Allopurinol hypersensitivity: investigating the cause and minimizing the risk.

Allopurinol is the most commonly prescribed urate-lowering therapy for the management of gout. Serious adverse reactions associated with allopurinol, while rare, are feared owing to the high mortality. Such reactions can manifest as a rash combined with eosinophilia, leukocytosis, fever, hepatitis and progressive kidney failure. Risk factors for allopurinol-related severe adverse reactions include the recent introduction of allopurinol, the presence of the HLA-B(*)58:01 allele, and factors that influence the drug concentration. The interactions between allopurinol, its metabolite, oxypurinol, and T cells have been studied, and evidence exists that the presence of the HLA-B(*)58:01 allele and a high concentration of oxypurinol function synergistically to increase the number of potentially immunogenic-peptide-oxypurinol-HLA-B(*)58:01 complexes on the cell surface, thereby increasing the risk of T-cell sensitization and a subsequent adverse reaction. This Review will discuss the above issues and place this in the clinical context of reducing the risk of serious adverse reactions.

Synergism of tapasin and human leukocyte antigens in resolving hepatitis C virus infection.

UNLABELLED: CD8+ T-cell responses to hepatitis C virus (HCV) are important in generating a successful immune response and spontaneously clearing infection. Human leukocyte antigen (HLA) class I presents viral peptides to CD8+ T cells to permit detection of infected cells, and tapasin is an important component of the peptide loading complex for HLA class I. We sought to determine if tapasin polymorphisms affected the outcome of HCV infection. Patients with resolved or chronic HCV infection were genotyped for the known G/C coding polymorphism in exon 4 of the tapasin gene. In a European, but not a US, Caucasian population, the tapasin G allele was significantly associated with the outcome of HCV infection, being found in 82.5% of resolvers versus 71.3% of persistently infected individuals (P = 0.02, odds ratio [OR] = 1.90 95% confidence interval [CI] = 1.11-3.23). This was more marked at the HLA-B locus at which heterozygosity of both tapasin and HLA-B was protective (P < 0.03). Individuals with an HLA-B allele with an aspartate at residue 114 and the tapasin G allele were more likely to spontaneously resolve HCV infection (P < 0.00003, OR = 3.2 95% CI = 1.6-6.6). Additionally, individuals with chronic HCV and the combination of an HLA-B allele with an aspartate at residue 114 and the tapasin G allele also had stronger CD8+ T-cell responses (P = 0.02, OR = 2.58, 95% CI-1.05-6.5). CONCLUSION: Tapasin alleles contribute to the outcome of HCV infection by synergizing with polymorphisms at HLA-B in a population-specific manner. This polymorphism may be relevant for peptide vaccination strategies against HCV infection.

Human leukocyte antigen (HLA)-B*57:01-restricted activation of drug-specific T cells provides the immunological basis for flucloxacillin-induced liver injury.

UNLABELLED: The role of the adaptive immune system in adverse drug reactions that target the liver has not been defined. For flucloxacillin, a delay in the reaction onset and identification of human leukocyte antigen (HLA)-B*57:01 as a susceptibility factor are indicative of an immune pathogenesis. Thus, we characterize flucloxacillin-responsive CD4+ and CD8+ T cells from patients with liver injury and show that naive CD45RA+CD8+ T cells from volunteers expressing HLA-B*57:01 are activated with flucloxacillin when dendritic cells present the drug antigen. T-cell clones expressing CCR4 and CCR9 migrated toward CCL17 and CCL 25, and secreted interferon-gamma (IFN-γ), T helper (Th)2 cytokines, perforin, granzyme B, and FasL following drug stimulation. Flucloxacillin bound covalently to selective lysine residues on albumin in a time-dependent manner and the level of binding correlated directly with the stimulation of clones. Activation of CD8+ clones with flucloxacillin was processing-dependent and restricted by HLA-B*57:01 and the closely related HLA-B*58:01. Clones displayed additional reactivity against ß-lactam antibiotics including oxacillin, cloxacillin, and dicloxacillin, but not abacavir or nitroso sulfamethoxazole. CONCLUSION: This work defines the immune basis for flucloxacillin-induced liver injury and links the genetic association to the iatrogenic disease.

High frequency of HLA B62 in fulminant type 1 diabetes with the drug-induced hypersensitivity syndrome.

