Crystal structures of N-myristoylated lipopeptide-bound HLA class I complexes indicate reorganization of B-pocket architecture upon ligand binding.

Rhesus monkeys have evolved MHC-encoded class I allomorphs such as Mamu-B∗098 that are capable of binding N-myristoylated short lipopeptides rather than conventional long peptides; however, it remains unknown whether such antigen-binding molecules exist in other species, including humans. We herein demonstrate that human leukocyte antigen (HLA)-A∗24:02 and HLA-C∗14:02 proteins, which are known to bind conventional long peptides, also have the potential to bind N-myristoylated short lipopeptides. These HLA class I molecules shared a serine at position 9 (Ser9) with Mamu-B∗098, in contrast to most MHC class I molecules that harbor a larger amino acid residue, such as tyrosine, at this position. High resolution X-ray crystallographic analyses of lipopeptide-bound HLA-A∗24:02 and HLA-C∗14:02 complexes indicated that Ser9 was at the bottom of the B pocket with its small hydroxymethyl side chain directed away from the B-pocket cavity, thereby contributing to the formation of a deep hydrophobic cavity suitable for accommodating the long-chain fatty acid moiety of lipopeptide ligands. Upon peptide binding, however, we found the hydrogen-bond network involving Ser9 was reorganized, and the remodeled B pocket was able to capture the second amino acid residue (P2) of peptide ligands. Apart from the B pocket, virtually no marked alterations were observed for the A and F pockets upon peptide and lipopeptide binding. Thus, we concluded that the structural flexibility of the large B pocket of HLA-A∗2402 and HLA-C∗1402 primarily accounted for their previously unrecognized capacity to bind such chemically distinct ligands as conventional peptides and N-myristoylated lipopeptides.

T cells discriminate between groups C1 and C2 HLA-C.

Dimorphic amino acids at positions 77 and 80 delineate HLA-C allotypes into two groups, C1 and C2, which associate with disease through interactions with C1 and C2-specific natural killer cell receptors. How the C1/C2 dimorphism affects T cell recognition is unknown. Using HLA-C allotypes that differ only by the C1/C2-defining residues, we found that KRAS-G12D neoantigen-specific T cell receptors (TCRs) discriminated between C1 and C2 presenting the same KRAS-G12D peptides. Structural and functional experiments, and immunopeptidomics analysis revealed that Ser77 in C1 and Asn77 in C2 influence amino acid preference near the peptide C-terminus (pΩ), including the pΩ-1 position, in which C1 favors small and C2 prefers large residues. This resulted in weaker TCR affinity for KRAS-G12D-bound C2-HLA-C despite conserved TCR contacts. Thus, the C1/C2 dimorphism on its own impacts peptide presentation and HLA-C-restricted T cell responses, with implications in disease, including adoptive T cell therapy targeting KRAS-G12D-induced cancers.

The New Kid on the Block: HLA-C, a Key Regulator of Natural Killer Cells in Viral Immunity.

The human leukocyte antigen system (HLA) is a cluster of highly polymorphic genes essential for the proper function of the immune system, and it has been associated with a wide range of diseases. HLA class I molecules present intracellular host- and pathogen-derived peptides to effector cells of the immune system, inducing immune tolerance in healthy conditions or triggering effective immune responses in pathological situations. HLA-C is the most recently evolved HLA class I molecule, only present in humans and great apes. Differentiating from its older siblings, HLA-A and HLA-B, HLA-C exhibits distinctive features in its expression and interaction partners. HLA-C serves as a natural ligand for multiple members of the killer-cell immunoglobulin-like receptor (KIR) family, which are predominately expressed by natural killer (NK) cells. NK cells are crucial for the early control of viral infections and accumulating evidence indicates that interactions between HLA-C and its respective KIR receptors determine the outcome and progression of viral infections. In this review, we focus on the unique role of HLA-C in regulating NK cell functions and its consequences in the setting of viral infections.

High-affinity oligoclonal TCRs define effective adoptive T cell therapy targeting mutant KRAS-G12D.

