Search

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.

Strategic planning and implementation of oral health care and disease prevention programmes after the 1990/91 Gulf war are discussed. The key concept was to develop access to care and disease prevention for all Kuwaiti children in government kindergarten/primary schools and to eliminate emphasis on extractions and restorations. Resources were restored to pre-war levels and then increased. Prevention programmes for 150 000 children were established. Prevention funds increased from 7% to 20% of the oral health budget. Prevention-based dentists increased from 9.7% to 28.0% of staff. Rising caries trends were stabilized or reduced by up to 36.8%. Percentage of caries-free primary dentition in children increased up to 37.6%, permanent dentition up to 27.0%. A dentistry school was established. [ABSTRACT FROM AUTHOR]

Surveys have shown increases in dental caries in young persons in Kuwait. Intake of sugar and carbohydrates has increased, as has the incidence of obesity. The objective of this study was to identify dietary practices and the potential for dental disease in primary, intermediate, and secondary school students in a health region. A random sample of 600 students aged 8, 13, and 17 yrs in 12 schools (6 boys' schools and 6 girls' schools) received a questionnaire. Responses were analyzed by age group, gender, and dietary practices at home and in school. The dietary composition of meals tended to be similar at all ages and in both genders. A total of 97% of 8-yr-old, 96% of 13-yr-old, and 92% of 17-yr-old students reported snacking at school. Sugar intake from identified snacks alone was calculated as 193.8 g/day or the estimated equivalent of 746 calories, and daily fat intake was 70.17 g. A total of 88.6% of respondents (56.6% of all children) used fluoridated toothpaste; 44.4% of all children (22.6% of 8-yr-old, 14.1% of 13-yr-old, and 7.7% of 17-yr-old students) reported brushing their teeth three times per day. Use of school snacks increased with age, while use of a toothbrush decreased. Fundamental oral hygiene procedures such as brushing and use of fluoridated toothpaste appear to have been implemented. Health workers need to provide new and pertinent oral health messages, nutrition counseling, attention to diet, and reinforcement with parents, food preparers, health facilities, and intermediate and secondary schools. [ABSTRACT FROM AUTHOR]

HLA-E is a nonclassical, nonpolymorphic, class Ib HLA molecule. Its primary function is to present a conserved nonamer peptide, termed VL9, derived from the signal sequence of classical MHC molecules to the NKG2x-CD94 receptors on NK cells and a subset of T lymphocytes. These receptors regulate the function of NK cells, and the importance of this role, which is conserved across mammalian species, probably accounts for the lack of genetic polymorphism. A second minor function is to present other, weaker binding, pathogen-derived peptides to T lymphocytes. Most of these peptides bind suboptimally to HLA-E, but this binding appears to be enabled by the relative stability of peptide-free, but receptive, HLA-E-β2m complexes. This, in turn, may favor nonclassical antigen processing that may be associated with bacteria infected cells. This review explores how the structure of HLA-E, bound to different peptides and then to NKG2-CD94 or T-cell receptors, relates to HLA-E cell biology and immunology. A detailed understanding of this molecule could open up opportunities for development of universal T-cell and NK-cell-based immunotherapies., (© 2025 The Author(s). Immunological Reviews published by John Wiley & Sons Ltd.)

Mutations in KRAS are some of the most common across multiple cancer types and are thus attractive targets for therapy. Recent studies demonstrated that mutant KRAS generates immunogenic neoantigens that are targetable by adoptive T-cell therapy in metastatic diseases. To expand mutant KRAS-specific immunotherapies, it is critical to identify additional HLA-I allotypes that can present KRAS neoantigens and their cognate T-cell receptors (TCR). Here, we identified a murine TCR specific to a KRAS-G12V neoantigen ( 7 VVVGAVGVGK 16 ) using a vaccination approach with transgenic mice expressing HLA-A*03:01 (HLA-A3). This TCR demonstrated exquisite specificity for mutant G12V and not WT KRAS peptides. To investigate the molecular basis for neoantigen recognition by this TCR, we determined its structure in complex with HLA-A3(G12V). G12V-TCR CDR3β and CDR1β formed a hydrophobic pocket to interact with p6 Val of the G12V but not the WT KRAS peptide. To improve the tumor sensitivity of this TCR, we designed rational substitutions to improve TCR:HLA-A3 contacts. Two substitutions exhibited modest improvements in TCR binding avidity to HLA-A3 (G12V) but did not sufficiently improve T-cell sensitivity for further clinical development. Our study provides mechanistic insight into how TCRs detect neoantigens and reveals the challenges in targeting KRAS-G12V mutations., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

HLA-B*27 was one of the first HLA alleles associated with an autoimmune disease, i.e., axial spondyloarthritis (axSpA) and acute anterior uveitis (B27AAU), which cause joint and eye inflammation, respectively. Gastrointestinal inflammation has been suggested as a trigger of axSpA. We recently identified a bacterial peptide (YeiH) that can be presented by HLA-B*27 to expanded public T cell receptors in the joint in axSpA and the eye in B27AAU. While YeiH is present in enteric microbiota and pathogens, additional evidence that pathogenic T cells in HLA-B*27-associated autoimmunity may have had a prior antigenic encounter within the gastrointestinal tract remains lacking. Here, we analyzed ocular, synovial, and blood T cells in B27AAU and axSpA, showing that YeiH-specific CD8+ T cells express a mucosal gene set and surface proteins consistent with intestinal differentiation, including CD161, integrin α4β7, and CCR6. In addition, we found an expansion of YeiH-specific CD8+ T cells in axSpA and B27AAU blood compared with that from individuals acting as healthy controls, whereas influenza-specific CD8+ T cells were equivalent across groups. Finally, we demonstrated the dispensability of TRBV9 for antigen recognition. Collectively, our data suggest that, in HLA-B27-associated autoimmunity, early antigen exposure and differentiation of pathogenic CD8+ T cells may occur in enteric organs.

