Selective Upregulation of CTLA-4 on CD8+ T Cells Restricted by HLA-B*35Px Renders them to an Exhausted Phenotype in HIV-1 infection.

HLA-B*35Px is associated with HIV-1 disease rapid progression to AIDS. However, the mechanism(s) underlying this deleterious effect of this HLA allele on HIV-1 infection outcome has not fully understood. CD8+ T cells play a crucial role to control the viral replication but impaired CD8+ T cells represent a major hallmark of HIV-1 infection. Here, we examined the effector functions of CD8+ T cells restricted by HLA-B*35Px (HLA-B*35:03 and HLA-B*35:02), HLA-B*27/B57 and non-HLA-B*27/B57 (e.g. HLA-A*01, A*02, A*03, A*11, A*24, A*26, B*40, B*08, B*38, B*44). CD8+ T cells restricted by HLA-B*35Px exhibited an impaired phenotype compared with those restricted by HLA-B*27/B57 and even non-HLA-B*27/B57. CD8+ T cells restricted by non-HLA-B*27/B57 when encountered their cognate epitopes upregulated TIM-3 and thus became suppressed by regulatory T cells (Tregs) via TIM-3: Galectin-9 (Gal-9). Strikingly, CD8+ T cells restricted by HLA-B*35Px expressed fewer TIM-3 and therefore did not get suppressed by Tregs, which was similar to CD8+ T cells restricted by HLA-B*27/B57. Instead, CD8+ T cells restricted by HLA-B*35Px upon recognition of their cognate epitopes upregulated CTLA-4. The transcriptional and impaired phenotype (e.g. poor effector functions) of HIV-specific CD8+ T cells restricted by HLA-B*35 was related to persistent CTLA-4, elevated Eomes and blimp-1 but poor T-bet expression. As such, anti-CTLA-4 antibody, Ipilimumab, reversed the impaired proliferative capacity of antigen-specific CD8+ T cells restricted by HLA-B*35Px but not others. This study supports the concept that CD8+ T resistance to Tregs-mediated suppression is related to allele restriction rather than the epitope specificity. Our results aid to explain a novel mechanism for the inability of HIV-specific CD8+ T cells restricted by HLA-B*35Px to control viral replication.

Platelets from donors with consistently low HLA-B8, -B12, or -B35 expression do not undergo antibody-mediated internalization.

Patients refractory to platelet transfusions because of alloimmunization require HLA-matched platelets, which is only possible if a large HLA-typed donor pool is available. However, even then, patients with broad immunization or rare haplotypes may not have suitable donors. In these patients, transfusions with platelets showing low HLA class I expression may be an alternative to fully HLA-matched transfusions. In this study, we quantified the proportion of donors with consistently low HLA-B8, -B12, and -B35 expression on platelets using human monoclonal antibodies specific for these antigens. Furthermore, as model for in vivo clearance, antibody-mediated internalization of these platelets by macrophages was investigated. The expression of HLA-B8, -B12, or -B35 on platelets was extremely variable between individuals (coefficients of variation, 41.4% to 73.6%). For HLA-B8, but not for HLA-B12 or -B35, this variation was in part explained by zygosity. The variation was most pronounced in, but not exclusive to, platelets. Expression within one donor was consistent over time. Remarkably, 32% of 113 HLA-B8, 34% of 98 HLA-B12, and 9% of 66 HLA-B35 donors showed platelet antigen expression that was not or only minimally above background. Antibody-mediated internalization of platelets by macrophages correlated with antibody opsonization and antigen expression and was absent in platelets with low or minimal HLA expression. In conclusion, our findings indicate that a substantial proportion of donors have platelets with consistently low expression of specific HLA class I antigens. These platelets may be used to treat refractory patients with antibodies directed against these particular antigens, despite HLA mismatches.

Contribution of ancient human remains analysis to the understanding of the variability in HLA-B gene variants in relation to the diagnosis of spondyloarthropathies.