Complete cancer regression occurs in a subset of patients following adoptive T cell therapy (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). However, the low success rate presents a great challenge to broader clinical application. To provide insight into TIL-based immunotherapy, we studied a successful case of ACT where regression was observed against tumors carrying the hotspot mutation G12D in the KRAS oncogene. Four T cell receptors (TCRs) made up the TIL infusion and recognized two KRAS-G12D neoantigens, a nonamer and a decamer, all restricted by human leukocyte antigen (HLA) C*08:02. Three of them (TCR9a, 9b, and 9c) were nonamer-specific, while one was decamer-specific (TCR10). We show that only mutant G12D but not the wild-type peptides stabilized HLA-C*08:02 due to the formation of a critical anchor salt bridge to HLA-C. Therapeutic TCRs exhibited high affinities, ranging from nanomolar to low micromolar. Intriguingly, TCR binding affinities to HLA-C inversely correlated with their persistence in vivo, suggesting the importance of antigenic affinity in the function of therapeutic T cells. Crystal structures of TCR-HLA-C complexes revealed that TCR9a to 9c recognized G12D nonamer with multiple conserved contacts through shared CDR2ß and CDR3α. This allowed CDR3ß variation to confer different affinities via a variable HLA-C contact, generating an oligoclonal response. TCR10 recognized an induced and distinct G12D decamer conformation. Thus, this successful case of ACT included oligoclonal TCRs of high affinity recognizing distinct conformations of neoantigens. Our study revealed the potential of a structural approach to inform clinical efforts in targeting KRAS-G12D tumors by immunotherapy and has general implications for T cell-based immunotherapies.

HLA-C variants associated with amino acid substitutions in the peptide binding groove influence susceptibility to Kawasaki disease.

Kawasaki disease (KD) is a pediatric vasculitis caused by an unknown trigger in genetically susceptible children. The incidence varies widely across genetically diverse populations. Several associations with HLA Class I alleles have been reported in single cohort studies. Using a genetic approach, from the nine single nucleotide variants (SNVs) associated with KD susceptibility in children of European descent, we identified SNVs near the HLA-C (rs6906846) and HLA-B genes (rs2254556) whose association was replicated in a Japanese descent cohort (rs6906846 p = 0.01, rs2254556 p = 0.005). The risk allele (A at rs6906846) was also associated with HLA-C*07:02 and HLA-C*04:01 in both US multi-ethnic and Japanese cohorts and HLA-C*12:02 only in the Japanese cohort. The risk A-allele was associated with eight non-conservative amino acid substitutions (amino acid positions); Asp or Ser (9), Arg (14), Ala (49), Ala (73), Ala (90), Arg (97), Phe or Ser (99), and Phe or Ser (116) in the HLA-C peptide binding groove that binds peptides for presentation to cytotoxic T cells (CTL). This raises the possibility of increased affinity to a "KD peptide" that contributes to the vasculitis of KD in genetically susceptible children.

HLA-B and HLA-C Differ in Their Nanoscale Organization at Cell Surfaces.

The particular HLA class I variants an individual carries influences their resistance and susceptibility to a multitude of diseases. Expression level and variation in the peptide binding region correlates with, for example, a person's progression to AIDS after HIV infection. One factor which has not yet been addressed is whether or not different HLA class I proteins organize differently in the cell membrane on a nanoscale. Here, we examined the organization of three HLA-B allotypes (B*2705, B*5301, and B*5701) and two HLA-C allotypes (C*0602 and C*0702) in the membrane of 721.221 cells which otherwise lack expression of HLA-B or HLA-C. All these allotypes are ligands for the T cell receptor and leukocyte immunoglobulin-like receptors, but additionally, the HLA-B allotypes are ligands for the killer-cell immunoglobulin-like receptor family member KIR3DL1, HLA-C*0602 is a ligand for KIR2DL1, and HLA-C*0702 is a ligand for KIR2DL2/3. Using super-resolution microscopy, we found that both HLA-B and HLA-C formed more clusters and a greater proportion of HLA contributed to clusters, when expressed at lower levels. Thus, HLA class I organization is a covariate in genetic association studies of HLA class I expression level with disease progression. Surprisingly, we also found that HLA-C was more clustered than HLA-B when expression level was controlled. HLA-C consistently formed larger and more numerous clusters than HLA-B and a greater proportion of HLA-C contributed to clusters than for HLA-B. We also found that the organization of HLA class I proteins varied with cell type. T cells exhibited a particularly clustered organization of HLA class I while B cells expressed a more uniform distribution. In summary, HLA class I variants are organized differently in the cell surface membrane which may impact their functions.