Natural killer (NK) cells kill target cells following triggering via germline-encoded receptors interacting with target cell-expressed ligands (direct killing), or via antibody-dependent cellular cytotoxicity (ADCC) mediated by FcγRIIIa. NK cytotoxicity is modulated by signaling through activating or inhibitory receptors. A major checkpoint is mediated by the NK inhibitory receptor NKG2A/CD94 and its target cell ligand, HLA-E, which is complexed with HLA signal sequence-derived peptides termed VL9 (HLA-E-VL9). We have previously reported the isolation of a murine HLA-E-VL9-specific IgM antibody 3H4 and the generation of a higher affinity IgG version (3H4v3). Here we have used phage display library selection to generate a high affinity version of 3H4v3, called 3H4v31, with an ∼700 fold increase in binding affinity. We show using an HLA-E-VL9+ K562 tumor model that, in vitro, the addition of 3H4v31 to target cells increased direct killing of targets by CD16-negative NK cell line NK-92 and also mediated ADCC by NK-92 cells transfected with CD16. Moreover, ADCC by primary NK cells was also enhanced in vitro by 3H4v31. 3H4v31 was also able to bind and enhance target cell lysis of endogenously expressed HLA-E-VL9 on human cervical cancer and human pancreatic cancer cell lines. In vivo, 3H4v31 slowed the growth rate of HLA-E-VL9+ K562 tumors implanted into NOD/SCID/IL2rγ null mice compared to isotype control when injected with NK-92 cells intratumorally. Together, these data demonstrate that mAb 3H4v31 can enhance NK cell killing of HLA-E-VL9-expressing tumor cells in vitro by both direct killing activity and by ADCC. Moreover, mAb 3H4v31 can enhance NK cell control of tumor growth in vivo. We thus identify HLA-E-VL9 monoclonal antibodies as a promising novel anti-tumor immunotherapy., One Sentence Summary: A high affinity monoclonal antibody against HLA-E-VL9 enhances natural killer cell anti-tumor killing by checkpoint inhibition and antibody dependent cellular cytotoxicity.

HLA-E is a non-classical class I MHC protein involved in innate and adaptive immune recognition. While recent studies have shown HLA-E can present diverse peptides to NK cells and T cells, the HLA-E repertoire recognized by CD94/NKG2x has remained poorly defined, with only a limited number of peptide ligands identified. Here we screen a yeast-displayed peptide library in the context of HLA-E to identify 500 high-confidence unique peptides that bind both HLA-E and CD94/NKG2A or CD94/NKG2C. Utilizing the sequences identified via yeast display selections, we train prediction algorithms and identify human and cytomegalovirus (CMV) proteome-derived, HLA-E-presented peptides capable of binding and signaling through both CD94/NKG2A and CD94/NKG2C. In addition, we identify peptides which selectively activate NKG2C + NK cells. Taken together, characterization of the HLA-E-binding peptide repertoire and identification of NK activity-modulating peptides present opportunities for studies of NK cell regulation in health and disease, in addition to vaccine and therapeutic design., (© 2023. Springer Nature Limited.)

Interest in MHC-E-restricted CD8+ T cell responses has been aroused by the discovery of their efficacy in controlling simian immunodeficiency virus (SIV) infection in a vaccine model. The development of vaccines and immunotherapies utilizing human MHC-E (HLA-E)-restricted CD8+ T cell response requires an understanding of the pathway(s) of HLA-E transport and antigen presentation, which have not been clearly defined previously. We show here that, unlike classical HLA class I, which rapidly exits the endoplasmic reticulum (ER) after synthesis, HLA-E is largely retained because of a limited supply of high-affinity peptides, with further fine-tuning by its cytoplasmic tail. Once at the cell surface, HLA-E is unstable and is rapidly internalized. The cytoplasmic tail plays a crucial role in facilitating HLA-E internalization, which results in its enrichment in late and recycling endosomes. Our data reveal distinctive transport patterns and delicate regulatory mechanisms of HLA-E, which help to explain its unusual immunological functions., (© 2023 He et al.)

Human leukocyte antigen (HLA)-E binds epitopes derived from HLA-A, HLA-B, HLA-C and HLA-G signal peptides (SPs) and serves as a ligand for CD94/NKG2A and CD94/NKG2C receptors expressed on natural killer and T cell subsets. We show that among 16 common classical HLA class I SP variants, only 6 can be efficiently processed to generate epitopes that enable CD94/NKG2 engagement, which we term 'functional SPs'. The single functional HLA-B SP, known as HLA-B/-21M, induced high HLA-E expression, but conferred the lowest receptor recognition. Consequently, HLA-B/-21M SP competes with other SPs for providing epitope to HLA-E and reduces overall recognition of target cells by CD94/NKG2A, calling for reassessment of previous disease models involving HLA-B/-21M. Genetic population data indicate a positive correlation between frequencies of functional SPs in humans and corresponding cytomegalovirus mimics, suggesting a means for viral escape from host responses. The systematic, quantitative approach described herein will facilitate development of prediction algorithms for accurately measuring the impact of CD94/NKG2-HLA-E interactions in disease resistance/susceptibility., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Pathogen-specific CD8 + T cell responses restricted by the nonpolymorphic nonclassical class Ib molecule human leukocyte antigen E (HLA-E) are rarely reported in viral infections. The natural HLA-E ligand is a signal peptide derived from classical class Ia HLA molecules that interact with the NKG2/CD94 receptors to regulate natural killer cell functions, but pathogen-derived peptides can also be presented by HLA-E. Here, we describe five peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that elicited HLA-E-restricted CD8 + T cell responses in convalescent patients with coronavirus disease 2019. These T cell responses were identified in the blood at frequencies similar to those reported for classical HLA-Ia-restricted anti-SARS-CoV-2 CD8 + T cells. HLA-E peptide-specific CD8 + T cell clones, which expressed diverse T cell receptors, suppressed SARS-CoV-2 replication in Calu-3 human lung epithelial cells. SARS-CoV-2 infection markedly down-regulated classical HLA class I expression in Calu-3 cells and primary reconstituted human airway epithelial cells, whereas HLA-E expression was not affected, enabling T cell recognition. Thus, HLA-E-restricted T cells could contribute to the control of SARS-CoV-2 infection alongside classical T cells.

Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU)) 1 . How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8 + T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public β-chain variable region-complementary-determining region 3β (BV9-CDR3β) motif 2-4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide-MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9-CDR3β TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

MHC-E regulates NK cells by displaying MHC class Ia signal peptides (VL9) to NKG2A:CD94 receptors. MHC-E can also present sequence-diverse, lower-affinity, pathogen-derived peptides to T cell receptors (TCRs) on CD8 + T cells. To understand these affinity differences, human MHC-E (HLA-E)-VL9 versus pathogen-derived peptide structures are compared. Small-angle X-ray scatter (SAXS) measures biophysical parameters in solution, allowing comparison with crystal structures. For HLA-E-VL9, there is concordance between SAXS and crystal parameters. In contrast, HLA-E-bound pathogen-derived peptides produce larger SAXS dimensions that reduce to their crystallographic dimensions only when excess peptide is supplied. Further crystallographic analysis demonstrates three amino acids, exclusive to MHC-E, that not only position VL9 close to the α2 helix, but also allow non-VL9 peptide binding with re-configuration of a key TCR-interacting α2 region. Thus, non-VL9-bound peptides introduce an alternative peptide-binding motif and surface recognition landscape, providing a likely basis for VL9- and non-VL9-HLA-E immune discrimination., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell receptors NKG2A-C/CD94 and regulate NK cell-mediated cytotoxicity. Here we report the isolation of 3H4, a murine HLA-E-VL9-specific IgM antibody that enhances killing of HLA-E-VL9-expressing cells by an NKG2A + NK cell line. Structural analysis reveal that 3H4 acts by preventing CD94/NKG2A docking on HLA-E-VL9. Upon in vitro maturation, an affinity-optimized IgG form of 3H4 showes enhanced NK killing of HLA-E-VL9-expressing cells. HLA-E-VL9-specific IgM antibodies similar in function to 3H4 are also isolated from naïve B cells of cytomegalovirus (CMV)-negative, healthy humans. Thus, HLA-E-VL9-targeting mouse and human antibodies isolated from the naïve B cell antibody pool have the capacity to enhance NK cell cytotoxicity., (© 2022. The Author(s).)

Persistence of HIV through integration into host DNA in CD4 + T cells presents a major barrier to virus eradication. Viral integration may be curtailed when CD8 + T cells are triggered to kill infected CD4 + T cells through recognition of histocompatibility leukocyte antigen (HLA) class I-bound peptides derived from incoming virions. However, this has been reported only in individuals with "beneficial" HLA alleles that are associated with superior HIV control. Through interrogation of the pre-integration immunopeptidome, we obtain proof of early presentation of a virion-derived HLA-A ∗ 02:01-restricted epitope, FLGKIWPSH (FH9), located in Gag Spacer Peptide 2 (SP2). FH9-specific CD8 + T cell responses are detectable in individuals with primary HIV infection and eliminate HIV-infected CD4 + T cells prior to virus production in vitro. Our data show that non-beneficial HLA class I alleles can elicit an effective antiviral response through early presentation of HIV virion-derived epitopes and also demonstrate the importance of SP2 as an immune target., Competing Interests: Declaration of interests L.D. is an employee of Immunocore Ltd. A.K. and D.B.L. were previously employees of Immunocore Ltd., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human leukocyte antigen-E (HLA-E) normally presents an HLA class Ia signal peptide to the NKG2A/C-CD94 regulatory receptors on natural killer (NK) cells and T cell subsets. Rhesus macaques immunized with a cytomegalovirus-vectored simian immunodeficiency virus (SIV) vaccine generated Mamu-E (HLA-E homolog)-restricted T cell responses that mediated post-challenge SIV replication arrest in >50% of animals. However, HIV-1-specific, HLA-E-restricted T cells have not been observed in HIV-1-infected individuals. Here, HLA-E-restricted, HIV-1-specific CD8 + T cells were primed in vitro. These T cell clones and allogeneic CD8 + T cells transduced with their T cell receptors suppressed HIV-1 replication in CD4 + T cells in vitro. Vaccine induction of efficacious HLA-E-restricted HIV-1-specific T cells should therefore be possible., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Diverse SIV and HIV epitopes that bind the rhesus homolog of HLA-E, Mamu-E, have recently been identified in SIVvaccine studies using a recombinant Rhesus cytomegalovirus (RhCMV 68-1) vector, where unprecedented protection against SIV challenge was achieved. Additionally, several Mycobacterial peptides identified both algorithmically and following elution from infected cells, are presented to CD8 + T cells by HLA-E in humans. Yet, a comparative and comprehensive analysis of relative HLA-E peptide binding strength via a reliable, high throughput in vitro assay is currently lacking. To address this, we developed and optimized a novel, highly sensitive peptide exchange ELISA-based assay that relatively quantitates peptide binding to HLA-E. Using this approach, we screened multiple peptides, including peptide panels derived from HIV, SIV, and Mtb predicted to bind HLA-E. Our results indicate that although HLA-E preferentially accommodates canonical MHC class I leader peptides, many non-canonical, sequence diverse, pathogen-derived peptides also bind HLA-E, albeit generally with lower relative binding strength. Additionally, our screens demonstrate that the majority of peptides tested, including some key Mtb and SIV epitopes that have been shown to elicit strong Mamu-E-restricted T cell responses, either bind HLA-E extremely weakly or give signals that are indistinguishable from the negative, peptide-free controls., (© 2020 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