CONTEXT: Fulminant type 1 diabetes (FT1D) is a subtype of type 1 diabetes characterized by an extremely abrupt onset. FT1D cases associated with the drug-induced hypersensitivity syndrome (DIHS) have recently been reported. OBJECTIVE: The clinical characteristics of FT1D associated with DIHS were investigated in this study. METHODS: Case reports of FT1D associated with DIHS in Japanese subjects were collected and analyzed by means of a questionnaire to the authors. A nationwide questionnaire survey was administered to dermatology specialists, concerning the frequency of FT1D associated with DIHS. RESULTS: In 15 case reports, the mean age at onset of FT1D was 53.4 yr and the mean time for its development from the onset of DIHS was 39.9 d. A higher frequency of human leukocyte antigen (HLA) B62, but not of HLA DR was found in FT1D with DIHS than that for cases without DIHS (P < 0.001). The reactivation of herpes virus 6 and cytomegalovirus was detected in 11 and four cases, respectively. Among 746 patients with DIHS in the nationwide survey, four developed FT1D during a 3-yr period. The frequency of FT1D in DIHS (0.54%) was much higher than that in the general Japanese population (0.010%). CONCLUSIONS: The clinical characteristics of FT1D with DIHS were similar to those without DIHS except for the high frequency of HLA B62, which may be involved in the pathogenesis of FT1D with DIHS. Because the frequency was much higher than that in the general Japanese population, FT1D should be kept in mind when DIHS develops.

NLRC5 controls basal MHC class I gene expression in an MHC enhanceosome-dependent manner.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play important roles in innate immune responses as pattern-recognition receptors. Although most NLR proteins act in cell autonomous immune pathways, some do not function as classical pattern-recognition receptors. One such NLR protein is the MHC class II transactivator, the master regulator of MHC class II gene transcription. In this article, we report that human NLRC5, which we recently showed to be involved in viral-mediated type I IFN responses, shuttles to the nucleus and activates MHC class I gene expression. Knockdown of NLRC5 in different human cell lines and primary dermal fibroblasts leads to reduced MHC class I expression, whereas introduction of NLRC5 into cell types with very low expression of MHC class I augments MHC class I expression to levels comparable to those found in lymphocytes. Expression of NLRC5 positively correlates with MHC class I expression in human tissues. Functionally, we show that both the N-terminal effector domain of NLRC5 and its C-terminal leucine-rich repeat domain are needed for activation of MHC class I expression. Moreover, nuclear shuttling and function depend on a functional Walker A motif. Finally, we identified a promoter sequence in the MHC class I promoter, the X1 box, to be involved in NLRC5-mediated MHC class I gene activation. Taken together, this suggested that NLRC5 acts in a manner similar to class II transactivator to drive MHC expression and revealed NLRC5 as an important regulator of basal MHC class I expression.

Multilayered defense in HLA-B51-associated HIV viral control.

Polymorphism in the HLA region of a chromosome is the major source of host genetic variability in HIV-1 outcome, but there is limited understanding of the mechanisms underlying the beneficial effect of protective class I alleles such as HLA-B57, -B27, and -B51. Taking advantage of a unique cohort infected with clade B' HIV-1 through contaminated blood, in which many variables such as the length of infection, the infecting viral strain, and host genetic background are controlled, we performed a comprehensive study to understand HLA-B51-associated HIV-1 control. We focused on the T cell responses against three dominant HLA-B51-restricted epitopes: Gag327-345(NI9) NANPDCKTI, Pol743-751(LI9) LPPVVAKEI, and Pol283-289(TI8) TAFTIPSI. Mutations in all three dominant epitopes were significantly associated with HLA-B51 in the cohort. A clear hierarchy in selection of epitope mutations was observed through epitope sequencing. L743I in position 1 of epitope LI9 was seen in most B51(+) individuals, followed by V289X in position 8 of the TI8, and then, A328S, in position 2 of the NI9 epitope, was also seen in some B51(+) individuals. Good control of viral load and higher CD4(+) counts were significantly associated with at least one detectable T cell response to unmutated epitopes, whereas lower CD4(+) counts and higher viral loads were observed in patients who had developed escape mutations in all three epitopes or who lacked T cell responses specific to these epitope(s). We propose that patients with HLA-B51 benefit from having multiple layers of effective defense against the development of immune escape mutations.

Killer Ig-like receptor ligand mismatch directs NK cell expansion in vitro.