Stable Frequencies of HLA-C*03:04/Peptide-Binding KIR2DL2/3+ Natural Killer Cells Following Vaccination.

Inhibitory KIRs play a central role in regulating NK cell activity. KIR2DL2/3 bind to HLA-C molecules, but the modulation of these interactions by viral infections and presentation of viral epitopes is not well-understood. We investigated whether the frequencies of KIR2DL2/3+ NK cells recognizing HLA-C*03:04/viral peptide complexes were impacted by YFV vaccination or HIV-1 and HCV infection. Ex vivo HLA class I tetramer staining of primary human NK cells derived from YFV-vaccinated individuals, or HIV-1- or HCV-infected individuals revealed that the YFV/HLA-C*03:04-NS2A4-13-tetramer bound to a larger proportion of KIR2DL2/3+ NK cells compared to HIV-1/HLA-C*03:04-Gag296-304- or HCV/HLA-C*03:04-Core136-144-tetramers. The YFV/HLA-C*03:04-NS2A4-13-tetramer also exhibited a stronger avidity to KIR2DL2/3 compared to the other tested tetramers. The proportional frequencies of KIR2DL2/3+ NK cells binding to the three tested HLA-C*03:04 tetramers were identical between YFV-vaccinated individuals or HIV-1- or HCV-infected individuals, and remained stable following YFV vaccination. These data demonstrate consistent hierarchies in the frequency of primary KIR2DL2/3+ NK cells binding HLA-C*03:04/peptide complexes that were determined by the HLA-C-presented peptide and not modulated by the underlying viral infection or vaccination.

HLA Class I Downregulation by HIV-1 Variants from Subtype C Transmission Pairs.

HIV-1 downregulates human leukocyte antigen A (HLA-A) and HLA-B from the surface of infected cells primarily to evade CD8 T cell recognition. HLA-C was thought to remain on the cell surface and bind inhibitory killer immunoglobulin-like receptors, preventing natural killer (NK) cell-mediated suppression. However, a recent study found HIV-1 primary viruses have the capacity to downregulate HLA-C. The goal of this study was to assess the heterogeneity of HLA-A, HLA-B, and HLA-C downregulation among full-length primary viruses from six chronically infected and six newly infected individuals from transmission pairs and to determine whether transmitted/founder variants exhibit common HLA class I downregulation characteristics. We measured HLA-A, HLA-B, HLA-C, and total HLA class I downregulation by flow cytometry of primary CD4 T cells infected with 40 infectious molecular clones. Primary viruses mediated a range of HLA class I downregulation capacities (1.3- to 6.1-fold) which could differ significantly between transmission pairs. Downregulation of HLA-C surface expression on infected cells correlated with susceptibility to in vitro NK cell suppression of virus release. Despite this, transmitted/founder variants did not share a downregulation signature and instead were more similar to the quasispecies of matched donor partners. These data indicate that a range of viral abilities to downregulate HLA-A, HLA-B, and HLA-C exist within and between individuals that can have functional consequences on immune recognition.IMPORTANCE Subtype C HIV-1 is the predominant subtype involved in heterosexual transmission in sub-Saharan Africa. Authentic subtype C viruses that contain natural sequence variations throughout the genome often are not used in experimental systems due to technical constraints and sample availability. In this study, authentic full-length subtype C viruses, including transmitted/founder viruses, were examined for the ability to disrupt surface expression of HLA class I molecules, which are central to both adaptive and innate immune responses to viral infections. We found that the HLA class I downregulation capacity of primary viruses varied, and HLA-C downregulation capacity impacted viral suppression by natural killer cells. Transmitted viruses were not distinct in the capacity for HLA class I downregulation or natural killer cell evasion. These results enrich our understanding of the phenotypic variation existing among natural HIV-1 viruses and how that might impact the ability of the immune system to recognize infected cells in acute and chronic infection.