T cell cross-reactivity ensures that diverse pathogen-derived epitopes encountered during a lifetime are recognized by the available TCR repertoire. A feature of cross-reactivity where previous exposure to one microbe can alter immunity to subsequent, non-related pathogens has been mainly explored for viruses. Yet cross-reactivity to additional microbes is important to consider, especially in HIV infection where gut-intestinal barrier dysfunction could facilitate T cell exposure to commensal/pathogenic microbes. Here we evaluated the cross-reactivity of a 'public', HIV-specific, CD8 T cell-derived TCR (AGA1 TCR) using MHC class I yeast display technology. Via screening of MHC-restricted libraries comprising ~2×10 8 sequence-diverse peptides, AGA1 TCR specificity was mapped to a central peptide di-motif. Using the top TCR-enriched library peptides to probe the non-redundant protein database, bacterial peptides that elicited functional responses by AGA1-expressing T cells were identified. The possibility that in context-specific settings, MHC class I proteins presenting microbial peptides influence virus-specific T cell populations in vivo is discussed., Competing Interests: JM, SF, LL, SB, FP, MB, AM, GG No competing interests declared, MG MHG is a co-founder of 3T Biosciences. KG KCG is a co-founder of 3T Biosciences., (© 2020, Mendoza et al.)

The original version of this Article contained an error in the spelling of the author Jonah B Sacha, which was incorrectly given as Jonah Sacha. These errors have now been corrected in both the PDF and HTML versions of the Article.

Through major histocompatibility complex class Ia leader sequence-derived (VL9) peptide binding and CD94/NKG2 receptor engagement, human leucocyte antigen E (HLA-E) reports cellular health to NK cells. Previous studies demonstrated a strong bias for VL9 binding by HLA-E, a preference subsequently supported by structural analyses. However, Mycobacteria tuberculosis (Mtb) infection and Rhesus cytomegalovirus-vectored SIV vaccinations revealed contexts where HLA-E and the rhesus homologue, Mamu-E, presented diverse pathogen-derived peptides to CD8 + T cells, respectively. Here we present crystal structures of HLA-E in complex with HIV and Mtb-derived peptides. We show that despite the presence of preferred primary anchor residues, HLA-E-bound peptides can adopt alternative conformations within the peptide binding groove. Furthermore, combined structural and mutagenesis analyses illustrate a greater tolerance for hydrophobic and polar residues in the primary pockets than previously appreciated. Finally, biochemical studies reveal HLA-E peptide binding and exchange characteristics with potential relevance to its alternative antigen presenting function in vivo.

The central nervous system (CNS) is an important target of HIV, and cerebrospinal fluid (CSF) can provide a window into host-virus interactions within the CNS. The goal of this study was to determine whether HIV-specific CD8(+) T cells are present in CSF of HIV controllers (HC), who maintain low to undetectable plasma viremia without antiretroviral therapy (ART). CSF and blood were sampled from 11 HC, defined based on plasma viral load (VL) consistently below 2,000 copies/ml without ART. These included nine elite controllers (EC, plasma VL <40 copies/ml) and two viremic controllers (VC, VL 40-2,000 copies/ml). All controllers had CSF VL <40 copies/ml. Three comparison groups were also sampled: six HIV noncontrollers (NC, VL >10,000 copies/ml, no ART); seven individuals with viremia suppressed due to ART (Tx, VL <40 copies/ml); and nine HIV-negative controls. CD4(+) and CD8(+) T cells in CSF and blood were analyzed by flow cytometry to assess expression of CCR5, activation markers CD38 and HLA-DR, and memory/effector markers CD45RA and CCR7. HIV-specific CD8(+) T cells were quantified by major histocompatibility complex class I multimer staining. HIV-specific CD8(+) T cells were detected ex vivo at similar frequencies in CSF of HC and noncontrollers; the highest frequencies were in individuals with CD4 counts below 500 cells/μl. The majority of HIV-specific CD8(+) T cells in CSF were effector memory cells expressing CCR5. Detection of these cells in CSF suggests active surveillance of the CNS compartment by HIV-specific T cells, including in individuals with long-term control of HIV infection in the absence of therapy.

Major histocompatibility complex E (MHC-E) is a highly conserved, ubiquitously expressed, nonclassical MHC class Ib molecule with limited polymorphism that is primarily involved in the regulation of natural killer (NK) cells. We found that vaccinating rhesus macaques with rhesus cytomegalovirus vectors in which genes Rh157.5 and Rh157.4 are deleted results in MHC-E-restricted presentation of highly varied peptide epitopes to CD8αβ(+) T cells, at ~4 distinct epitopes per 100 amino acids in all tested antigens. Computational structural analysis revealed that MHC-E provides heterogeneous chemical environments for diverse side-chain interactions within a stable, open binding groove. Because MHC-E is up-regulated to evade NK cell activity in cells infected with HIV, simian immunodeficiency virus, and other persistent viruses, MHC-E-restricted CD8(+) T cell responses have the potential to exploit pathogen immune-evasion adaptations, a capability that might endow these unconventional responses with superior efficacy., (Copyright © 2016, American Association for the Advancement of Science.)