NK cell alloreactivity is governed largely through failure to detect self-HLA class I ligands by the clonally distributed inhibitory killer Ig-like receptors (KIR) expressed on the NK cell surface. In this study, we investigated the extent to which HLA class I-KIR interactions influence human NK cell proliferation in the allogeneic setting. NK cells were cultured with feeder cells either matched or mismatched for inhibitory KIR ligands, the latter lacking one or more ligands present in the NK cell donor. In postculture cytotoxicity assays, the ability of polyclonal NK cells to kill KIR ligand-mismatched targets was enhanced by exposure to appropriately mismatched feeder cells in prior culture. This corresponded with an increased frequency of postculture donor NK cells expressing a given inhibitory KIR if the allogeneic feeder cells used in the culture lacked its ligand. Similar skewing of KIR distribution was seen in clonally expanded NK cells. Finally, a flow cytometry-based proliferation assay was used to show KIR-specific NK cell division in response to missing self. The findings demonstrate that KIR distribution among a population of alloresponding peripheral blood NK cells is shaped by the HLA class I environment.

Killer immunoglobulin-like receptor ligand HLA-Bw4 protects against multiple sclerosis.

OBJECTIVE: Multiple sclerosis (MS) is a chronic inflammatory disease affecting the central nervous system. A human leukocyte antigen (HLA) class II association is well established (DRB1*1501-DQB1*0602), but more recently HLA class II-independent associations with HLA class I variants have also been reported. The HLA class I (HLA-A, -B, -C) molecules serve as ligands for both T-cell receptors and killer immunoglobulin-like receptors (KIRs). We investigated the HLA class I alleles defined by their KIR binding motifs and the KIR genes to evaluate whether these genes could influence MS susceptibility or severity, alone or in combination. METHODS: We typed Norwegian MS patients (n = 631) and controls (n = 555) for HLA-A, -B, -C and -DRB1 alleles as well as the presence or absence of genes encoding inhibitory (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3) and activating (KIR2DS1, KIR2DS2, KIR2DS3, KIR2DL4, KIR2DS4, KIR2DS5, KIR3DS1) KIRs. RESULTS: The frequency of the HLA-Bw4 specificity, which is the ligand for the inhibitory KIR3DL1, was significantly reduced in MS patients as compared with controls (41.4% vs 55.1%, p(uncorrected (uc)) = 4.6 x 10(-6)). Also after stratifying for known HLA class II associations, the HLA-Bw4 association was seen (p(uc) = 0.002). No significant differences in gene carrier frequencies of inhibitory and activating KIRs were observed. However, our data indicate that MS patients who carry the activating KIR2DS2 and the inhibitory KIR2DL2 genes have more severe disease than patients not carrying these genes. INTERPRETATION: Carriage of the ligand of the inhibitory KIR3DL1 receptor, HLA-Bw4, was found to protect against MS in an HLA-DRB1 independent manner.

Phenotypic and functional analyses of KIR3DL1+ and KIR3DS1+ NK cell subsets demonstrate differential regulation by Bw4 molecules and induced KIR3DS1 expression on stimulated NK cells.

Recently, the Z27 mAb was shown to recognize the NK cell-activating receptor KIR3DS1, and several genetic studies suggest that the most probable ligands of KIR3DS1 are HLA class I molecules with the Bw4 motif. Despite these findings, the attempts to establish a functional interaction between KIR3DS1 and its potential ligand have been unsuccessful. Here, we study the proliferation and cytotoxicity of KIR3DS1(+) NK cells, compared with KIR3DL1(+) NK cells, according to the Bw4(+) or Bw4(-) allogeneic environment. Our results show for the first time that KIR3DS1 expression on NK cells can be induced after exposure to stimulator cells (221, K562, EBV-B cell lines, and B cells), polyinosinic-polycytidylic acid, IL-15, or IL-2. Furthermore, whereas KIR3DL1(+) NK cell proliferation and cytotoxicity were inhibited in a Bw4(+) but not a Bw4(-) context, KIR3DS1(+) NK cell functions were not influenced by the presence of Bw4 on target cells. Nevertheless, despite the absence of demonstrated regulation of KIR3DS1(+) NK cell functions by HLA-Bw4 molecules, we found a higher KIR3DS1(+) NK cell frequency and higher levels of KIR3DS1 expression in Bw4(+) compared with Bw4(-) individuals. Altogether, these results suggest that KIR3DS1 does not recognize HLA-Bw4 molecules in a physiological context, and they highlight the induced expression of KIR3DS1 observed on stimulated NK cells and the higher frequency of KIR3DS1(+) NK cells in Bw4(+) individuals. Because a protective KIR3DS1-Bw4 association has been reported in viral infections, our results further the understanding of the role of KIR3DS1(+) NK cells in controlling viral infections.