Stability and Expression Levels of HLA-C on the Cell Membrane Modulate HIV-1 Infectivity.

HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes and the triggering of activating killer immunoglobulin-like receptors on NK cells, whereas lower levels may provide poor HIV-1 control and rapid progression to AIDS. We characterized the relative amounts of HLA-C heterotrimers (heavy chain/ß2 microglobulin [ß2m]/peptide) and HLA-C free heavy chains on peripheral blood mononuclear cells (PBMCs) from healthy blood donors harboring both alleles with stable or unstable binding to ß2m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMCs with various allotypes. We observed significant differences in HLA-C heterotrimer stability and in expression levels. We found that R5 HIV-1 virions produced by PBMCs harboring unstable HLA-C alleles were more infectious than those produced by PBMCs carrying the stable variants. We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low-expression HLA-C alleles and unstable binding to ß2m/peptide might have worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication.IMPORTANCE Following HIV-1 infection, some people advance rapidly to AIDS while others have slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control. Here we reveal how HLA-C variants contribute to the modulation of viral infectivity. HLA-C is present on the cell surface in two different conformations. The immunologically active conformation is part of a complex that includes ß2 microglobulin/peptide; the other conformation is not bound to ß2 microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display stronger immunity to HIV-1, reduced viral infectivity and effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from ß2 microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions. This study provides new information that could be useful in the design of novel vaccine strategies and therapeutic approaches to HIV-1.

The molecular basis for peptide repertoire selection in the human leucocyte antigen (HLA) C*06:02 molecule.

Human leukocyte antigen (HLA)-C*06:02 is identified as the allele associated with the highest risk for the development of the autoimmune skin disease psoriasis. However, the diversity and mode of peptide presentation by the HLA-C*06:02 molecule remains unclear. Here, we describe the endogenous peptide repertoire of ∼3,000 sequences for HLA-C*06:02 that defines the peptide-binding motif for this HLA allomorph. We found that HLA-C*06:02 predominantly presents nonamer peptides with dominant arginine anchors at the P2 and P7 positions and a preference for small hydrophobic residues at the C terminus (PΩ). To determine the structural basis of this selectivity, we determined crystal structures of HLA-C*06:02 in complex with two self-peptides (ARTELYRSL and ARFNDLRFV) and an analogue of a melanocyte autoantigen (ADAMTSL5, VRSRR-abu-LRL) implicated in psoriasis. These structures revealed that HLA-C*06:02 possesses a deep peptide-binding groove comprising two electronegative B- and E-pockets that coincide with the preference for P2 and P7 arginine anchors. The ADAMTSL5 autoantigen possessed a P7-Leu instead of the P7-Arg residue, but nevertheless was accommodated within the HLA-C*06:02 antigen-binding cleft. Collectively, our results provide the structural basis for understanding peptide repertoire selection in HLA-C*06:02.

Characterization of a novel HLA-C*04 allele, HLA-C*04:277.

HLA-C*04:277 has one nucleotide difference from HLA-C*04:01:01:01 at position 1034 (G>A).

KIR2DS5 allotypes that recognize the C2 epitope of HLA-C are common among Africans and absent from Europeans.