Polymorphic differences distinguishing MHC class I subtypes often permit the presentation of shared epitopes in conformationally identical formats but can affect T-cell repertoire selection, differentially impacting autoimmune susceptibilities and viral clearance in vivo. The molecular mechanisms underlying this effect are not well understood. We performed structural, thermodynamic, and functional analyses of a conserved T-cell receptor (TCR) which is frequently expanded in response to a HIV-1 epitope when presented by HLA-B*5701 but is not selected by HLA-B*5703, which differs from HLA-B*5701 by two concealed polymorphisms. Our findings illustrate that although both HLA-B*57 subtypes display the epitope in structurally conserved formats, the impact of their polymorphic differences occurs directly as a consequence of TCR ligation, primarily because of peptide adjustments required for TCR binding, which involves the interplay of polymorphic residues and water molecules. These minor differences culminate in subtype-specific differential TCR-binding kinetics and cellular function. Our data demonstrate a potential mechanism whereby the most subtle MHC class I micropolymorphisms can influence TCR use and highlight their implications for disease outcomes.

A large body of functional and epidemiological evidence have previously illustrated the impact of specific MHC class I subtypes on clinical outcome during HIV-1 infection, and these observations have recently been re-iterated in genome wide association studies (GWAS). Yet because of the complexities surrounding GWAS-based approaches and the lack of knowledge relating to the identity of rarer single nucleotide polymorphism (SNP) variants, it has proved difficult to discover independent causal variants associated with favourable immune control. This is especially true of the candidate variants within the HLA region where many of the recently proposed disease influencing SNPs appear to reflect linkage with 'protective' MHC class I alleles. Yet causal MHC-linked SNPs may exist but remain overlooked owing to the complexities associated with their identification. Here we focus on the ancestral TNFα promoter -237A variant (rs361525), shown historically to be in complete linkage disequilibrium with the 'protective' HLA-B*5701 allele. Many of the ancestral SNPs within the extended TNFα promoter have been associated with both autoimmune conditions and disease outcomes, however, the direct role of these variants on TNFα expression remains controversial. Yet, because of the important role played by TNFα in HIV-1 infection, and given the proximity of the -237 SNP to the core promoter, its location within a putative repressor region previously characterized in mice, and its disruption of a methylation-susceptible CpG dinucleotide motif, we chose to carefully evaluate its impact on TNFα production. Using a variety of approaches we now demonstrate that carriage of the A SNP is associated with lower TNFα production, via a mechanism not readily explained by promoter methylation nor the binding of transcription factors or repressors. We propose that the -237A variant could represent a minor causal SNP that additionally contributes to the HLA-B*5701-mediated 'protective' effect during HIV-1 infection.

Mutations within MHC class I-restricted epitopes have been studied in relation to T cell-mediated immune escape, but their impact on NK cells via interaction with killer Ig-like receptors (KIRs) during early HIV infection is poorly understood. In two patients acutely infected with HIV-1, we observed the appearance of a mutation within the B*57-restricted TW10 epitope (G9E) that did not facilitate strong escape from T cell recognition. The NK cell receptor KIR3DL1, carried by these patients, is known to recognize HLA-B*5703 and is associated with good control of HIV-1. Therefore, we tested whether the G9E mutation influenced the binding of HLA-B*5703 to soluble KIR3DL1 protein by surface plasmon resonance, and while the wild-type sequence and a second (T3N) variant were recognized, the G9E variant abrogated KIR3DL1 binding. We extended the study to determine the peptide sensitivity of KIR3DL1 interaction with epitopes carrying mutations near the C termini of TW10 and a second HLA-B*57-restricted epitope, IW9. Several amino acid changes interfered with KIR3DL1 binding, the most extreme of which included the G9E mutation commonly selected by HLA-B*57. Our results imply that during HIV-1 infection, some early-emerging variants could affect KIR-HLA interaction, with possible implications for immune recognition.

A major challenge to developing a successful HIV vaccine is the vast diversity of viral sequences, yet it is generally assumed that an epitope conserved between different strains will be recognised by responding T-cells. We examined whether an invariant HLA-B8 restricted Nef₉₀₋₉₇ epitope FL8 shared between five high titre viruses and eight recombinant vaccinia viruses expressing Nef from different viral isolates (clades A-H) could activate antiviral activity in FL8-specific cytotoxic T-lymphocytes (CTL). Surprisingly, despite epitope conservation, we found that CTL antiviral efficacy is dependent on the infecting viral isolate. Only 23% of Nef proteins, expressed by HIV-1 isolates or as recombinant vaccinia-Nef, were optimally recognised by CTL. Recognition of the HIV-1 isolates by CTL was independent of clade-grouping but correlated with virus-specific polymorphisms in the epitope flanking region, which altered immunoproteasomal cleavage resulting in enhanced or impaired epitope generation. The finding that the majority of virus isolates failed to present this conserved epitope highlights the importance of viral variance in CTL epitope flanking regions on the efficiency of antigen processing, which has been considerably underestimated previously. This has important implications for future vaccine design strategies since efficient presentation of conserved viral epitopes is necessary to promote enhanced anti-viral immune responses.

T-cell receptors (TCRs) are membrane proteins which recognize antigens with high specificity forming the basis of the cellular immune response. The study of these receptors has been limited by the challenges in expressing sufficient quantities of stable soluble protein. Here we report our systematic approach for generating soluble, (alpha)(beta)-TCRs, for X-ray crystallographic studies. By using small-scale expression screens, novel standardized quality control mechanisms and crystallization and imaging robots we were able to add significantly to the current TCR structural database. Our success in crystallizing both isolated TCRs and Major histocompatibility complex (MHC):TCR complexes has provided us with sufficient data to develop focused crystallization screens, which have proved generically useful for the crystallization of this family of proteins and complexes.

