgenic mice expressing the common human HLA MHC-I allele B*07:02 (HLA-B7) with HMPV and screened a robust library of overlapping and computationally predicted HLA-B7 binding peptides. Six HLA-B7-restricted CD8 + T cell epitopes were identified using ELISPOT screening in the F, M, and N proteins, with M 195-203 (M195) eliciting the strongest responses. MHC-tetramer flow cytometric staining confirmed HLA-B7 epitope-specific CD8 + T cells migrated to lungs and spleen of HMPV-immune mice. Immunization with pooled HLA-B7-restricted peptides reduced viral titer and protected mice from virulent infection. Finally, we confirmed that CD8 + T cells from HLA-B7 positive humans also recognize the identified epitopes. These results enable identification of HMPV-specific CD8 + T cells in humans and help to inform future HMPV vaccine design., (© 2021. The Author(s).)

Efforts are being made worldwide to understand the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic, including the impact of T cell immunity and cross-recognition with seasonal coronaviruses. Screening of SARS-CoV-2 peptide pools revealed that the nucleocapsid (N) protein induced an immunodominant response in HLA-B7 + COVID-19-recovered individuals that was also detectable in unexposed donors. A single N-encoded epitope that was highly conserved across circulating coronaviruses drove this immunodominant response. In vitro peptide stimulation and crystal structure analyses revealed T cell-mediated cross-reactivity toward circulating OC43 and HKU-1 betacoronaviruses but not 229E or NL63 alphacoronaviruses because of different peptide conformations. T cell receptor (TCR) sequencing indicated that cross-reactivity was driven by private TCR repertoires with a bias for TRBV27 and a long CDR3β loop. Our findings demonstrate the basis of selective T cell cross-reactivity for an immunodominant SARS-CoV-2 epitope and its homologs from seasonal coronaviruses, suggesting long-lasting protective immunity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis (SJS/TEN) are part of a disease continuum of vesiculobullous mucocutaneous reactions affecting the skin and mucous membranes including the ocular surface. Manifestations of disease range from mild dry eye to progressive conjunctival cicatrisation, limbal epithelial stem cell failure and corneal blindness. In Far Eastern and South East Asian populations where SJS/TEN is prevalent, numerous human leukocyte antigen (HLA) gene variants at the A, B and C loci have been identified as risk factors for developing SJS/TEN with severe ocular complications (SOC). By contrast, the incidence of SJS/TEN with SOC in European countries is relatively low. To date, ocular SJS/TEN risk altering alleles have not been widely investigated in European populations. In this study, we analysed the association of HLA -A, -B and -C alleles with SJS/TEN in 33 patients residing in the UK with age matched controls. The data showed statistically significant novel negative allele association with HLA-B*0702 and a trend with HLA-C*0702 in the patient group, indicating these alleles are protective. Further characterisation of protective and risk alleles in other ethnic groups is required to fully elucidate the putative role of these alleles in the susceptibility of SJS/TEN with or without severe ocular complications in patients in the UK.

Frontal fibrosing alopecia (FFA) is a recently described inflammatory and scarring type of hair loss affecting almost exclusively women. Despite a dramatic recent increase in incidence the aetiopathogenesis of FFA remains unknown. We undertake genome-wide association studies in females from a UK cohort, comprising 844 cases and 3,760 controls, a Spanish cohort of 172 cases and 385 controls, and perform statistical meta-analysis. We observe genome-wide significant association with FFA at four genomic loci: 2p22.2, 6p21.1, 8q24.22 and 15q2.1. Within the 6p21.1 locus, fine-mapping indicates that the association is driven by the HLA-B*07:02 allele. At 2p22.1, we implicate a putative causal missense variant in CYP1B1, encoding the homonymous xenobiotic- and hormone-processing enzyme. Transcriptomic analysis of affected scalp tissue highlights overrepresentation of transcripts encoding components of innate and adaptive immune response pathways. These findings provide insight into disease pathogenesis and characterise FFA as a genetically predisposed immuno-inflammatory disorder driven by HLA-B*07:02.

Adenoviruses are a major cause of infectious mortality in children following allogeneic hematopoietic stem cell transplantation, with adoptive transfer of adenovirus-specific T cells being an effective therapeutic approach. We have previously shown that T cells specific for the peptide epitope LTDLGQNLLY were protective. In this study, we aimed to establish a viral dissemination assay to measure the antiviral capacity of T cells specific for this and other peptide epitopes in an infectious setting. We used replication-competent adenovirus 11 (Ad11pGFP) and adenovirus 5 containing adenovirus 35 fiber (Ad5F35GFP) viruses and T cells specific for HLA-A*01-restricted LTDLGQNLLY, HLA-B*07-restricted KPYSGTAYNAL, and HLA-A*02-restricted LLDQLIEEV peptide epitopes. T cells in PBMC from healthy donors were expanded with peptide and IL-2 or treated with IL-2 alone to serve as nonstimulated control cells, and then these expanded or nonstimulated CD8 + cells were purified and cocultured with autologous monocytes infected with adenovirus at low multiplicity of infection. After 3 d, the number of infected GFP + monocytes and, hence, viral dissemination was quantified by flow cytometry. T cells expanded with LTDLGQNLLY peptide from multiple HLA-A*01 + donors prevented adenovirus dissemination, and nonstimulated T cells did not prevent dissemination, thus, indicating that LTDLGQNLLY-specific T cells have high antiviral capacity. Similarly, expanded KPYSGTAYNAL- and LLDQLIEEV-specific T cells could prevent viral dissemination. However, the frequency of expanded T cells specific for these last two epitopes was variable between donors with consequent variable prevention of adenoviral dissemination. Taken together, we demonstrate that T cells specific for three peptide epitopes, from both structural and nonstructural proteins, can prevent adenoviral dissemination and provide a novel method to measure the antiviral capacity of adenovirus-specific T cell responses., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Tick-borne encephalitis virus (TBEV) is a leading cause of viral meningoencephalitis in many parts of Europe and eastwards in Asia, with high morbidity and often long-term neurologic sequelae. With no treatment available, studies of the immune response to TBEV are essential for the understanding of the immunopathogenesis of tick-borne encephalitis and for the development of therapeutics. We have previously demonstrated that CD8 + T cell responses in peripheral blood in patients with acute TBEV peak at around 7 d after hospitalization in the neuroinvasive phase of the disease. In this study, we identified six novel TBEV HLA-A2- and HLA-B7-restricted epitopes, all derived from the nonstructural proteins of TBEV. This identification allowed for a comprehensive phenotypic and temporal analysis of the HLA-A2- and HLA-B7-restricted Ag-specific CD8 + T cell response during the acute stages of human TBEV infection. HLA-A2- and HLA-B7-restricted TBEV epitope-specific effector cells predominantly displayed a CD45RA - CCR7 - CD27 + CD57 - phenotype at day 7, which transitioned into separate distinct phenotypes for HLA-A2- and HLA-B7-restricted TBEV-specific CD8 + T cells, respectively. At day 21, the most prevalent phenotype in the HLA-A2-restricted CD8 + T cell populations was CD45RA - CCR7 - CD27 + CD57 + , whereas the HLA-B7-restricted CD8 + T cell population was predominantly CD45RA + CCR7 - CD27 + CD57 + Almost all TBEV epitope-specific CD8 + T cells expressed α4 and β1 integrins at days 7 and 21, whereas the bulk CD8 + T cells expressed lower integrin levels. Taken together, human TBEV infection elicits broad responses to multiple epitopes, predominantly derived from the nonstructural part of the virus, establishing distinct maturation patterns for HLA-A2- and HLA-B7-restricted TBEV epitope-specific CD8 + T cells., (Copyright © 2018 The Authors.)