INTRODUCTION: KIR2DS5 is an activating human NK cell receptor of lineage III KIR. These include both inhibitory KIR2DL1, 2 and 3 and activating KIR2DS1 that recognize either the C1 or C2 epitope of HLA-C. In Europeans KIR2DS5 is essentially monomorphic, with KIR2DS5*002 being predominant. Pioneering investigations showed that KIR2DS5*002 has activating potential, but cannot recognize HLA-A, -B, or -C. Subsequent studies have shown that KIR2DS5 is highly polymorphic in Africans, and that KIR2DS5*006 protects pregnant Ugandan women from preeclampsia. Because inhibitory C2-specific KIR2DL1 correlates with preeclampsia, whereas activating C2-specific KIR2DS1 protects, this association pointed to KIR2DS5*006 being an activating C2-specific receptor. To test this hypothesis we made KIR-Fc fusion proteins from all ten KIR2DS5 allotypes and tested their binding to a representative set of HLA-A, -B and -C allotypes. RESULTS: Six African-specific KIR2DS5 bound to C2+ HLA-C but not to other HLA class I. Their avidity for C2 is ∼20% that of C2-specific KIR2DL1 and ∼40% that of C2-specific KIR2DS1. Among the African C2 receptors is KIR2DS5*006, which protected a cohort of pregnant Ugandans from pre-eclampsia. Three African KIR2DS5 allotypes and KIR2DS5*002, bound no HLA-A, -B or -C. As a group the C2-binding KIR2DS5 allotypes protect against pre-eclampsia compared to the non-binding KIR2DS5 allotypes. Natural substitutions that contribute to loss or reduction of C2 receptor function are at positions 127, 158, and 176 in the D2 domain. CONCLUSIONS: KIR2DS5*005 has the KIR2DS5 consensus sequence, is the only allele found at both centromeric and telomeric locations of KIR2DS5, and is likely the common ancestor of all KIR2DS5 alleles. That KIR2DS5*005 has C2 receptor activity, points to KIR2DS5*002, and other allotypes lacking C2 receptor function, being products of attenuation, a characteristic feature of most KIR B haplotype genes. Alleles encoding attenuated and active KIR2DS5 are present in both centromeric and telomeric locations.

Structural and regulatory diversity shape HLA-C protein expression levels.

Expression of HLA-C varies widely across individuals in an allele-specific manner. This variation in expression can influence efficacy of the immune response, as shown for infectious and autoimmune diseases. MicroRNA binding partially influences differential HLA-C expression, but the additional contributing factors have remained undetermined. Here we use functional and structural analyses to demonstrate that HLA-C expression is modulated not just at the RNA level, but also at the protein level. Specifically, we show that variation in exons 2 and 3, which encode the α1/α2 domains, drives differential expression of HLA-C allomorphs at the cell surface by influencing the structure of the peptide-binding cleft and the diversity of peptides bound by the HLA-C molecules. Together with a phylogenetic analysis, these results highlight the diversity and long-term balancing selection of regulatory factors that modulate HLA-C expression.

KIR2DL2/2DL3-E(35) alleles are functionally stronger than -Q(35) alleles.

KIR2DL2 and KIR2DL3 segregate as alleles of a single locus in the centromeric motif of the killer cell immunoglobulin-like receptor (KIR) gene family. Although KIR2DL2/L3 polymorphism is known to be associated with many human diseases and is an important factor for donor selection in allogeneic hematopoietic stem cell transplantation, the molecular determinant of functional diversity among various alleles is unclear. In this study we found that KIR2DL2/L3 with glutamic acid at position 35 (E(35)) are functionally stronger than those with glutamine at the same position (Q(35)). Cytotoxicity assay showed that NK cells from HLA-C1 positive donors with KIR2DL2/L3-E(35) could kill more target cells lacking their ligands than NK cells with the weaker -Q(35) alleles, indicating better licensing of KIR2DL2/L3(+) NK cells with the stronger alleles. Molecular modeling analysis reveals that the glutamic acid, which is negatively charged, interacts with positively charged histidine located at position 55, thereby stabilizing KIR2DL2/L3 dimer and reducing entropy loss when KIR2DL2/3 binds to HLA-C ligand. The results of this study will be important for future studies of KIR2DL2/L3-associated diseases as well as for donor selection in allogeneic stem cell transplantation.

A novel allele HLA-C*07:445 identified in a French hematopoietic stem cell donor.

The novel allele HLA-C*07:445 has 1 nucleotide change from HLA-C*07:01 at nucleotide 277 C>A in exon 2.

Allele and Haplotype Frequencies of Human Leukocyte Antigen-A, -B, -C, -DRB1, and -DQB1 From Sequence-Based DNA Typing Data in Koreans.

