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.

Collaboration of a Detrimental HLA-B*35:01 Allele with HLA-A*24:02 in Coevolution of HIV-1 with T Cells Leading to Poorer Clinical Outcomes.

Although mutant-specific T cells are elicited in some individuals infected with HIV-1 mutant viruses, the detailed characteristics of these T cells remain unknown. A recent study showed that the accumulation of strains expressing Nef135F, which were selected by HLA-A*24:02-restricted T cells, was associated with poor outcomes in individuals with the detrimental HLA-B*35:01 allele and that HLA-B*35:01-restricted NefYF9 (Nef135-143)-specific T cells failed to recognize target cells infected with Nef135F mutant viruses. Here, we investigated HLA-B*35:01-restricted T cells specific for the NefFF9 epitope incorporating the Nef135F mutation. Longitudinal T-cell receptor (TCR) clonotype analysis demonstrated that 3 types of HLA-B*35:01-restricted T cells (wild-type [WT] specific, mutant specific, and cross-reactive) with different T cell repertoires were elicited during the clinical course. HLA-B*35:01+ individuals possessing wild-type-specific T cells had a significantly lower plasma viral load (pVL) than those with mutant-specific and/or cross-reactive T cells, even though the latter T cells effectively recognized the mutant virus-infected cells. These results suggest that mutant-specific and cross-reactive T cells could only partially suppress HIV-1 replication in vivo. An ex vivo analysis of the T cells showed higher expression of PD-1 on cross-reactive T cells and lower expression of CD160/2B4 on the mutant-specific T cells than other T cells, implying that these inhibitory and stimulatory molecules are key to the reduced function of these T cells. In the present study, we demonstrate that mutant-specific and cross-reactive T cells do not contribute to the suppression of HIV-1 replication in HIV-1-infected individuals, even though they have the capacity to recognize mutant virus-infected cells. Thus, the collaboration of HLA-A*24:02 with the detrimental allele HLA-B*35:01 resulted in the coevolution of HIV-1 alongside virus-specific T cells, leading to poorer clinical outcomes. IMPORTANCE HIV-1 escape mutations are selected under pressure from HIV-1-specific CD8+ T cells. Accumulation of these mutations in circulating viruses impairs the control of HIV-1 by HIV-1-specific T cells. Although it is known that HIV-1-specific T cells recognizing mutant virus were elicited in some individuals infected with a mutant virus, the role of these T cells remains unclear. Accumulation of phenylalanine at HIV-1 Nef135 (Nef135F), which is selected by HLA-A*24:02-restricted T cells, led to poor clinical outcome in individuals carrying the detrimental HLA-B*35:01 allele. In the present study, we found that HLA-B*35:01-restricted mutant-specific and cross-reactive T cells were elicited in HLA-B*35:01+ individuals infected with the Nef135F mutant virus. These T cells could not effectively suppress HIV-1 replication in vivo even though they could recognize mutant virus-infected cells in vitro. Mutant-specific and cross-reactive T cells expressed lower levels of stimulatory molecules and higher levels of inhibitory molecules, respectively, suggesting a potential mechanism whereby these T cells fail to suppress HIV-1 replication in HIV-1-infected individuals.

Can Glycosylation Mask the Detection of MHC Expressing p53 Peptides by T Cell Receptors?

Proteins of the major histocompatibility complex (MHC) class I, or human leukocyte antigen (HLA) in humans interact with endogenous peptides and present them to T cell receptors (TCR), which in turn tune the immune system to recognize and discriminate between self and foreign (non-self) peptides. Of especial importance are peptides derived from tumor-associated antigens. T cells recognizing these peptides are found in cancer patients, but not in cancer-free individuals. What stimulates this recognition, which is vital for the success of checkpoint based therapy? A peptide derived from the protein p53 (residues 161-169 or p161) was reported to show this behavior. T cells recognizing this unmodified peptide could be further stimulated in vitro to create effective cancer killing CTLs (cytotoxic T lymphocytes). We hypothesize that the underlying difference may arise from post-translational glycosylation of p161 in normal individuals, likely masking it against recognition by TCR. Defects in glycosylation in cancer cells may allow the presentation of the native peptide. We investigate the structural consequences of such peptide glycosylation by investigating the associated structural dynamics.

Identification of an HLA-A*24:02-restricted α-fetoprotein signal peptide-derived antigen and its specific T-cell receptor for T-cell immunotherapy.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer with limited treatments. Asia has the highest HCC incidence rates; China accounts for over 50% of all HCC cases worldwide. T-cell receptor (TCR) -engineered T-cell immunotherapies specific for human leukocyte antigen (HLA) -A*02:01-restricted α-fetoprotein (AFP) peptide have shown encouraging results in clinics. HLA-A*24:02 is more common than HLA-A*02:01 in Asian countries, including China. Here we identified a novel HLA-A*24:02-restricted peptide KWVESIFLIF (AFP2-11 ) located in AFP signal peptide domain by mass spectrometric analysis of HLA-bound peptides from HepG2 cells. A TCR (KWV3.1) specific for AFP2-11 -HLA-A*24:02 was isolated from peripheral blood mononuclear cells of a healthy donor. The binding affinity of soluble KWV3.1 to its antigen was determined to be ~55 µm, within the affinity range of native TCRs for self-antigens. KWV3.1-transfected T cells could specifically activate and kill AFP2-11 pulsed T2-A24 cells and AFP+  HLA-A*24:02+ tumor cell lines, demonstrating that AFP2-11 can be naturally presented on the surface of AFP+ tumor cell lines. The newly identified antigenic peptide can provide a novel target for immunotherapeutic strategies for patients with AFP+  HLA-A*24:02+ HCC.