Escape from HLA-B*08-restricted CD8 T cells by hepatitis C virus is associated with fitness costs.

The inherent sequence diversity of the hepatitis C virus (HCV) represents a major hurdle for the adaptive immune system to control viral replication. Mutational escape within targeted CD8 epitopes during acute HCV infection has been well documented and is one possible mechanism for T-cell failure. HLA-B*08 was recently identified as one HLA class I allele associated with spontaneous clearance of HCV replication. Selection of escape mutations in the immunodominant HLA-B*08-restricted epitope HSKKKCDEL(1395-1403) was observed during acute infection. However, little is known about the impact of escape mutations in this epitope on viral replication capacity. Their previously reported reversion back toward the consensus residue in patients who do not possess the B*08 allele suggests that the consensus sequence in this epitope is advantageous for viral replication in the absence of immune pressure. The aim of this study was to determine the impact of mutational escape from this immunodominant epitope on viral replication. We analyzed it with a patient cohort with chronic HCV genotype 1b infection and in a single-source outbreak (genotype 1b). Sequence changes in this highly conserved region are rare and selected almost exclusively in the presence of the HLA-B*08 allele. When tested in the subgenomic replicon (Con1), the observed mutations reduce viral replication compared with the prototype sequence. The results provide direct evidence that escape mutations in this epitope are associated with fitness costs and that the antiviral effect of HLA-B*08-restricted T cells is sufficiently strong to force the virus to adopt a relatively unfavorable sequence.

HLA class I molecules regulate IFN-gamma production induced in NK cells by target cells, viral products, or immature dendritic cells through the inhibitory receptor ILT2/CD85j.

Recent advances support an important role for NK cells in determining immune responses beyond their cytolytic functions, which is supported by their capacity to secrete several cytokines and chemokines. In particular, NK-derived IFN-gamma has proven to be fundamental in shaping adaptive immune responses. Although the role of inhibitory NK receptors (iNKR) in the regulation of cytotoxicity has been widely explored, their involvement in the control of cytokine production has been scarcely analyzed. Specifically, no data are available referring to the role of the iNKR ILT2/CD85j in the regulation of IFN-gamma secretion by NK cells. Published data support a differential regulation of cytotoxicity and cytokine expression. Thus, formal proof of the involvement of HLA class I in regulating the production of cytokines through binding to ILT2/CD85j has been missing. We have determined the response of human NK-92 and primary human ILT2/CD85j(+) NK cells from healthy donors to target cells expressing or not HLA class I. We found specificities of HLA class I-mediated inhibition of IFN-gamma mRNA expression, protein production, and secretion consistent with the specific recognition by ILT2/CD85j. We also found inhibition of IFN-gamma production by ILT2/CD85j(+) T cells in response to superantigen stimulation. Furthermore, ligation of ILT2/CD85j inhibited the production of IFN-gamma in response to poly(I:C), and blocking of ILT2/CD85j-HLA class I interactions increased the secretion of IFN-gamma in NK/immature dendritic cell cocultures. The data support a role for self HLA class I in the regulation of IFN-gamma secretion at the mRNA and protein levels by interacting with the iNKR ILT2/CD85j.

Killer cell immunoglobulin-like receptors on NK cells: the how, where and why.

Natural killer (NK) cells have killer cell immunoglobulin-like receptors (KIR) that recognize and interact with HLA class I antigen. The KIRs are a multigene family and its members are often highly polymorphic. Evidence is emerging from disease-association studies that KIR receptors can play beneficial roles in viral infections, such as HIV, HCV, but may also predispose to certain autoimmune diseases. Knowledge regarding expression and function of KIR on human NK cells is lagging behind the rapid expansion of sequencing and genetic data already generated. This review focuses on recent discoveries that have been made, which help bridge this gap. We now appreciate the importance of phenotypic diversity of KIR receptor expression in NK cells and are starting to unravel some of the mysteries surrounding control of their complex expression patterns. In particular, the role that HLA ligand contributes to KIR receptor expression will be discussed. It is also becoming increasingly clear that genetic factors, such as promoters and epi-genetic mechanisms such as methylation, are hugely important in controlling NK cell receptor expression and function. The relevance of phenotypic diversity of NK cell receptors will be discussed in light of these recent findings.