Genetic investigations on ancient human remains affected by rheumatological pathologies are a research field of particular interest for identifying the pathogenesis of diseases, especially those having an autoimmune background such as spondyloarthopaties (SpA). Reliable studies concerning this topic require collaboration between multiple disciplines, usually starting from paleopathologic observations up to molecular genetic screening. Here, we focused our investigation in a medieval necropolis in the Basque Country (13th-15th century, N = 163), which presents a high frequency of joint pathologies through two approaches: on the one hand, the analysis of joint manifestations for the differential diagnosis of the SpA and, on the other hand, the determination of the alleles of the HLA-B gene. The morphological analysis allowed determining that 30% of the individuals had rheumatic bone manifestations, with SpA being the most frequent (45%). The genetic analysis of individuals with and without pathologies, based on the study of the HLA-B gene, allowed finding 17 alleles for this gene, with HLA-B40, HLA-B27 and HLA-B35 being the most frequent. Although these alleles have been traditionally described as genetic markers associated to the development of SpA, in this study they were also found in individuals with other rheumatic diseases (osteoarthritis and rheumatoid arthritis) and even in individuals without pathologies. These data confirm the complexity of the relationship of the HLA-B gene variants with SpA, since it is not possible to establish a diagnosis of SpA with these variants alone. However, we suggest that allele HLA-B40, in combination with some specific rheumatic bone manifestations, facilitates the diagnosis of SpA.

Immunodominant Dengue Virus-Specific CD8+ T Cell Responses Are Associated with a Memory PD-1+ Phenotype.

UNLABELLED: Dengue disease is a large public health problem that mainly afflicts tropical and subtropical regions. Understanding of the correlates of protection against dengue virus (DENV) is poor and hinders the development of a successful human vaccine. The present study aims to define DENV-specific CD8(+)T cell responses in general and those of HLA alleles associated with dominant responses in particular. In human blood donors in Nicaragua, we observed a striking dominance of HLA B-restricted responses in general and of the allele B*35:01 in particular. Comparing these patterns to those in the general population of Sri Lanka, we found a strong correlation between restriction of the HLA allele and the breadth and magnitude of CD8(+)T cell responses, suggesting that HLA genes profoundly influence the nature of responses. The majority of gamma interferon (IFN-γ) responses were associated with effector memory phenotypes, which were also detected in non-B*35:01-expressing T cells. However, only the B*35:01 DENV-specific T cells were associated with marked expression of the programmed death 1 protein (PD-1). These cells did not coexpress other inhibitory receptors and were able to proliferate in response to DENV-specific stimulation. Thus, the expression of particular HLA class I alleles is a defining characteristic influencing the magnitude and breadth of CD8 responses, and a distinct, highly differentiated phenotype is specifically associated with dominant CD8(+)T cells. These results are of relevance for both vaccine design and the identification of robust correlates of protection in natural immunity. IMPORTANCE: Dengue is an increasingly significant public health problem as its mosquito vectors spread over greater areas; no vaccines against the virus have yet been approved. An important step toward vaccine development is defining protective immune responses; toward that end, we here characterize the phenotype of the immunodominant T cell responses. These DENV-reactive T cells express high levels of the receptor programmed death 1 protein (PD-1), while those from disease-susceptible alleles do not. Not only does this represent a possible correlate of immunodominance, but it raises the hypothesis that PD-1 might be a regulator that prevents excessive damage while preserving antiviral function. Further, as this study employs distinct populations (Nicaraguan and Sri Lankan donors), we also confirmed that this pattern holds despite geographic and ethnic differences. This finding indicates that HLA type is the major determinant in shaping T cell responses.

Confirmation and next-generation sequencing of allele HLA-B*35:279 found in a family of a leukaemia patient with Western Asia origin.

The confirmation of novel allele HLA-B*35:279 in a family of a leukaemia patient with Western Asia origin is reported. Moreover, next-generation sequencing (NGS) resulted in whole-gene sequence data and revealed the inheritance of HLA-B*35:279 on the paternal haplotype.

Understanding the obstacle of incompatibility at residue 156 within HLA-B*35 subtypes.