HIV-1-specific cytotoxic T cells (CTLs) play an important role in the control of HIV-1 subtype B or C infection. However, the role of CTLs in HIV-1 subtype A/E infection still remains unclear. Here we investigated the association of HLA class I alleles with clinical outcomes in treatment-naive Vietnamese infected with subtype A/E virus. We found that HLA-C*12:02 was significantly associated with lower plasma viral loads (pVL) and higher CD4 counts and that the HLA-A*29:01-B*07:05-C*15:05 haplotype was significantly associated with higher pVL and lower CD4 counts than those for individuals without these respective genotypes. Nine Pol and three Nef mutations were associated with at least one HLA allele in the HLA-A*29:01-B*07:05-C*15:05 haplotype, with a strong negative correlation between the number of HLA-associated Pol mutations and CD4 count as well as a positive correlation with pVL for individuals with these HLA alleles. The results suggest that the accumulation of mutations selected by CTLs restricted by these HLA alleles affects HIV control. IMPORTANCE Most previous studies on HLA association with disease progression after HIV-1 infection have been performed on cohorts infected with HIV-1 subtypes B and C, whereas few such population-based studies have been reported for cohorts infected with the Asian subtype A/E virus. In this study, we analyzed the association of HLA class I alleles with clinical outcomes for 536 HIV-1 subtype A/E-infected Vietnamese individuals. We found that HLA-C*12:02 is protective, while the HLA haplotype HLA-A*29:01-B*07:05-C*15:05 is deleterious. The individuals with HIV-1 mutations associated with at least one of the HLA alleles in the deleterious HLA haplotype had higher plasma viral loads and lower CD4 counts than those of individuals without the mutations, suggesting that viral adaptation and escape from HLA-mediated immune control occurred. The present study identifies a protective allele and a deleterious haplotype for HIV-1 subtype A/E infection which are different from those identified for cohorts infected with HIV-1 subtypes B and C., (Copyright © 2018 American Society for Microbiology.)

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.

T-cell receptor (TCR) immunotherapy uses T cells engineered with new TCRs to enable detection and killing of cancer cells. Efficacy of TCR immunotherapy depends on targeting antigenic peptides that are efficiently presented by the best-suited major histocompatibility complex (MHC) molecules of cancer cells. However, efficient strategies are lacking to easily identify TCRs recognizing immunodominant peptide-MHC (pMHC) combinations utilizing any of the six possible MHC class I alleles of a cancer cell. We generated an MHC cell library and developed a platform approach to detect, isolate, and re-express TCRs specific for immunodominant pMHCs. The platform approach was applied to identify a human papillomavirus (HPV16) oncogene E5-specific TCR, recognizing a novel, naturally processed pMHC (HLA-B*15:01) and a cytomegalovirus-specific TCR targeting an immunodominant pMHC (HLA-B*07:02). The platform provides a useful tool to isolate in an unbiased manner TCRs specific for novel and immunodominant pMHC targets for use in TCR immunotherapy.