BACKGROUND: Data on allele frequencies (AFs) and haplotype frequencies (HFs) of HLA-C and -DQB1 are limited in Koreans. We investigated AFs and HFs of HLA-A, -B, -C, -DRB1, and -DQB1 in Koreans by high-resolution sequence-based typing (SBT). METHODS: Hematopoietic stem cells were obtained from 613 healthy, unrelated donors to analyze HLA-A, -B, -C, -DRB1, and -DQB1 genotypes by using AlleleSEQR HLA-A, -B, -C, -DRB1, and -DQB1 SBT kits (Abbott Molecular, USA), respectively. Alleles belonging to HLA-C*07:01/07:06 group were further discriminated by using PCR-sequence specific primer analysis. AFs and HFs were calculated by direct counting and maximum likelihood method, respectively. RESULTS: In all, 24 HLA-A, 46 HLA-B, 24 HLA-C, 29 HLA-DRB1, and 15 HLA-DQB1 alleles were identified. AFs and HFs of HLA-A, -B, and -DRB1 were similar to those reported previously. For the HLA-C locus, C*01:02 was the most common allele, followed by C*03:03, C*03:04, C*14:02, C*03:02, and C*07:02 (AF ≥7%). AFs of C*07:01 and C*07:06 were 0.16% and 3.18%, respectively. For the HLA-DQB1 locus, DQB1*03:01 was the most common allele, followed by DQB1*03:03, *03:02, *06:01, *05:01, *04:01, and *06:02 (AF ≥7%). AFs of DQB1*02:01 and DQB1*02:02 were 2.12% and 6.69%, respectively. HFs of A*33:03-C*07:06 and C*07:06-B*44:03 were 3.09% and 3.10%, respectively, while those of DRB1*07:01-DQB1*02:02 and DRB1*03:01-DQB1*02:01 were 6.61% and 2.04%, respectively. CONCLUSIONS: This study reported AFs and HFs of HLA, including HLA-C and -DQB1, in Koreans by using high-resolution SBT. These data can be used to resolve ambiguous results of HLA typing for organ and hematopoietic stem cell transplantations.

Peptide-induced HLA-E expression in human PBMCs is dependent on peptide sequence and the HLA-E genotype.

Human Leukocyte Antigen (HLA)-E is a low-polymorphic non-classical HLA class I molecule which plays a crucial role in immune surveillance by presentation of peptides to T and natural killer (NK) cells. HLA-E polymorphism is related to HLA-E surface expression and is associated with patient outcome after stem cell transplantation. We aim to investigate the regulation of HLA-E expression level in peripheral blood mononuclear cells (PBMCs) of healthy individuals homozygous for HLA-E*01:01 or HLA-E*01:03, by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. Basal and peptide-induced HLA-E surface expression levels were higher in PBMC from HLA-E*01:03 homozygous subjects as compared to PBMC from HLA-E*01:01 homozygous subjects. HLA-E mRNA levels were comparable between the two genotypes and remained constant after peptide stimulation. HLA-E surface expression seemed to be not only dependent on the HLA-E genotype, but also on the sequence of the peptide as evidenced by the profound difference in HLA-E upregulation with the Hsp60 and the B7 peptide. Our results showed that peptide-induced HLA-E expression is regulated at the posttranscriptional level as extracellular peptide stimulation did not influence RNA expression. This study provides new insights in the mechanism by which HLA-E expression is regulated and underlines a new role for extracellular peptides in inducing HLA-E translation, which may represent a defense mechanism against lytic viral infections and necrosis.

Uncovering the peptide-binding specificities of HLA-C: a general strategy to determine the specificity of any MHC class I molecule.