Defining permissive and non-permissive mismatches for transplantation is a demanding challenge. Single mismatches at amino acid (AA) position 156 of human leucocyte antigen (HLA) class I have been described to alter the peptide motif, repertoire, or mode of peptide loading through differential interaction with the peptide-loading complex. Hence, a single mismatch can tip the balance and trigger an immunological reaction. HLA-B*35 subtypes have been described to evade the loading complex, 156 mismatch distinguishing B*35:01 and B*35:08 changes the binding groove sufficiently to alter the sequence features of the selected peptide repertoire. To understand the functional influences of residue 156 in B*35 variants, we analyzed the peptide binding profiles of HLA-B*35:01(156Leu), B*35:08(156Arg) and B*35:62(156Trp). The glycoprotein tapasin represents a target for immune evasions and functions within the multimeric peptide-loading complex to stabilize empty class I molecules and promote acquisition of high-affinity peptides. All three B*35 subtypes showed a tapasin-independent mode of peptide acquisition. HLA-B*35-restricted peptides of low- and high-binding affinities were recovered in the presence and absence of tapasin and subsequently sequenced utilizing mass spectrometry. The peptides derived from B*35 variants differ substantially in their features dependent on their mode of recruitment; all peptides were preferentially anchored by Pro at p2 and Tyr, Phe, Leu, or Lys at pΩ. However, the Trp at residue 156 altered the p2 motif to an Ala and restricted the pΩ to a Trp. Our results highlight the importance of understanding the impact of key micropolymorphism and how a single AA mismatch orchestrates the neighboring AAs.

Design and validation of conditional ligands for HLA-B*08:01, HLA-B*15:01, HLA-B*35:01, and HLA-B*44:05.

We designed conditional ligands restricted to HLA-B*08:01, -B*35:01, and -B*44:05 and proved the use of a conditional ligand previously designed for HLA-B*15:02 together with HLA-B*15:01. Furthermore, we compared the detection capabilities of specific HLA-B*15:01-restricted T cells using the HLA-B*15:01 and HLA-B*15:02 major histocompatibility complex (MHC) multimers and found remarkable differences in the staining patterns detected by flow cytometry. These new conditional ligands greatly add to the application of MHC-based technologies in the analyses of T-cell recognition as they represent frequently expressed HLA-B molecules. This expansion of conditional ligands is important to allow T-cell detection over a wide range of HLA restrictions, and provide comprehensive understanding of the T-cell recognition in a given context.

KIR3DL2 binds to HLA-B27 dimers and free H chains more strongly than other HLA class I and promotes the expansion of T cells in ankylosing spondylitis.

The human leukocyte Ag HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of APC as both classical ß2-microglobulin-associated B27 and B27 free H chain forms (FHC), including disulfide-bonded H chain homodimers (termed B27(2)). B27 FHC forms, but not classical B27, bind to KIR3DL2. HLA-A3, which is not associated with spondyloarthritis (SpA), is also a ligand for KIR3DL2. In this study, we show that B27(2) and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B27(2) tetramers bound KIR3DL2-transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric, and monomeric FHC from HLA-B27-expressing cell lines. Binding to B27(2) and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B27(2) and B27 FHC stimulated KIR3DL2CD3ε-transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFN-γ secretion and promoted greater survival of KIR3DL2(+) CD4 T and NK cells than binding to other HLA-class I. KIR3DL2(+) T cells from B27(+) SpA patients proliferated more in response to Ag presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27(+) SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC.

Highly divergent T-cell receptor binding modes underlie specific recognition of a bulged viral peptide bound to a human leukocyte antigen class I molecule.

Human leukocyte antigen (HLA)-I molecules can present long peptides, yet the mechanisms by which T-cell receptors (TCRs) recognize featured pHLA-I landscapes are unclear. We compared the binding modes of three distinct human TCRs, CA5, SB27, and SB47, complexed with a "super-bulged" viral peptide (LPEPLPQGQLTAY) restricted by HLA-B*35:08. The CA5 and SB27 TCRs engaged HLA-B*35:08(LPEP) similarly, straddling the central region of the peptide but making limited contacts with HLA-B*35:08. Remarkably, the CA5 TCR did not contact the α1-helix of HLA-B*35:08. Differences in the CDR3ß loop between the CA5 and SB27 TCRs caused altered fine specificities. Surprisingly, the SB47 TCR engaged HLA-B*35:08(LPEP) using a completely distinct binding mechanism, namely "bypassing" the bulged peptide and making extensive contacts with the extreme N-terminal end of HLA-B*35:08. This docking footprint included HLA-I residues not observed previously as TCR contact sites. The three TCRs exhibited differing patterns of alloreactivity toward closely related or distinct HLA-I allotypes. Thus, the human T-cell repertoire comprises a range of TCRs that can interact with "bulged" pHLA-I epitopes using unpredictable strategies, including the adoption of atypical footprints on the MHC-I.