The novel HLA-B*07:294 allele differs from the closest matching allele, HLA-B*07:02:01 by 1 nucleotide substitution., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Antibody identification by a bead array assay in a kidney patient revealed several HLA-specific antibodies including one directed against the HLA-B7 antigen. Low-resolution typing of the patient indicated the presence of an HLA-B*07 allele. To rule out an HLA-specific autoantibody the HLA-typing of the patient was further refined by nucleotide sequencing on a next-generation sequencing platform and eventually showed an HLA-B*39:01:01:03 and HLA-B*07:181N genotype. Thereby the allospecific nature of the antibody was proven. The HLA-B7-specific antibody could be explained by an immunization during the first kidney-transplantation in 1996 with an HLA-B*07 positive donor. When assessing the plausibility of antibodies, the presence of nonexpressed alleles should be taken into consideration., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Leukemias are highly immunogenic, but they have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I-associated peptide antigens with O-linked β- N -acetylglucosamine ( O -GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O -GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O -GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these posttranslationally modified neoantigens provide logical targets for cancer immunotherapy. Cancer Immunol Res; 5(5); 376-84. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Full-length sequences of HLA-B*07:05:01:01, B*14:01:01 and B*18:02, confirmed by cloning and sequencing in Chinese donors., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The assignment of human leukocyte antigen (HLA) null alleles is clinically relevant in the setting of stem cell transplantation. Cell surface expression profiling and mRNA processing analysis of the HLA-B allele previously designated as B*07:44, have been performed., Materials and Methods: Cell surface expression of HLA-B*07:44 was determined using flow cytometry. Genomic full-length and HLA-B*07-specific cDNA sequencing were carried out by Sanger procedure., Results: Flow cytometric analysis confirmed previous serologic results and demonstrated a lack of cell membrane expression of the HLA-B protein. The mRNA processing, studied using direct HLA-B*07-specific cDNA sequencing, revealed the presence of a unique, aberrantly spliced mRNA, with a deletion of the last 43 bp on the 5'-end of exon 4. The substitution from T to G at genomic position 1799 compared to B*07:02:01 introduced a new and stronger splice donor site at exon 4. This alternative splicing produced an mRNA containing a premature stop codon at position 280, explaining the absence of mature HLA-B7 protein on the cell surface., Conclusion: These findings led us to consider this HLA-B variant as a HLA null allele. The World Health Organization (WHO) Nomenclature Committee has since renamed this variant B*07:44N ., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Luminex panel reactive antibody (PRA) screening assays using microbeads are widely used for organ transplantation. Anti-HLA serum reactivity is calculated by correcting for non-specific binding to the negative control (NC) beads. High mean fluorescence intensity (MFI) value of NC beads are observed in some patients and can result in false negative results in the PRA screening assay. We analyzed the clinical characteristics and HLA types of those patients with high MFI values of NC beads., Methods: Sixty-six patients with high MFI values of NC beads (> 300) in the PRA LABScreen Mixed assay (One Lambda) tested were included as the high NC group. Age and gender matched controls with low MFI values of NC beads (< 100) (n = 132), tested with PRA, were selected as the low NC group and 207 healthy Koreans were used as normal controls. Association of clinical characteristics and HLA types with the high NC group were analyzed using Chi-square test or Fischer's exact test, as appropriate., Results: The proportion of patients with underlying liver disease was higher in the high NC group compared to the low NC group (18.1% vs. 1.5%, p < 0.001, OR = 14.2). The seropositivity of anti-nuclear antibody and rheumatoid factor, the frequency of use of intravenous immunoglobulin G, anti-thymocyte globulin, and rituximab showed no difference between two groups. The phenotype frequency (PF) of HLA-B46 was higher in the high NC group than in the low NC group (8.0% vs. 3.2%, p = 0.036, OR = 2.8). The PF of HLA-B7 was lower in the high NC group than in the healthy controls (0.0% vs. 6.5%, p = 0.008, OR = 0.1). The PF of HLA-DR1 was lower in the high NC group than in the low NC group (0.8% vs. 6.6%, p = 0.015, OR = 0.1) or healthy controls (0.8% vs. 7.4%, p = 0.003, Pc = 0.042, OR = 0.1)., Conclusions: Increased non-specific binding to NC beads was associated with underlying liver disease and HLAB46. HLA-B7 and HLA-DR1 were related to a lower chance of non-specific binding to NC beads. The mechanism of those associations, such as differences in non-specific antibody response according to HLA phenotype or underlying disease, needs to be elucidated in further studies.

One nucleotide replacement at residue 559 of HLA-B*07:05:01:01 or B*07:06:01 results in a novel allele, HLA-B*07:269., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) has been reported to interact with a wide spectrum of HLA class I (HLA-I) molecules, albeit with different affinities determined by allelic polymorphisms and conformational features. HLA-G dimerization and the presence of intracellular Cys residues in HLA-B7 have been shown to be critical for their recognition by LILRB1. We hypothesized that dimerization of classical HLA class Ia molecules, previously detected in exosomes, might enhance their interaction with LILRB1. A soluble LILRB1-Fc fusion protein and a sensitive cellular reporter system expressing a LILRB1-ζ chimera were employed to assess receptor interaction with different HLA class Ia molecules transfected in the human lymphoblastoid 721.221 cell line. Under these conditions, intracellular Cys residues and HLA-I dimerization appeared associated with increased LILRB1 recognition. On the other hand, a marginal interaction of LILRB1 with primary monocytic cells, irrespective of their high HLA-I expression, was enhanced by type I interferon (IFN). This effect appeared disproportionate to the cytokine-induced increase of surface HLA-I expression and was accompanied by detection of HLA class Ia dimers. Altogether, the results support that a regulated assembly of these noncanonical HLA-I conformers during the immune response may enhance the avidity of their interaction with LILRB1., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Prior work has demonstrated that HIV-1-specific CD8 T cells can cross-recognize variant epitopes. However, most of these studies were performed in the context of chronic infection, where the presence of viral quasispecies makes it difficult to ascertain the true nature of the original antigenic stimulus. To overcome this limitation, we evaluated the extent of CD8 T cell cross-reactivity in patients with acute HIV-1 clade B infection. In each case, we determined the transmitted founder virus sequence to identify the autologous epitopes restricted by individual HLA class I molecules. Our data show that cross-reactive CD8 T cells are infrequent during the acute phase of HIV-1 infection. Moreover, in the uncommon instances where cross-reactive responses were detected, the variant epitopes were poorly recognized in cytotoxicity assays. Molecular analysis revealed that similar antigenic structures could be cross-recognized by identical CD8 T cell clonotypes mobilized in vivo, yet even subtle differences in a single TCR-accessible peptide residue were sufficient to disrupt variant-specific reactivity. These findings demonstrate that CD8 T cells are highly specific for autologous epitopes during acute HIV-1 infection. Polyvalent vaccines may therefore be required to provide optimal immune cover against this genetically labile pathogen., (Copyright © 2016 by The American Association of Immunologists, Inc.)