MHC class I molecules (HLA-I in humans) present peptides derived from endogenous proteins to CTLs. Whereas the peptide-binding specificities of HLA-A and -B molecules have been studied extensively, little is known about HLA-C specificities. Combining a positional scanning combinatorial peptide library approach with a peptide-HLA-I dissociation assay, in this study we present a general strategy to determine the peptide-binding specificity of any MHC class I molecule. We applied this novel strategy to 17 of the most common HLA-C molecules, and for 16 of these we successfully generated matrices representing their peptide-binding motifs. The motifs prominently shared a conserved C-terminal primary anchor with hydrophobic amino acid residues, as well as one or more diverse primary and auxiliary anchors at P1, P2, P3, and/or P7. Matrices were used to generate a large panel of HLA-C-specific peptide-binding data and update our pan-specific NetMHCpan predictor, whose predictive performance was considerably improved with respect to peptide binding to HLA-C. The updated predictor was used to assess the specificities of HLA-C molecules, which were found to cover a more limited sequence space than HLA-A and -B molecules. Assessing the functional significance of these new tools, HLA-C*07:01 transgenic mice were immunized with stable HLA-C*07:01 binders; six of six tested stable peptide binders were immunogenic. Finally, we generated HLA-C tetramers and labeled human CD8(+) T cells and NK cells. These new resources should support future research on the biology of HLA-C molecules. The data are deposited at the Immune Epitope Database, and the updated NetMHCpan predictor is available at the Center for Biological Sequence Analysis and the Immune Epitope Database.

The immunodominant influenza A virus M158-66 cytotoxic T lymphocyte epitope exhibits degenerate class I major histocompatibility complex restriction in humans.

UNLABELLED: Cytotoxic T lymphocytes recognizing conserved peptide epitopes are crucial for protection against influenza A virus (IAV) infection. The CD8 T cell response against the M158-66 (GILGFVFTL) matrix protein epitope is immunodominant when restricted by HLA-A*02, a major histocompatibility complex (MHC) molecule expressed by approximately half of the human population. Here we report that the GILGFVFTL peptide is restricted by multiple HLA-C*08 alleles as well. We observed that M158-66 was able to elicit cytotoxic T lymphocyte (CTL) responses in both HLA-A*02- and HLA-C*08-positive individuals and that GILGFVFTL-specific CTLs in individuals expressing both restriction elements were distinct and not cross-reactive. The crystal structure of GILGFVFTL-HLA-C*08:01 was solved at 1.84 Å, and comparison with the known GILGFVFTL-HLA-A*02:01 structure revealed that the antigen bound both complexes in near-identical conformations, accommodated by binding pockets shaped from shared as well as unique residues. This discovery of degenerate peptide presentation by both HLA-A and HLA-C allelic variants eliciting unique CTL responses to IAV infection contributes fundamental knowledge with important implications for vaccine development strategies. IMPORTANCE: The presentation of influenza A virus peptides to elicit immunity is thought to be narrowly restricted, with a single peptide presented by a specific HLA molecule. In this study, we show that the same influenza A virus peptide can be more broadly presented by both HLA-A and HLA-C molecules. This discovery may help to explain the differences in immunity to influenza A virus between individuals and populations and may also aid in the design of vaccines.

HLA-C antibodies are associated with irreversible rejection in kidney transplantation: Shared molecular eplets characterization.

We report an interesting case concerning an irreversible antibody-mediated rejection (AMR), associated with anti-HLA-C DSA, which occurred after a second kidney transplantation despite having determined a low number of antibodies directed against HLA-C antigens (MFI<1000) in the previous transplantation (which was then considered to be an indicator of low risk of AMR). A 63-year-old woman was re-transplanted with pre-transplant (PrT) sensitization. On day 7 post-transplantation, oligoanuria occurred and increased MFIs for the detected PrT antibodies and other antibodies (non-detected or detected with very low PrT MFI) were observed. SAB assay also showed antibodies against the second donor HLA-C mismatches and other HLA-C antigens. Nephrologists suspected AMR and the patient was therefore treated with methylprednisolone/plasmapheresis/IVIG/anti-CD20 without improvement, which led to transplantectomy. Histologic analysis confirmed acute AMR. Interestingly, it was possible to define exactly the potential immunizing epitopes whose recognition determines the specific antibody production. So, 1st donor DSAs (detected PrT with low MFI), 2nd donor DSAs (detected PTP), and non-DSA detected PTP have several shared eplets, being the 11AVR eplet the only one present on all alleles. Thus, the recognition of 11AVR eplet in the first transplant modeled the patient's antibody response. Therefore, we propose that donor HLA-C typing should always be performed for recipients with anti-HLA-C antibodies, and specific shared-eplets should be investigated in order to determine previous transplant mismatches.