Molecular modelling of HLA-B*35:132.

Here we describe the molecular modelling of the new variant HLA-B*35:132. This allele shows one mismatch with B*35:01:01:01 in exon 3 at position 575 where a T is substituted by a C, which implies an amino acidic change from Leucine to Proline. This seems not to alter the molecular structure and not to compromise the HLA complex and T-cell receptor interaction.

Differential clade-specific HLA-B*3501 association with HIV-1 disease outcome is linked to immunogenicity of a single Gag epitope.

The strongest genetic influence on immune control in HIV-1 infection is the HLA class I genotype. Rapid disease progression in B-clade infection has been linked to HLA-B*35 expression, in particular to the less common HLA-B*3502 and HLA-B*3503 subtypes but also to the most prevalent subtype, HLA-B*3501. In these studies we first demonstrated that whereas HLA-B*3501 is associated with a high viral set point in two further B-clade-infected cohorts, in Japan and Mexico, this association does not hold in two large C-clade-infected African cohorts. We tested the hypothesis that clade-specific differences in HLA associations with disease outcomes may be related to distinct targeting of critical CD8(+) T-cell epitopes. We observed that only one epitope was significantly targeted differentially, namely, the Gag-specific epitope NPPIPVGDIY (NY10, Gag positions 253 to 262) (P = 2 × 10(-5)). In common with two other HLA-B*3501-restricted epitopes, in Gag and Nef, that were not targeted differentially, a response toward NY10 was associated with a significantly lower viral set point. Nonimmunogenicity of NY10 in B-clade-infected subjects derives from the Gag-D260E polymorphism present in ∼90% of B-clade sequences, which critically reduces recognition of the Gag NY10 epitope. These data suggest that in spite of any inherent HLA-linked T-cell receptor repertoire differences that may exist, maximizing the breadth of the Gag-specific CD8(+) T-cell response, by the addition of even a single epitope, may be of overriding importance in achieving immune control of HIV infection. This distinction is of direct relevance to development of vaccines designed to optimize the anti-HIV CD8(+) T-cell response in all individuals, irrespective of HLA type.

Survivin-specific T-cell reactivity correlates with tumor response and patient survival: a phase-II peptide vaccination trial in metastatic melanoma.

BACKGROUND: Therapeutic vaccination directed to induce an anti-tumoral T-cell response is a field of extensive investigation in the treatment of melanoma. However, many vaccination trials in melanoma failed to demonstrate a correlation between the vaccine-specific immune response and therapy outcome. This has been mainly attributed to immune escape by antigen loss, rendering us in the need of new vaccination targets. PATIENTS AND METHODS: This phase-II trial investigated a peptide vaccination against survivin, an oncogenic inhibitor-of-apoptosis protein crucial for the survival of tumor cells, in HLA-A1/-A2/-B35-positive patients with treatment-refractory stage-IV metastatic melanoma. The study endpoints were survivin-specific T-cell reactivity (SSTR), safety, response, and survival (OS). RESULTS: Sixty-one patients (ITT) received vaccination therapy using three different regimens. 55 patients (PP) were evaluable for response and survival, and 41/55 for SSTR. Patients achieving progression arrest (CR + PR + SD) more often showed SSTRs than patients with disease progression (p = 0.0008). Patients presenting SSTRs revealed a prolonged OS (median 19.6 vs. 8.6 months; p = 0.0077); multivariate analysis demonstrated SSTR as an independent predictor of survival (p = 0.013). The induction of SSTRs was associated with gender (female vs. male; p = 0.014) and disease stage (M1a/b vs. M1c; p = 0.010), but not with patient age, HLA type, performance status, or vaccination regimen. CONCLUSION: Survivin-specific T-cell reactivities strongly correlate with tumor response and patient survival, indicating that vaccination with survivin-derived peptides is a promising treatment strategy in melanoma.

Cell type-specific proteasomal processing of HIV-1 Gag-p24 results in an altered epitope repertoire.