One nucleotide replacement at residue 398 of HLA-B*07:05:01 or HLA-B*07:06 results in a novel allele, HLA-B*07:249., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Adenovirus infections of immunocompromised patients, particularly following allogeneic hematopoietic stem cell transplantation, are associated with morbidity and mortality. Immunotherapy by adoptive transfer of hexon-specific and penton-specific T cells has been successfully applied, but many approaches are impeded by the low number of HLA class I-restricted adenoviral peptide epitopes described to date. We use a novel method to identify naturally presented adenoviral peptide epitopes from infected human cells, ectopically expressing defined HLA, using peptide elution and liquid chromatography-mass spectrometry analysis. We show that the previously described HLA-A*01:01-restricted peptide epitope LTDLGQNLLY from hexon protein is naturally presented, and demonstrate the functionality of LTDLGQNLLY-specific T cells. We further identify a novel immunodominant HLA-B*07:02-restricted peptide epitope VPATGRTLVL from protein 13.6 K, and demonstrate the high proliferative, cytotoxic, and IFN-γ-producing capacity of peptide-specific T cells. Lastly, LTDLGQNLLY-specific T cells can be detected ex vivo following adoptive transfer therapy, and LTDLGQNLLY-specific and VPATGRTLVL-specific T cells have memory phenotypes ex vivo. Given their proliferative and cytotoxic capacity, such epitope-specific T cells are promising candidates for adoptive T-cell transfer therapy of adenovirus infection.

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.

Immunotherapy of B-cell malignancies using CD19-targeted chimeric antigen receptor-transduced T cells or CD20-targeted therapeutic monoclonal antibodies has shown clinical efficacy. However, refractory disease and the emergence of antigen-loss tumor escape variants after treatment demonstrate the need to target additional antigens. Here we aimed to target the B-cell receptor-associated protein CD79b by a T-cell receptor (TCR)-based approach. Because thymic selection depletes high-avidity T cells recognizing CD79b-derived peptides presented in self-HLA molecules, we aimed to isolate T cells recognizing these peptides presented in allogeneic HLA. Peptide-HLA tetramers composed of CD79b peptides bound to either HLA-A2 or HLA-B7 were used to isolate T-cell clones from HLA-A*0201 and B*0702-negative individuals. For 3 distinct T-cell clones, CD79b specificity was confirmed through CD79b gene transduction and CD79b-specific shRNA knockdown. The CD79b-specific T-cell clones were highly reactive against CD79b-expressing primary B-cell malignancies, whereas no recognition of nonhematopoietic cells was observed. Although lacking CD79b-cell surface expression, intermediate reactivity toward monocytes, hematopoietic progenitor cells, and T-cells was observed. Quantitative reverse transcriptase polymerase chain reaction revealed low CD79b gene expression in these cell types. Therefore, aberrant gene expression must be taken into consideration when selecting common, apparently lineage-specific self-antigens as targets for TCR-based immunotherapies., (© 2015 by The American Society of Hematology.)

Human leukocyte antigen (HLA) class I molecules generally present peptides (p) of 8 to 11 amino acids (aa) in length. Although an increasing number of examples with lengthy (>11 aa) peptides, presented mostly by HLA-B alleles, have been reported. Here we characterize HLA-A*02:01 restricted, in addition to the HLA-B*0702 and HLA-B*4402 restricted, lengthy peptides (>11 aa) arising from the B-cell ligandome. We analyzed a number of 15-mer peptides presented by HLA-A*02:01, and confirmed pHLA-I formation by HLA folding and thermal stability assays. Surprisingly the binding affinity and stability of the 15-mer epitopes in complex with HLA-A*02:01 were comparable with the values observed for canonical length (8 to 11 aa) HLA-A*02:01-restricted peptides. We solved the structures of two 15-mer epitopes in complex with HLA-A*02:01, within which the peptides adopted distinct super-bulged conformations. Moreover, we demonstrate that T-cells can recognize the 15-mer peptides in the context of HLA-A*02:01, indicating that these 15-mer peptides represent immunogenic ligands. Collectively, our data expand our understanding of longer epitopes in the context of HLA-I, highlighting that they are not limited to the HLA-B family, but can bind the ubiquitous HLA-A*02:01 molecule, and play an important role in T-cell immunity., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.)

T cells have been proven to be therapeutically effective in patients with relapsed leukemias, although target antigens on leukemic cells as well as T-cell receptors (TCRs), potentially recognizing those antigens, are mostly unknown. We have applied an immunopeptidomic approach and isolated human leukocyte antigen (HLA) ligands from primary leukemia cells. We identified a number of ligands derived from different genes that are restrictedly expressed in the hematopoietic system. We exemplarily selected myeloperoxidase (MPO) as a potential target and isolated a high-avidity TCR with specificity for a HLA-B*07:02-(HLA-B7)-restricted epitope of MPO in the single HLA-mismatched setting. T cells transgenic for this TCR demonstrated high peptide and antigen specificity as well as leukemia reactivity in vitro and in vivo. In contrast, no significant on- and off-target toxicity could be observed. In conclusion, we here demonstrate, exemplarily for MPO, that leukemia-derived HLA ligands can be selected for specific effector tool development to redirect T cells to be used for graft manipulation or adoptive T-cell therapies in diverse transplant settings. This approach can be extended to other HLA ligands and HLA molecules in order to provide better treatment options for this life-threatening disease.