In HIV-1 infection, the synergistic association of a subset of Bw4 MHC class I molecules and the activating killer inhibitory receptor (KIR), KIR3DS1, with prolonged AIDS-free survival has been reported. As KIRs represent a diverse group of MHC class I receptors, we questioned whether Bw4 MHC class I molecules expressing isoleucine at position 80 (Bw4Ile80) and in complex with HIV-1-derived T cell epitopes represented KIR3DS1 ligands. MHC class I tetramers are powerful tools for the detection of T cell receptor-MHC class I interactions, and have recently been used to evaluate KIR-MHC class I binding ex vivo. Specifically, this approach has been successfully utilized to assess binding of Bw4 MHC class I tetramers to KIR3DL1, an inhibitory KIR and allele of KIR3DS1. In this study we generated a diverse panel of HIV-1-specific Bw4Ile80 MHC class I tetramers and tested its ability to bind transiently expressed KIR3DS1 on 293-T cells. Using flow cytometry analysis, the expression of KIR3DS1 on 293-T cells was confirmed by anti-FLAG BioM2 staining, prior to incubation with PE-conjugated MHC class I tetramers. Despite choosing a broad array of peptide epitopes and diverse Bw4Ile80 MHC class I molecules, we were unable to detect tetramer binding to KIR3DS1. We speculate that our negative finding may be a consequence of the MHC class I molecules and peptide epitopes chosen, but could also relate to key amino acid differences that distinguish KIR3DS1 from KIR3DL1.

HLA-B*57 is associated with slower disease progression to AIDS, and CD8+ T cell responses to B*57-restricted epitopes are thought to contribute to this protective effect. In this study, we evaluate the B*57-restricted p24 KAFSPEVIPMF (KF11) immune response which is immunodominant during chronic infection. Previously, we observed that the KF11 clade variants KGFNPEVIPMF [A2G,S4N] and KAFNPEIIMPF [S4N,V7I], sharing a position 4 mutation, are differentially recognized by KF11-specific T cells. By combining structural and cellular studies, we now demonstrate that the KF11 and [A2G,S4N] epitopes induce distinct functional responses in [A2G,S4N] and KF11-specific T cells, respectively, despite minimal structural differences between the individual B*57-peptide complexes. Recently, we also elucidated the highly distinct structure of KF11 in complex with B*5703, and have now characterized the CD8+ T cell repertoire recognizing this epitope. We now report striking features of TCR conservation both in terms of TCR Valpha and Vbeta chain usage, and throughout the hypervariable region. Collectively, our findings highlight unusual features of the B*5701/B*5703-KF11-specific immune responses which could influence disease progression and that might be important to consider when designing future vaccine regimens.

The mechanisms underlying the relatively slow progression of human immunodeficiency virus type 2 (HIV-2) compared with HIV-1 infection are undefined and could be a result of more effective immune responses. We used HIV-2 and HIV-1 IFN-gamma enzyme-linked immunospot assays to evaluate CD8(+) T cell responses in antiretroviral-naive HIV-2- ('HIV-2(+)') and HIV-1-infected ('HIV-1(+)') individuals. Gag-specific responses were detected in the majority of HIV-2(+) and HIV-1(+) subjects. Overlapping gag peptide analysis indicated a significantly greater magnitude and breadth of responses in the HIV-1(+) cohort, and this difference was attributable to low responses in HIV-2(+) subjects with undetectable viral load (medians 2107 and 512 spot-forming units per 10(6) PBMC, respectively, p=0.007). We investigated the phenotype of viral epitope-specific CD8(+) T cells identified with HLA-B53- and HLA-B58-peptide tetramers (8 HIV-2(+), 11 HIV-1(+) subjects). HIV-2-specific CD8(+) T cells were predominantly CD27(+) CD45RA(-), and only a minority expressed perforin. The limited breadth and low frequency of CD8(+) T cell responses to HIV-2 gag in aviremic HIV-2(+) subjects suggests that these responses reflect antigen load in plasma, as is the case in HIV-1 infection. Immune control of HIV-2 does not appear to be related to the frequency of perforin-expressing virus-specific CD8(+) T cells.

The cost of salt fluoridation in a given country depends primarily on the number of salt factories and on the technical level available in the country. Equipment required may cost U.S. dollars 400,000 for large plants producing at least 20,000 tons/year providing salt for populations of several millions. Reliable batch mixers have been built locally for U.S. dollars 3000 to U.S. dollars 10,000, with one such mixer capable of producing 10 batches of one metric ton/day or 2000 to 3000 tons a year for a population of 350,000 to 500,000. Frequently 85-90% of the costs are devoted to infrastructure; in combination with salt iodization, the cost for fluoride equipment is 30-50% less. loIization is promoted by WHO, UNICEF, other international organizations and national aid agencies which can indirectly support salt fluoridation. With respect to running costs, the expense for the fluoride chemical is the major factor in small plants producing for example 6000 tons of salt, i. e U.S. dollars 0.015 to 0.03 per year and capita. The cost for personnel necessary for addition of fluoride and quality control is approximately U.S. dollars 0.008/capita/year in small plants and even less in large ones. With adequate implementation, salt fluoridation affords a cariostatic effectiveness equal to that of water fluoridation. When its cost is compared to that of water fluoridation, there may not be much difference regarding initial cost for equipment except in the case of small salt factories where local production of batch mixers may lower initial expenses substantially. Running costs for salt fluoridation are 10 to 100 times lower because the amount of fluoride chemical needed and its handling are up to 100 times less than with water fluoridation. In practice, the cost of salt fluoridation is often so low that many producers did not raise the price of fluoridated salt; this has been the case in Switzerland since 1955 and also in several countries in the Americas today.