Proteasomes are critical for the processing of antigens for presentation through the major histocompatibility complex (MHC) class I pathway. HIV-1 Gag protein is a component of several experimental HIV-1 vaccines. Therefore, understanding the processing of HIV-1 Gag protein and the resulting epitope repertoire is essential. Purified proteasomes from mature dendritic cells (DC) and activated CD4(+) T cells from the same volunteer were used to cleave full-length Gag-p24 protein, and the resulting peptide fragments were identified by mass spectrometry. Distinct proteasomal degradation patterns and peptide fragments were unique to either mature DC or activated CD4(+) T cells. Almost half of the peptides generated were cell type specific. Two additional differences were observed in the peptides identified from the two cell types. These were in the HLA-B35-Px epitope and the HLA-B27-KK10 epitope. These epitopes have been linked to HIV-1 disease progression. Our results suggest that the source of generation of precursor MHC class I epitopes may be a critical factor for the induction of relevant epitope-specific cytotoxic T cells.

HLA-B35 correlates with a favorable outcome following adjuvant administration of an HLA-matched allogeneic melanoma vaccine.

This work presents survival data of 42 melanoma patients at high risk for disease recurrence who received an allogeneic melanoma vaccine composed of three cell lines, each matching at least one allele of the recipient's human leukocyte antigen (HLA)-A and -B loci. The 5-year overall survival (OS) rate and disease-free survival (DFS) compared favorably with the standard interferon-α regimen. Interestingly, patients bearing HLA-B35 had significantly better OS and DFS (OS of 100% and DFS of 90% for HLA-B35 vs 56% and 23%, for the non-B35 patients). In contrast, patients expressing HLA-B07 did not fare well with the vaccine. Although the data include a relatively small cohort of patients, it strongly hints toward a correlation between HLA types and potential benefit from anticancer immunotherapy.

Hepatitis C virus replication in Caucasian HIV controllers.

Whether HIV controllers, patients who spontaneously control HIV viraemia, are able to control hepatitis C virus (HCV) infection, in terms of spontaneous clearance or lower HCV replication, is not well understood. To assess to what extent Caucasian HIV controllers are able to control HCV replication and potential associated factors, plasma HIV-1 and HCV RNA levels, anti-HCV antibodies, HCV genotype and human leucocyte antigens (HLA) typing were determined in samples from 75 HIV controllers (33 viraemic controllers, <1000 HIV-1 RNA copies/mL, and 42 elite controllers, <40 HIV-1 RNA copies/mL) and compared with 261 HIV-infected noncontrollers. We did not find differences in the HCV spontaneous clearance rates between groups. However, we interestingly found a lower HCV viral load in HIV controllers, alongside a different distribution of HCV genotypes in relation to the comparison group. In addition, HLA-B57 was associated with a lower HCV viral load in the control group and HIV controllers, and conversely, HLA-B35 with higher HCV viral load in HIV controllers. The subrepresentation of HCV genotype 1 and the overrepresentation of HLA-B57 only partly explained the lower HCV viral load found in HIV controllers. In fact, HIV controller status was independently associated with lower HCV viral load, together with HCV genotype non-1, the presence of HLA-B57 and absence of HLA-B35. Caucasian HIV controllers are able to better control HCV replication, in terms of lower HCV viral load levels. These findings support the idea that some common host mechanisms are involved in the defence against these two persistent infections.

HIV-specific cytotoxic T-cells in HIV-exposed but uninfected Gambian women.

A crucial requirement in the rational design of a prophylactic vaccine against the human immunodeficiency virus (HIV) is to establish whether or not protective immunity can occur following natural infection. The immune response to HIV infection is characterized by very vigorous HIV-specific cytotoxic T-lymphocyte (CTL) activity. We have identified four HIV-1 and HIV-2 cross-reactive peptide epitopes, presented to CTL from HIV-infected Gambians by HLA-B35 (the most common Gambian class I HLA molecule). These peptides were used to elicit HIV-specific CTLs from three out of six repeatedly exposed but HIV-seronegative female prostitutes with HLA-B35. These women remain seronegative with no evidence of HIV infection by polymerase chain reaction or viral culture. Their CTL activity may represent protective immunity against HIV infection.