Endoplasmic reticulum-associated aminopeptidase-1 (ERAP1) plays a critical role in the processing of peptides prior to binding to MHC class I molecules. In this article, we show for the first time, to our knowledge, that the HLA-B27 immunodominant influenza nucleoprotein (NP) 383-391 epitope is made as an N-terminally extended 14-mer before it is trimmed by ERAP. In the absence of ERAP, there is a significant reduction in the CTL response to the B27/NP383-391 epitope in influenza A (flu)-infected B27/ERAP(-/-) mice. With the use of tetramer staining, the number of naive CD8(+) T cells expressing TCR Vβ8.1 in B27/ERAP(-/-) transgenic mice is significantly lower than that seen in B27/ERAP(+/+) mice. HLA-B27 surface expression in naive and flu-infected B27/ERAP(-/-) mice is also lower than the expression seen for the same allele in naive and flu-infected B27/ERAP(+/+) mice. In contrast, surface expression of HLA-B7 was unaffected by the absence of ERAP in B7/ERAP(-/-) transgenic mice. The B7-restricted NP418-426 CTL response in flu-infected B7/ERAP(-/-) and B7/ERAP(+/+) mice was also similar. These results provide, to our knowledge, the first in vivo demonstration of ERAP functionally influencing host immune response in an HLA allele-specific manner. This principle has relevance to diseases such as ankylosing spondylitis, in which HLA-B27 and ERAP jointly contribute to disease predisposition., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Purpose: Survivin is a member of the inhibitor-of-apoptosis family. Essential for tumor cell survival and overexpressed in most cancers, survivin is a promising target for anti-cancer immunotherapy. Immunogenicity has been demonstrated in multiple cancers. Nonetheless, few clinical trials have demonstrated survivin-vaccine-induced immune responses., Experimental Design: This phase I trial was conducted to test whether vaccine EMD640744, a cocktail of five HLA class I-binding survivin peptides in Montanide(®) ISA 51 VG, promotes anti-survivin T-cell responses in patients with solid cancers. The primary objective was to compare immunologic efficacy of EMD640744 at doses of 30, 100, and 300 μg. Secondary objectives included safety, tolerability, and clinical efficacy., Results: In total, 49 patients who received ≥2 EMD640744 injections with available baseline- and ≥1 post-vaccination samples [immunologic-diagnostic (ID)-intention-to-treat] were analyzed by ELISpot- and peptide/MHC-multimer staining, revealing vaccine-activated peptide-specific T-cell responses in 31 patients (63 %). This cohort included the per study protocol relevant ID population for the primary objective, i.e., T-cell responses by ELISpot in 17 weeks following first vaccination, as well as subjects who discontinued the study before week 17 but showed responses to the treatment. No dose-dependent effects were observed. In the majority of patients (61 %), anti-survivin responses were detected only after vaccination, providing evidence for de novo induction. Best overall tumor response was stable disease (28 %). EMD640744 was well tolerated; local injection-site reactions constituted the most frequent adverse event., Conclusions: Vaccination with EMD640744 elicited T-cell responses against survivin peptides in the majority of patients, demonstrating the immunologic efficacy of EMD640744.

Prediction of HLA binding affinity is widely used to identify candidate T cell epitopes, and an affinity of 500 nM is routinely used as a threshold for peptide selection. However, the fraction (percentage) of peptides predicted to bind with affinities of 500 nM varies by allele. For example, of a large collection of ~30,000 dengue virus-derived peptides only 0.3% were predicted to bind HLA A*0101, whereas nearly 5% were predicted for A*0201. This striking difference could not be ascribed to variation in accuracy of the algorithms used, as predicted values closely correlated with affinity measured in vitro with purified HLA molecules. These data raised the question whether different alleles would also vary in terms of epitope repertoire size, defined as the number of associated epitopes or, alternatively, whether alleles vary drastically in terms of the affinity threshold associated with immunogenicity. To address this issue, strains of HLA transgenic mice with wide (A*0201), intermediate (B*0702), or narrow (A*0101) repertoires were immunized with peptides of varying binding affinity and relative percentile ranking. The results show that absolute binding capacity is a better predictor of immunogenicity, and analysis of epitopes from the Immune Epitope Database revealed that predictive efficacy is increased using allele-specific affinity thresholds. Finally, we investigated the genetic and structural basis of the phenomenon. Although no stringent correlate was defined, on average HLA B alleles are associated with significantly narrower repertoires than are HLA A alleles.

It is recognized that host response following viral infection is characterized by immunodominance, but deciphering the different factors contributing to immunodominance has proved a challenge due to concurrent expression of multiple MHC class I alleles. To address this, we generated H2-K(-/-)/D(-/-) double-knockout transgenic mice expressing either one or two human MHC-I alleles. We hypothesized that co-expression of different allele combinations figures critically in immunodominance and examined this in influenza-infected, double Tg MHC-I mice. In A2/B7 or A2/B27 mice, using ELISpot assays with the A2-restricted matrix I.58-66, the B7-restricted NP418-426 or the B27-restricted NP383-391 influenza A (flu) epitopes, we observed the expected recognition of both peptides for both alleles. In contrast, in flu-infected B7/B27 mice, a significantly reduced level of B27/NP383-restricted CTL response was detected while there was no change in the B7/NP418-restricted CTL response. Flu-specific tetramer studies revealed a partial deletion of Vβ8.1(+) NP383/B27-restricted CD8(+) T cells, and a diminished Vβ12(+) CD8(+) T-cell expansion in B7/B27 Tg mice. Using HLA Tg chimeric mice, we confirmed these findings. These findings shed light on the immune consequences of co-dominant expression of MHC-I alleles for host immune response to pathogens., (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.)