Fluoridation of water supplies has proven to be an effective preventive measure for dental caries. Many developing countries in the Americas have multiple water systems and economies that do not permit the viable application of this approach. Some of the highest dental caries prevalence in the world was evident in the Americas. Fluoridated salt was considered as a potential solution on account of the urgent need for dental caries prevention to millions of people with limited access to routine dental services. A fluoridated salt trial was initiated in Colombia (1963) and upon successful completion with preventive results comparable to water fluoridation, the approach was introduced to other countries and was supported by resolutions of WHO, PAHO, regional health groups and the FDI. The procedures for addition of fluoride were comparable to those for iodization and the two elements were compatible. In the period 1972-2004, ten countries introduced national or localized programmes and five more initiated programmes. Results, based on addition of F ion at 200-250 mg/kg salt, indicated caries prevalence reductions in 12 year olds ranging from 84% in Jamaica, 73% in Costa Rica to 40% in Uruguay at an average cost of 0.06 U.S. dollars /capita/year. This paper provides a background to the situation in the Americas, illustrates the approaches and feasibility of implementing viable fluoridated salt programmes in countries, and demonstrates the results obtainable at minimum cost.

Immunization of newborns against viral infections may be hampered by ineffective CD8(+) T cell responses. To characterize the function of CD8(+) T lymphocytes in early life, we studied newborns with congenital human cytomegalovirus (HCMV) infection. We demonstrate that HCMV infection in utero leads to the expansion and the differentiation of mature HCMV-specific CD8(+) T cells, which have similar characteristics to those detected in adults. High frequencies of HCMV-specific CD8(+) T cells were detected by ex vivo tetramer staining as early as after 28 weeks of gestation. During the acute phase of infection, these cells had an early differentiation phenotype (CD28(-)CD27(+)CD45RO(+), perforin(low)), and they acquired a late differentiation phenotype (CD28(-)CD27(-)CD45RA(+), perforin(high)) during the course of the infection. The differentiated cells showed potent perforin-dependent cytolytic activity and produced antiviral cytokines. The finding of a mature and functional CD8(+) T cell response to HCMV suggests that the machinery required to prime such responses is in place during fetal life and could be used to immunize newborns against viral pathogens.

The mechanisms underlying non-progression in HIV-1 infection are not well understood; however, this state has been associated previously with strong HIV-1-specific CD8+ T cell responses and the preservation of proliferative CD4+ T cell responses to HIV-1 antigens. Using a combination of interferon-gamma (IFN-gamma) ELISpot assays and tetramer staining, the HIV-1-specific CD8+ T cell populations were quantified and characterized in untreated long-term HIV-1-infected non-progressors and individuals with slowly progressive disease, both in relation to CD4+ T cell responses, and in comparison with responses to cytomegalovirus (CMV) antigens. High levels of CD8+ T cell responses specific for HIV-1 or CMV were observed, but neither their frequency nor their phenotype seemed to differ between the two patient groups. Moreover, while CMV-specific CD4+ T cell responses were preserved in these donors, IFN-gamma release by HIV-1-specific CD4+ T cells was generally low. These data raise questions with regard to the role played by CD8+ T cells in the establishment and maintenance of long-term non-progression.

We describe the long-term survival of an individual infected with HIV-1 during extrauterine life as a premature newborn. In the absence of viral attenuation in the Nef/LTR structure or significant co-receptor polymorphisms, slow progression was associated with the strong HIV-1-specific broadly cross-reactive CD8 T cell responses. HIV-1 infection as early as 25 weeks' gestation may thus results in the development of immune responses that control viral replication and lead to prolonged survival.

Objectives: To determine whether CD8 T lymphocytes from HIV-1-infected patients expressing B*5701 and B*5703 show broad cross-reactivity against different variants of a conserved p24 epitope, which might account for the good prognosis of HIV-1-infected individuals with HLA-B*57., Design: B*5701+ and B*5703+ were recruited from Nairobi, Kenya and from Oxford, UK. All patients had been HIV positive for at least 8 years and could be categorized as slow progressors., Methods: CD8 cytotoxic T cell clones were generated from B*5701+ and B*5703+ donors and tested for their ability to recognize clade variants of an index p24 epitope in standard cytolytic assays. Cross-reactive responses in freshly isolated peripheral blood mononuclear cells (PBMC) were assessed by interferon-gamma (IFNgamma) production and tetramer binding., Results: Broad cross-clade reactivity for both cytolysis and tetramer binding was observed in CD8 T cell clones from patients harbouring the index epitope sequence. Patterns of cross-reactivity were similar in freshly isolated PBMC but varied between individuals in terms of strength and breath of responses generated. One common variant induced an unusual response with tetramer binding but often failed to induce IFNgamma production, and another was a weak stimulator of both IFNgamma and cytolytic activity., Conclusion: B*5701+ and B5703+ donors demonstrate broad functional cross-reactivity to both common and rare variants of a dominant p24 epitope, which could be relevant to the association of B*57 alleles with slow progression to AIDS.

The viruses HIV-1, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and hepatitis C virus (HCV) are characterized by the establishment of lifelong infection in the human host, where their replication is thought to be tightly controlled by virus-specific CD8+ T cells. Here we present detailed studies of the differentiation phenotype of these cells, which can be separated into three distinct subsets based on expression of the costimulatory receptors CD28 and CD27. Whereas CD8+ T cells specific for HIV, EBV and HCV exhibit similar characteristics during primary infection, there are significant enrichments at different stages of cellular differentiation in the chronic phase of persistent infection according to the viral specificity, which suggests that distinct memory T-cell populations are established in different virus infections. These findings challenge the current definitions of memory and effector subsets in humans, and suggest that ascribing effector and memory functions to subsets with different differentiation phenotypes is no longer appropriate.