PIP: A crucial requirement in the rational design of a prophylactic vaccine against HIV is to establish whether or not protective immunity can occur following natural infection. The immune response to HIV infection is characterized by very vigorous HIV-specific cytotoxic T-lymphocyte (CTL) activity. Four HIV-1 and HIV-2 cross-reactive peptide epitopes were identified, presented to CTL from HIV-infected Gambian women by HLA-B35 (the most common Gambian class 1 HLA molecule). The study population consisted of 20 women: 14 had been prostitutes for more than 5 years and reported little condom usage and 6 were long-term sexual partners of HIV-infected men. Peptide-stimulated cultures were also set up from 8 known seropositive donors with HLA-B35 or B53, and from a control group of volunteers at low-risk of HIV infection with HLA-B35 (12 Gambian and 7 European) and 2 Gambians with HLA-B53. Specific CTL activity against one or more peptides was repeatedly detected after 10-14 days in the peptide-stimulated cultures from 3 of the 6 high-risk seronegative women with HLA-B35, but not in their three counterparts with HLA-B53 nor in any of the low-risk volunteers. The strongest responses were generated toward the HIV-1 pol peptide, which lies close to the active site of reverse transcriptase, and to the nef peptide, which is conserved between HIV-1 and -2. HIV-specific CTL in seronegative subjects could potentially be a response to acute HIV infection, before the development of antibodies, but the women were still seronegative and virus-culture negative 3 months after the CTL were first detected, making recent infection extremely unlikely. These women remain seronegative with no evidence of HIV infection by polymerase chain reaction or viral culture. Their CTL activity may represent protective immunity against HIV infection.

Polymorphism of human minor histocompatibility antigens: T cell recognition of human minor histocompatibility peptides presented by HLA-B35 subtype molecules.

To investigate the polymorphism of human minor histocompatibility (mH) antigens, PBLs from 23 Japanese individuals and 25 German individuals with HLA-B35 were studied by using four human mH antigen-specific, HLA-B35-restricted CTL clones. The CTL clones killed PHA-stimulated PBLs from all 23 Japanese individuals. On the other hand, they killed the PHA-stimulated PBLs from 19 of 25 German individuals and partially killed the PHA-stimulated PBLs from three German individuals (CTL weakly sensitive cell line); those from another three individuals (CTL-resistant cell line) were not killed by the CTL clones. All of three CTL weakly sensitive cell lines carry HLA-B*3503 molecules, whereas the three CTL-resistant cell lines carry HLA-B*3502, B*3507, and B*3508 molecules. The cytotoxicity of the CTL clones for three CTL weakly sensitive cell lines was enhanced by stimulation of human mH peptides isolated from HLA-B*3501 molecules purified from C1R-B*3501 cells. Small amounts of human mH peptides were isolated from B*3503 molecules purified from these three CTL weakly sensitive cell lines. Taken together, these results indicate that weak recognition by the CTL clones of three CTL weakly sensitive cell line results from a small amount of the human mH peptides presented by B*3503 molecules. The CTL-resistant cell line carrying B*3507 loaded with the human mH peptides was killed by four CTL clones, whereas the cell lines carrying B*3502 or B*3508 loaded with the peptides were not. The human mH peptides were not isolated from B*3507 molecules purified from the cell lines expressing this subtype, whereas small amounts of the human mH peptides were isolated from B*3502 and B*3508 molecules purified from the cell lines expressing the subtypes. These results indicate that failure of the CTL recognition of the cell line carrying B*3507 is due to a lack of human mH antigens in this cell line. The failure of the CTL recognition of the cell lines carrying B*3502 and B*3508 is not explained by only the amount of the human mH peptides binding to these B35 subtype molecules because the amount of the human mH peptides eluted from B*3502 and B*3508 molecules purified from the cell lines carrying these B35 subtypes is almost the same as that eluted from B*3503 molecules purified from the cell lines carrying B*3503.(ABSTRACT TRUNCATED AT 400 WORDS)

A single nine-amino acid peptide induces virus-specific, CD8+ human cytotoxic T lymphocyte clones of heterogeneous serotype specificities.