Distinct Polymorphisms in HLA Class I Molecules Govern Their Susceptibility to Peptide Editing by TAPBPR.

Understanding how peptide selection is controlled on different major histocompatibility complex class I (MHC I) molecules is pivotal for determining how variations in these proteins influence our predisposition to infectious diseases, cancer, and autoinflammatory conditions. Although the intracellular chaperone TAPBPR edits MHC I peptides, it is unclear which allotypes are subjected to TAPBPR-mediated peptide editing. Here, we examine the ability of 97 different human leukocyte antigen (HLA) class I allotypes to interact with TAPBPR. We reveal a striking preference of TAPBPR for HLA-A, particularly for supertypes A2 and A24, over HLA-B and -C molecules. We demonstrate that the increased propensity of these HLA-A molecules to undergo TAPBPR-mediated peptide editing is determined by molecular features of the HLA-A F pocket, specifically residues H114 and Y116. This work reveals that specific polymorphisms in MHC I strongly influence their susceptibility to chaperone-mediated peptide editing, which may play a significant role in disease predisposition.

Impact of a single HLA-A*24:02-associated escape mutation on the detrimental effect of HLA-B*35:01 in HIV-1 control.

BACKGROUND: HLA-B*35 is an HLA allele associated with rapid progression to AIDS. However, a mechanism underlying the detrimental effect of HLA-B*35 on disease outcome remains unknown. Recent studies demonstrated that most prevalent subtype HLA-B*35:01 is a detrimental allele in HIV-1 clade B-infected individuals. We here investigated the effect of mutations within the epitopes on HLA-B*35:01-restricted CD8+ T cells having abilities to suppress HIV-1 replication. METHODS: We analyzed 16 HLA-B*35:01-restricted epitope-specific T cells in 63 HIV-1 clade B-infected Japanese B*35:01+ individuals and identified HLA-B*35:01-restricted CD8+ T cells having abilities to suppress HIV-1 replication. We further analyzed the effect of HLA-associated mutations on the ability of these T cells. FINDINGS: The breadth of T cell responses to 4 epitopes was inversely associated with plasma viral load (pVL). However, the accumulation of an Y135F mutation in NefYF9 out of the 4 epitopes, which is selected by HLA-A*24:02-restricted T cells, affected the ability of YF9-specific T cells to suppress HIV-1 replication. HLA-B*35:01+ individuals harboring this mutation had much higher pVL than those without it. YF9-specific T cells failed to suppress replication of the Y135F mutant in vitro. These results indicate that this mutation impairs suppression of HIV-1 replication by YF9-specific T cells. INTERPRETATION: These findings indicate that the Y135F mutation is a key factor underlying the detrimental effect of HLA-B*35:01 on disease outcomes in HIV-1 clade B-infected individuals. FUND: Grants-in-aid for AIDS Research from AMED and for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan.

Detection of a novel allele, HLA-A*24:198, by sequence-based typing in a Chinese individual.

HLA-A*24:198 has one nucleotide change from HLA-A*24:02:01 where aspartic acid (29) is changed to asparagine.

Predictive biomarkers for the efficacy of peptide vaccine treatment: based on the results of a phase II study on advanced pancreatic cancer.

BACKGROUND: The purpose of the present study was to explore novel biomarkers that can predict the clinical outcome of patients before treatment or during vaccination. These would be useful for the selection of appropriate patients who would be expected to exhibit better treatment outcomes from vaccination, and for facilitating the development of cancer vaccine treatments. METHODS: From a single-arm, non-randomized, human leukocyte antigen (HLA)-A-status-blind phase II trial of a vaccine treatment using three HLA-A*2402-restricted peptides for advanced pancreatic cancer (PC), we obtained peripheral blood samples from 36 patients of an HLA-A*2402-matched group and 27 patients of an HLA-A*2402-unmatched group. RESULTS: Multivariate analysis (HR = 2.546; 95% CI = 1.138 to 5.765; p = 0.0231) and log-rank test (p = 0.0036) showed that a high expression level of programmed death-1 (PD-1) on CD4+ T cells was a negative predictive biomarker of overall survival in the HLA-A*2402-matched group . Moreover, a high expression level of PD-1 on CD4+ T cells was a negative predictor for the induction of cytotoxic T lymphocytes (p = 0.0007). After treatment, we found that the upregulation of PD-1 and T cell immunoglobulin mucin-3 (Tim-3) expression on CD4+ and CD8+ T cells was significantly associated with a poor clinical outcome in the HLA-A*2402-matched group (p = 0.0330, 0.0282, 0.0046, and 0.0068, respectively). In contrast, there was no significant difference for these factors in the HLA-A*2402-unmatched group. CONCLUSIONS: Our results indicate that the upregulation of PD-1 and Tim-3 expression on CD4+ and CD8+ T cells may restrict T cell responses in advanced PC patients; therefore, combination immunotherapy with blockade of PD-1 and Tim-3 to restore T cell responses may be a potential therapeutic approach for advanced PC patients. TRIAL REGISTRATION: Clinical-Trail-Registration: UMIN000008082 .

Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions.

The αß T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D Kd of ∼5,000 molecules/µm(2) This value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination.

Superimposed epitopes restricted by the same HLA molecule drive distinct HIV-specific CD8+ T cell repertoires.

Superimposed epitopes, in which a shorter epitope is embedded within a longer one, can be presented by the same HLA class I molecule. CD8(+) CTL responses against such epitopes and the contribution of this phenomenon to immune control are poorly characterized. In this study, we examined HLA-A*24:02-restricted CTLs specific for the superimposed HIV Nef epitopes RYPLTFGWCF (RF10) and RYPLTFGW (RW8). Unexpectedly, RF10-specific and RW8-specific CTLs from HIV-1-infected HLA-A*24:02+ individuals had no overlapping Ag reactivity or clonotypic compositions. Single-cell TCR sequence analyses demonstrated that RF10-specific T cells had a more diverse TCR repertoire than did RW8-specific T cells. Furthermore, RF10-specific CTLs presented a higher Ag sensitivity and HIV suppressive capacity compared with RW8-specific CTLs. Crystallographic analyses revealed important structural differences between RF10- and RW8-HLA-A*24:02 complexes as well, with featured and featureless conformations, respectively, providing an explanation for the induction of distinct T cell responses against these epitopes. The present study shows that a single viral sequence containing superimposed epitopes restricted by the same HLA molecule could elicit distinct CD8+ T cell responses, therefore enhancing the control of HIV replication. This study also showed that a featured epitope (e.g., RF10) could drive the induction of T cells with high TCR diversity and affinity.

Structure of TCR and antigen complexes at an immunodominant CTL epitope in HIV-1 infection.

We investigated the crystal structure of an HLA-A*2402-restricted CTL epitope in the HIV-1 nef gene (Nef134-10) before (pHLA) or after TCR docking. The wild type epitope and two escape mutants were included in the study. Y135F was an early-appearing major mutation, while F139L was a late-appearing mutation which was selected in the patients without Y135F. F139 was an eminent feature of the Nef134-10 epitope. Wild type-specific TCR was less fit to F139L mutant suggesting that F139L is an escape from the CTL against the wild type epitope. Although Y135F mutation disrupted the hydrogen bond to HLA-A*2402 His70, newly formed hydrogen bond between T138 and His70 kept the conformation of the epitope in the reconstituted pMHC. TCR from Y135F- or dually-specific CTL had unique mode of binding to the mutant epitope. Y135F has been reported as a processing mutant but CTL carrying structurally adequate TCR can be found in the patients.

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

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

Identification of HLA-A24-restricted novel T Cell epitope peptides derived from P-cadherin and kinesin family member 20A.

We here identified human leukocyte antigen-(HLA-)A(∗)2402-restricted epitope peptides from Cadherin 3, type 1, P-cadherin (CDH3) and kinesin family member 20A (KIF20A) that were found to be specifically expressed in cancer cells through genome-wide expression profile analysis. CDH3-10-807 peptide and KIF20A-10-66 peptide successfully induced specific CTL clones, and these selectively responded to COS7 cells expressing both HLA-A(∗)2402 and respective protein while did not respond to parental cells or COS7 cells expressing either HLA-A(∗)2402 or respective protein. Furthermore, CTL clones responded to cancer cells that endogenously express HLA-A(∗)2402 and respective protein, suggesting that CDH3-10-807 peptide and KIF20A-10-66 peptide are naturally presented on HLA-A(∗)2402 molecule of human cancer cells. Our results demonstrated that CDH3-10-807 peptide and KIF20A-10-66 peptide are novel HLA-A24-restricted tumor-associated antigens and would be applicable for CTL-inducing cancer therapies.

[Two conformations of pHLA-A*2402: a supplement to Wolynes' theory].

Wolynes argued that the track of a protein's folding was directed by the tendency of lowering its energy, and thus when a local minimum of its energy was reached, a relatively stable conformation was formed. However not all of the local minimums will lead the protein to a biologically useful conformation, for those otherwise are called energy traps. Wolynes energy landscape theory and natural selection have well explained the high efficiency of protein folding in vivo, instead of being stuck in energy traps. As to whether a protein can assume different conformations with the same bioactivity, there is no clear answer yet. In this paper, two conformational states of a pHLA-A*2402 are discovered after refolding, and by studying their interactions with TCR and CD8alphaalpha, two conformations of pHLA-A*2402 are confirmed of having escaped from natural selection.