The low frequency of antigen-specific naïve T cells has challenged numerous laboratories to develop various techniques to study the naïve T-cell repertoire. Here, we combine the generation of naïve repertoire-derived antigen-specific T-cell lines based on MHC-tetramer staining and magnetic-bead enrichment with in-depth functional assessment of the isolated T cells. Cytomegalovirus (CMV) specific T-cell lines were generated from seronegative individuals. Generated T-cell lines consisted of a variety of immunodominant CMV-epitope-specific oligoclonal T-cell populations restricted to various HLA-molecules (HLA-A1, A2, B7, B8, and B40), and the functional and structural avidity of the CMV-specific T cells was studied. Although all CMV-specific T cells were isolated based on their reactivity toward a specific peptide-MHC complex, we observed a large variation in the functional avidity of the MHC-tetramer positive T-cell populations, which correlated with the structural avidity measured by the recently developed Streptamer koff -rate assay. Our data demonstrate that MHC-tetramer staining is not always predictive for specific T-cell reactivity, and challenge the sole use of MHC-tetramers as an indication of the peripheral T-cell repertoire, independent of the analysis of functional activity or structural avidity parameters., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The increased susceptibility of patients with atopic dermatitis (AD) to disseminated viral skin infections such as eczema herpeticum (ADEH+) is poorly understood., Objectives: The primary goal of the current study was to determine whether ADEH+ subjects have identifiable defects in cell-mediated immunity that reduce their ability to control viral infections., Materials and Methods: In this study, we evaluated cytokine expression by various subsets of peripheral blood mononuclear cells from ADEH+ (n = 24) compared with AD without a history of viral infections (ADEH-) (n = 20) before and after treatment with herpes simplex virus (HSV)., Results: We found that interferon (IFN)-γ expression after HSV treatment was lower in the CD8+ T cells and monocytes from patients with ADEH+ compared with patients who are ADEH- or nonatopic. Given the induction of CD8+ T cells as the result of antigen presentation by human leucocyte antigen (HLA) class I, consistent with the findings described above we also found that the HLA B7 allele was significantly associated with risk of the ADEH+ phenotype (odds ratio = 1·91, P = 0·02, 125 ADEH+ and 161 ADEH- subjects)., Conclusions: These data suggest that defects in viral-induced IFN-γ from CD8+ T cells contribute to the ADEH+ phenotype., (© 2013 British Association of Dermatologists.)

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.

Allovectin (velimogene aliplasmid) is a cancer immunotherapeutic currently completing a pivotal phase 3 study for metastatic melanoma. Consisting of a bicistronic plasmid encoding both major histocompatibility complex (MHC) class I heavy and light chains (HLA-B7 and β2-microglobulin, respectively) formulated with a cationic lipid-based system, it is designed for direct intratumoral administration. Following injection into a single lesion, the product is intended to induce anti-tumor immune responses against both treated and distal lesions. Both the plasmid and lipid components of Allovectin contribute to the biological activity of the drug product, and its therapeutic activity is hypothesized to derive from multiple mechanisms of actions (MOAs). These include the induction of both cytotoxic T-cell and innate immune responses directed against allogeneic as well as tumor-derived targets, consequences of both an increased MHC class I expression on tumor cells and the induction of a localized immune/inflammatory response. In this paper, we review Allovectin's proposed MOAs, placing their contributions in the context of anti-tumor immunity and highlighting both preclinical and clinical supporting data.

Human leukocyte antigen HLA-B alleles have better protective activity against HIV-1 than HLA-A alleles, possibly due to differences in HLA-restricted HIV-1-specific CD8+ cytotoxic T lymphocyte (CTL) function, but the mechanism is unknown. HIV-1 negative regulatory factor (Nef) mediates down-regulation of surface expression of class I HLA (HLA-I) and may therefore impair immune recognition by CTL. Because of sequence differences in the cytoplasmic domains, HLA-A and -B are down-regulated by Nef but HLA-C and -E are not affected. However, the latter are expressed at low levels and are not of major importance in the CTL responses to HIV-1. Here, we compared the role of the cytoplasmic domains of HLA-A and -B in Nef-mediated escape from CTL. We found HLA-B cytoplasmic domains were more resistant to Nef-mediated down-regulation than HLA-A cytoplasmic domains and demonstrated that these differences affect CTL recognition of virus-infected cells in vitro. We propose that the relative resistance to Nef-mediated down-regulation by the cytoplasmic domains of HLA-B compared with HLA-A contributes to the better control of HIV-1 infection associated with HLA-B-restricted CTLs.

It was hypothesized that the EBV-specific CD8(+) T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8(+) T cell response in greater detail, we analyzed the HLA-A2-, HLA-B7-, and HLA-B8-restricted EBV- and CMV-specific CD8(+) T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8(+) T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7(+) MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBV(RPP)-specific CD8(+) T cells ex vivo. However, the magnitude of the HLA-B*0702/EBV(RPP)-specific and HLA-B*0702/CMV(TPR)-specific CD8(+) T cell response (i.e., the percentage of tetramer(+) CD8(+) T cells in a study subject harboring CD8(+) T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBV(RPP) peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBV(RPP)-specific CD8(+) T cells were decreased. Altogether, our findings suggest that the HLA-B*0702-restricted viral (in particular the EBV one)-specific CD8(+) T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS.

The transporter associated with antigen processing (TAP) translocates the viral proteolytic peptides generated by the proteasome and other proteases in the cytosol to the endoplasmic reticulum lumen. There, they complex with nascent human leukocyte antigen (HLA) class I molecules, which are subsequently recognized by the CD8(+) lymphocyte cellular response. However, individuals with nonfunctional TAP complexes or tumor or infected cells with blocked TAP molecules are able to present HLA class I ligands generated by TAP-independent processing pathways. Herein, using a TAP-independent polyclonal vaccinia virus-polyspecific CD8(+) T cell line, two conserved vaccinia-derived TAP-independent HLA-B*0702 epitopes were identified. The presentation of these epitopes in normal cells occurs via complex antigen-processing pathways involving the proteasome and/or different subsets of metalloproteinases (amino-, carboxy-, and endoproteases), which were blocked in infected cells with specific chemical inhibitors. These data support the hypothesis that the abundant cellular proteolytic systems contribute to the supply of peptides recognized by the antiviral cellular immune response, thereby facilitating immunosurveillance. These data may explain why TAP-deficient individuals live normal life spans without any increased susceptibility to viral infections.