We have shown that the dominance of CD8+ T cells expressing TCR Vbeta17 in the adult HLA-A*0201-restricted influenza A/M1(58-66)-specific response is acquired following first antigen exposure. Despite the acquired dominance of Vbeta17+ cells, subdominant M1(58-66)-specific clones expressing non-Vbeta17+ TCR persist in all individuals. To determine whether the affinity of the expressed TCR for the HLA-A*0201/M1(58-66) complex could influence functional properties, M1(58-66)-specific clones expressing subdominant (non-Vbeta17+) TCR were compared to cytotoxic T lymphocyte (CTL) clones expressing dominant (Vbeta17+) TCR. The Vbeta17+ CTL required up to 10,000-fold lower amounts of M1 peptide to mediate lysis compared to CTL clones expressing other Vbeta gene segments. All Vbeta17+ CTL clones tested bound HLA-A*0201/M1(58-66) tetramer, but two of three CTL clones expressing other TCR did not bind tetramer. The inability of non-Vbeta17+ CTL to bind tetramer did not correlate with phenotype, CD8 dependence or with cytokine production profiles. This suggests a limitation for the use of tetramers in examining subdominant T cell responses. Together these findings suggest that Vbeta17+ CTL which dominate the HLA-A*0201-restricted CTL response against influenza A are not functionally distinct from subdominant non-Vbeta17+ CTL. The dominance of Vbeta17+ CTL is likely to result from a competitive advantage due to superior CTL avidity for the HLA-A*0201/M1(58-66) complex.

Human cytomegalovirus (HCMV) infection is largely asymptomatic in the immunocompetent host, but remains a major cause of morbidity in immunosuppressed individuals. Using the recently described technique of staining antigen-specific CD8(+) T cells with peptide-HLA tetrameric complexes, we have demonstrated high levels of antigen-specific cells specific for HCMV peptides and show that this may exceed 4% of CD8(+) T cells in immunocompetent donors. Moreover, by staining with tetramers in combination with antibodies to cell surface markers and intracellular cytokines, we demonstrate functional heterogeneity of HCMV-specific populations. A substantial proportion of these are effector cytotoxic T lymphocytes, as demonstrated by their ability to lyse peptide-pulsed targets in "fresh" killing assays. These data suggest that the immune response to HCMV is periodically boosted by a low level of HCMV replication and that sustained immunological surveillance contributes to the maintenance of host-pathogen homeostasis. These observations should improve our understanding of the immunobiology of persistent viral infection.

Background: CMV infection remains a significant clinical problem in the context of LT. Changes in the magnitude of the CMV-specific CTL response after LT have not previously been assessed but may be important in determining the outcome of CMV infection., Method: We used a fluorescent HLA-B*0702-CMV peptide tetrameric complex to directly visualize and quantitate CMV-specific CD8+ CTL both in immunosuppressed patients after LT and in immunocompetent controls., Results: CMV-specific CD8+ CTL, at a frequency ranging from 0.1 to 5.8% of CD8+, were detected in the peripheral blood of 22 of 25 B*0702, CMV immunoglobulin G seropositive individuals, with no difference observed between immunocompetent controls and patients >3 years after LT. In CMV seropositive LT recipients who did not have symptomatic CMV infection during the first 3 months after LT, CMV-specific CD8+ CTL magnitude initially decreased, then increased up to 5 times higher than pre-LT levels within 3 months. Two CMV seronegative recipients of seropositive donors had symptomatic CMV infection in association with high viral load. In both patients, no CD8+ CTL response was detected before the onset of symptoms, and a reduction in viral load was observed during antiviral therapy. However, polymerase chain reaction negativity was achieved only when a demonstrable CMV-specific CD8+ CTL response was generated. Responses were never observed in asymptomatic CMV seronegative patients., Conclusions: We suggest that the generation of CMV-specific CD8+ CTL may be driven by, and seems to coincide with the suppression of, viral reactivation. Direct monitoring of CMV-specific CD8+ CTL using an HLA-peptide tetramer may prove to be of value in the management of patients after LT.

Objective: To look for evidence of T lymphocyte expansions in the blood and synovial fluid (SF) of patients with reactive arthritis (ReA)., Methods: Paired peripheral blood and synovial samples from 10 patients with ReA were studied by dual color flow cytometry using T cell receptor (TCR) V beta specific and CD4 or CD8 specific antibodies. Two synovial CD8 expansions were studied by 3 color flow cytometry. Peripheral blood samples from 13 healthy, age matched individuals were studied as controls., Results: Statistically significant expansions were observed in all patients, occurring in blood and SF CD4 and CD8 compartments, but were most common in the synovial CD8 compartment. Expansions studied in further detail displayed an activated "memory" phenotype. A synovial BV22S1/CD8 expansion was seen in 5/6 patients with sexually acquired ReA., Conclusion: Multiple T lymphocyte expansions are found in both the blood and SF of patients with ReA. Expansions were most commonly found in the synovial CD8 compartment, where they appeared to express both activation and memory markers. This indicates that T lymphocytes (and in particular cytotoxic T cells) may play a pathogenic role in ReA. These findings are consistent with either an antigen or a superantigen driven response.

Primary infection with Epstein-Barr virus often results in the clinical syndrome of acute infectious mononucleosis (glandular fever). This illness is characterized by a striking lymphocytosis, the nature of which has been controversial. We show that large monoclonal or oligoclonal populations of CD8+ T cells account for a significant proportion of the lymphocytosis and provide molecular evidence that these populations have been driven by antigen. The results suggest that the selective and massive expansion of a few dominant clones of CD8+ T cells is an important feature of the primary response to this virus.