It is generally accepted that virus-specific CD8+ cytotoxic T lymphocytes (CTLs) recognize nine-amino acid peptides in conjunction with HLA class I molecules. We recently reported that dengue virus-specific CD8+ CTLs of two different serotype specificities, which were established by stimulation with dengue virus, recognize a single nine-amino acid peptide of the nonstructural protein NS3 of dengue virus type 4 (D4V) in an HLA-B35-restricted fashion. To further analyze the relationships between the serotype specificities of T cells and the amino acid sequence of the recognized peptides, we examined the ability of this viral peptide D4.NS3.500-508 (TPEGIIPTL) to stimulate T lymphocytes of an HLA-B35-positive, dengue virus type 4-immune donor. Peptide stimulation of the PBMC generated dengue virus-specific, HLA-B-35-restricted CD8+ CTL clones. These clones lysed dengue virus-infected autologous cells, as well as autologous target cells pulsed with this peptide. Four patterns of dengue virus serotype specificities were demonstrated on target cells infected with dengue-vaccinia recombinant viruses or pulsed with synthetic peptides corresponding to amino acid sequences of four dengue virus serotypes. Two serotype-specific clones recognized only D4V. Three dengue virus subcomplex-specific clones recognized D1V, D3V, and D4V, and one subcomplex-specific clone recognized D2V and D4V. Three dengue virus serotype-cross-reactive clones recognized D1V-D4V. Thus, a single nine-amino acid peptide induces proliferation of a heterogeneous panel of dengue virus-specific CD8+ CTL clones that are all restricted by HLA-B35 but have a variety of serotype specificities. Peptides that contain a single amino acid substitution at each position of D4.NS3.500-508 were recognized differently by the T cell clones. These results indicate that a single epitope can be recognized by multiple CD8+ CTLs that have a variety of serotype specificities, but the manner of recognition by these multiple CTLs is heterogeneous.

In vivo expansion of HLA-B35 alloreactive T cells sharing homologous T cell receptors: evidence for maintenance of an oligoclonally dominated allospecificity by persistent stimulation with an autologous MHC/peptide complex.

The nature of alloantigens seen by T lymphocytes, in particular the role of peptides in allorecognition, has been studied intensively whereas knowledge about the in vivo emergence, diversity, and the structural basis of specificity of alloreactive T cells is very limited. Here we describe human T cell clones that recognize HLA-B35 alloantigens in a peptide-dependent manner. TCR sequence analysis revealed that several of these allospecific clones utilize homologous TCR: they all express TCRAV2S3J36C1 and TCRBV4S1J2S7C2 chains with highly related CDR3 sequences. Thus peptide-specific alloreactivity is reflected in homologous CDR3 sequences in a manner similar to that described for T cells that recognize nominal peptide/self-MHC complexes. The in vivo frequency of this TCR specificity was studied in unstimulated PBL of the responding cell donor who was not sensitized against HLA-B35. The vast majority (approximately 75%) of the VA2S3J36 junctional regions obtained from two samples of PBL, isolated at a 9-yr interval, encode CDR3 identical or homologous to those of the functionally characterized HLA-B35 allospecific T cells. These data are most easily explained by a model of alloreactivity in which persistent or recurrent exposure to a foreign peptide/self-MHC complex led to the in vivo expansion and long-term maintenance of specific T cells that show fortuitous crossrecognition of an HLA-B35/peptide complex and dominate the alloresponse against HLA-B35.

Design, expression, and processing of epitomized hepatitis C virus-encoded CTL epitopes.

Hepatitis C virus (HCV) vaccine efficacy may crucially depend on immunogen length and coverage of viral sequence diversity. However, covering a considerable proportion of the circulating viral sequence variants would likely require long immunogens, which for the conserved portions of the viral genome, would contain unnecessarily redundant sequence information. In this study, we present the design and in vitro performance analysis of a novel "epitome" approach that compresses frequent immune targets of the cellular immune response against HCV into a shorter immunogen sequence. Compression of immunological information is achieved by partial overlapping shared sequence motifs between individual epitopes. At the same time, sequence diversity coverage is provided by taking advantage of emerging cross-reactivity patterns among epitope variants so that epitope variants associated with the broadest variant cross-recognition are preferentially included. The processing and presentation analysis of specific epitopes included in such a compressed, in vitro-expressed HCV epitome indicated effective processing of a majority of tested epitopes, although re-presentation of some epitopes may require refined sequence design. Together, the present study establishes the epitome approach as a potential powerful tool for vaccine immunogen design, especially suitable for the induction of cellular immune responses against highly variable pathogens.