The TCR plays a critical role in recognizing intracellular pathogens and initiating pathways leading to the destruction of infected cells by the immune system. Although genetic variability is known to greatly impact on the human immune system and the outcome of infection, the influence of sequence variation leading to the inactivation or deletion of TCR gene segments is unknown. To investigate this issue, we examined the CD8(+) T cell response to an HLA-B7-restricted epitope ((265)RPHERNGFTVL(275)) from the pp65 Ag of human CMV that was highly biased and frequently dominated by a public TCR β-chain encoded by the variable gene segment TRBV4-3. Approximately 40% of humans lack T cells expressing TRBV4-3 because of a 21.5-kb insertion/deletion polymorphism, but these individuals remain responsive to this epitope, using a diverse T cell repertoire characterized by private TCR usage. Although most residues within the bulged 11-mer peptide were accessible for TCR contact, the public and private TCRs showed distinct patterns of sensitivity to amino acid substitution at different positions within the peptide, thereby suggesting that the repertoire diversity generated in the absence of the dominant public TRBV4-3(+) TCR could lead to better protection from viral escape mutation. Thus, variation in the size of the TRBV repertoire clearly contributes toward interindividual variability in immune responses and is presumably maintained in many ethnic groups to enhance the diversity of Ag-specific T cell responses.

The ability of CD8(+) T cells to act as cytolytic effectors and produce interferon-γ (IFN-γ) was demonstrated to mediate resistance to Toxoplasma gondii in murine models because of the recognition of peptides restricted by murine major histocompatibility complex (MHC) class I molecules. However, no T gondii-specific HLA-B07-restricted peptides were proven protective against T gondii. Recently, 2 T gondii-specific HLA-B*0702-restricted T cell epitopes, GRA7(20-28) (LPQFATAAT) and GRA3(27-35) (VPFVVFLVA), displayed high-affinity binding to HLA-B*0702 and elicited IFN-γ from peripheral blood mononuclear cells of seropositive HLA-B*07 persons. Herein, these peptides were evaluated to determine whether they could elicit IFN-γ in splenocytes of HLA-B*0702 transgenic mice when administered with adjuvants and protect against subsequent challenge. Peptide-specific IFN-γ-producing T cells were identified by enzyme-linked immunosorbent spot and proliferation assays utilizing splenic T lymphocytes from human lymphocyte antigen (HLA) transgenic mice. When HLA-B*0702 mice were immunized with one of the identified epitopes, GRA7(20-28) in conjunction with a universal CD4(+) T cell epitope (PADRE) and adjuvants (CD4(+) T cell adjuvant, GLA-SE, and TLR2 stimulatory Pam(2)Cys for CD8(+) T cells), this immunization induced CD8(+) T cells to produce IFN-γ and protected mice against high parasite burden when challenged with T gondii. This work demonstrates the feasibility of bioinformatics followed by an empiric approach based on HLA binding to test this biologic activity for identifying protective HLA-B*0702-restricted T gondii peptides and adjuvants that elicit protective immune responses in HLA-B*0702 mice., (Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Autoreactive cytotoxic CD8 T-cells (CTLs) play a key pathogenic role in the destruction of insulin-producing beta-cells resulting in type 1 diabetes. However, knowledge regarding their targets is limited, restricting the ability to monitor the course of the disease and immune interventions. In a multi-step discovery process to identify novel CTL epitopes in human preproinsulin (PPI), PPI was digested with purified human proteasomes, and resulting COOH-fragments aligned with algorithm-predicted HLA-binding peptides to yield nine potential HLA-A1, -A2, -A3 or -B7-restricted candidates. An UV-exchange method allowed the generation of a repertoire of multimers including low-affinity HLA-binding peptides. These were labeled with quantum dot-fluorochromes and encoded in a combinatorial fashion, allowing parallel and sensitive detection of specific, low-avidity T-cells. Significantly increased frequencies of T-cells against four novel PPI epitopes (PPI(4-13)/B7, PPI(29-38)/A2, PPI(76-84)/A3 and PPI(79-88)/A3) were detected in stored blood of patients with recent onset diabetes but not in controls. Changes in frequencies of circulating CD8 T-cells against these novel epitopes were detected in blood of islet graft recipients at different time points after transplantation, which correlated with clinical outcome. In conclusion, our novel strategy involving a sensitive multiplex detection technology and requiring minimal volumes of stored blood represents a major improvement in the direct ex-vivo characterization and enumeration of immune cells in the pathogenesis of type 1 diabetes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Mammaglobin-A (MGBA), a 10-kD protein, is over expressed in 80% of primary and metastatic human breast cancers. Breast cancer patients demonstrate high frequencies of CD8(+) cytotoxic T lymphocytes (CTL) specific to MGBA. Defining CD8(+) CTL responses to HLA class I-restricted MGBA-derived epitopes assumes significance in the context of our ongoing efforts to clinically translate vaccine strategies targeting MGBA for prevention and/or treatment of human breast cancers. In this study, we define the CD8(+) CTL response to MGBA-derived candidate epitopes presented in the context of HLA-B7, which has a frequency of 17.7% in Caucasian and 15.5% in African American populations. We identified seven MGBA-derived candidate epitopes with high predicted binding scores for HLA-B7 using a computer algorithm. Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-B7 indicated that MGBA B7.3 (VSKTEYKEL), B7.6 (KLLMVLMLA), B7.7 (NPQVSKTEY), and B7.1 (YAGSGCPLL) have the highest HLA-B7 binding affinities. Further, two CD8(+) CTL cell lines generated in vitro against T2.B7 cells individually loaded with MGBA-derived candidate epitopes showed significant cytotoxic activity against MGBA B7.1, B7.3, B7.6, and B7.7. In addition, the same CD8(+) CTL lines lysed the HLA-B7(+)/MGBA(+) human breast cancer cell line DU-4475 but had no significant cytotoxicity against HLA-B7(-) or MGBA(-) breast cancer cell lines. Cold-target inhibition studies strongly suggest that MGBA B7.3 is an immunodominant epitope. In summary, our results define HLA-B7-restriced, MGBA-derived, CD8(+) CTL epitopes with all of the necessary features for developing novel vaccine strategies against HLA-B7 expressing breast cancer patients.

In the USA, the incidence of cutaneous melanoma is increasing rapidly. It has been shown to be responsive to immune-stimulating drugs. Allovectin-7 allows the immune system to recognize metastatic melanoma lesions as foreign by incorporating a MHC class I complex into the tumor through direct injection. Once transfected, tumor biopsies have shown an increased presence of cytotoxic T lymphocytes in the tumor beds themselves. Phase I and II trials have shown local and systemic response to these tumors, with an excellent safety profile. Currently, a Phase III trial has completed enrollment and is set to determine the safety and efficacy of treatment in comparison with standard chemotherapy.

Importance of the Field: Immunotherapy for cancer has been investigated for several decades, achieving limited success. The development of effective new immunotherapeutic agents has reignited interest in the filed. Intralesional injection of plasmids in order to transfect genes capable of stimulating or augmenting immune recognition and destruction of tumors is a relatively new approach., Areas Covered in This Review: Our objective is to discuss the role velimogene aliplasmid (Allovectin-7, Vical Incorporated), a plasmid-lipid complex containing the DNA sequences encoding HLA-B7 and beta2 microglobulin, as an immunotherapeutic agent., What the Reader Will Gain: Intralesional velimogene aliplasmid induces anti-tumor responses in a proportion of melanoma patients with locoregional and limited distant metastases. Preclinical data and the results of Phase I, II and III clinical trials with this drug are reviewed. The limited data in other malignancies is also reviewed. Velimogene aliplasmid in humans appears safe, with minimal drug-related adverse events., Take Home Message: Velimogene aliplasmid has activity in melanoma with local and limited distant disease associated with an excellent safety profile. The activity of this approach is also being investigated in other malignancies.

Human cytomegalovirus (HCMV) encodes four glycoproteins, termed gpUS2, gpUS3, gpUS6 and gpUS11 that interfere with MHC class I biosynthesis and antigen presentation. Despite gpUS2-11 expression, however, HCMV infection is efficiently controlled by cytolytic CD8 T lymphocytes (CTL). To address the role of gpUS2 and gpUS11 in antigen presentation during viral infection, HCMV mutants were generated that expressed either gpUS2 or gpUS11 alone without coexpression of the three other proteins. Fibroblasts infected with these viruses showed reduced HLA-A2 and HLA-B7 surface expression. Surprisingly, however, CTL directed against the tegument protein pp65 and the regulatory IE1 protein still recognized and lysed mutant virus infected fibroblasts. Yet, suppression of IE1 derived peptide presentation by gpUS2 or gpUS11 was far more pronounced. The results show that gpUS2 and gpUS11 alone only incompletely protect HCMV infected fibroblasts from CTL recognition and underline the importance of studying infected cells to elucidate HCMV immune evasion.

Purpose: To determine the role of gene-environmental interactions between the Class I and Class II HLA alleles and the humoral anti-Epstein-Barr Virus (EBV) responses in the development of brain injury and clinical disability in multiple sclerosis (MS) patients., Methods: A total of 93 MS patients (62 females; 31 males) and 122 healthy controls underwent HLA typing and testing for antibodies against EBV. The MS patients underwent brain MRI and quantitative measurements of T1- and T2-lesion volumes (LVs) and brain parenchymal fraction (BPF) were obtained. There were 54 MS cases that underwent MRI and EBV-antibody assessments at the 3-year follow-up. The anti-EBV panel included measurements of the levels of anti-EBV early antigen (EA) IgG, anti-EBV nuclear antigen (EBNA) IgG and anti-EBV viral capsid antigen (VCA) IgM and anti-EBV VCA IgG. The relationships between HLA alleles, anti-EBV antibody levels, MRI and clinical parameters were assessed in regression analysis., Results: The presence of HLA B7 was associated with increased T1-LV and trends indicating increased anti-EBV VCA IgG levels, higher disability (EDSS) and more destructive MRI parameters (increased T2-LV and decreased BPF). The presence of HLA A2 was associated with lower EDSS and a trend toward decreased anti-EBV VCA IgG levels; the associations with MRI variables were not significant. The HLA B7-A2 haplotype was significantly associated with higher T2-LV and T1-LV and a trend toward lower BPF was observed., Conclusions: Our data suggest that gene-environment interactions between specific HLA Class I loci and EBV exposure are associated with MRI markers of lesion injury and brain atrophy in MS patients.

Seronegative spondarthritis (SSA) is a group of inflammatory disorders that shares certain clinical features and has a strong association with the human leucocyte antigen (HLA)-B27 allele. Serologically, HLA-B27, HLA-B22, HLA-B7, HLA-B40 and HLA-B42 antigens belong to the HLA-B cross-reacting antigen group (CREG). In addition to B27, other B locus antigens are associated with B27-negative American black, Brazilian, French and Chinese SSA patients. Many B27-negative individuals in India have developed SSA with severe clinical and radiological findings. This stimulated the evaluation of the involvement of HLA-B7 CREG antigens among B27-negative SSA patients from western India. A total of 276 SSA patients who were B27-negative and fitted the modified New York criteria for AS and the European Spondyloarthropathy Study Group (ESSG) criteria for spondarthritis from western India were studied and compared with 637 normal, healthy individuals who were B27-negative and of the same ethnic background. A significantly increased phenotype frequency of HLA-B7 (PF = 57.24% vs. 22.44%; P < 0.001) and a significant decreased phenotype frequency of HLA-B40 (PF = 18.11% vs. 31.86%; P < 0.001) was observed when compared to the controls. These results suggest that HLA-B7 antigen may be associated with B27-negative SSA in patients from western India.