HLA-B*2704, an allotype associated with ankylosing spondylitis, is critically dependent on transporter associated with antigen processing and relatively independent of tapasin and immunoproteasome for maturation, surface expression, and T cell recognition: relationship to B*2705 and B*2706.

B*2704 is strongly associated to ankylosing spondylitis in Asian populations. It differs from the main HLA-B27 allotype, B*2705, in three amino acid changes. We analyzed the influence of tapasin, TAP, and immunoproteasome induction on maturation, surface expression, and T cell allorecognition of B*2704 and compared some of these features with B*2705 and B*2706, allotypes not associated to disease. In the tapasin-deficient .220 cell line, this chaperone significantly influenced the extent of folding of B*2704 and B*2705, but not their egress from the endoplasmic reticulum. In contrast, B*2706 showed faster folding and no accumulation in the endoplasmic reticulum in the absence of tapasin. Surface expression of B*2704 was more tapasin dependent than B*2705. However, expression of free H chain decreased in the presence of this chaperone for B*2705 but not B*2704, suggesting that more suboptimal ligands were loaded on B*2705 in the absence of tapasin. Despite its influence on surface expression, tapasin had little effect on allorecognition of B*2704. Both surface expression and T cell recognition of B*2704 were critically dependent on TAP, as established with TAP-deficient and TAP-proficient T2 cells. Both immunoproteasome and surface levels of B*2704 were induced by IFN-gamma, but this had little effect on allorecognition. Thus, except for the differential effects of tapasin on surface expression, the tapasin, TAP, and immunoproteasome dependency of B*2704 for maturation, surface expression, and T cell recognition are similar to B*2705, indicating that basic immunological features are shared by the two major HLA-B27 allotypes associated to ankylosing spondylitis in human populations.

HLA B44 is associated with decreased severity of autoimmune lymphoproliferative syndrome in patients with CD95 defects (ALPS type Ia).

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte apoptosis characterized by non-malignant lymphadenopathy and splenomegaly, expansion of T cells without either CD4 or CD8 surface markers, and increased incidence of autoimmune diseases and lymphoma. Most patients with ALPS have dominant, heterozygous mutations in tumor necrosis factor receptor superfamily member 6 (TNFRSF6), which encodes CD95, also known as Fas, a mediator of apoptosis. Penetrance and range of disease manifestations in ALPS are highly variable, even among family members who share the same dominant TNFRSF6 mutation. To evaluate HLA as a candidate modifier locus, we typed HLA A, B (including subtypes), and DQB alleles in 356 individuals from 63 unrelated families with defined TNFRSF6 mutations associated with ALPS. We also developed a quantitative severity score and performed statistical analysis. Among the healthier, mutation-bearing individuals, transmission of HLA B44 was significantly overrepresented (nominal P<0.0074) as compared to transmission in patients with severe clinical features of ALPS. The B44 allele may exert a protective role in ALPS.

Two HLA-B14 subtypes (B*1402 and B*1403) differentially associated with ankylosing spondylitis differ substantially in peptide specificity but have limited peptide and T-cell epitope sharing with HLA-B27.

The peptide specificity of HLA-B*1403, an allotype associated with ankylosing spondylitis (Lopez-Larrea, C., Mijiyawa, M., Gonzalez, S., Fernandez-Morera, J. L., Blanco-Gelaz, M. A., Martinez-Borra, J., and Lopez-Vazquez, A. (2002) Arthritis Rheum. 46, 2968-2971) was compared with those of the non-associated B*1402 and the prototypic disease-associated B*2705 allotypes. Although differing by a single residue (L156R), B*1402 and B*1403 shared only 32-35% of their peptide repertoires. Subtype-related differences observed in multiple peptide positions, including P3 and P7, were largely explained by a direct effect of the L156R change on peptide specificity. The HLA-B14 subtypes shared only approximately 3% of their peptide repertoires with B*2705. This was due to distinct residue usage at most positions, as revealed by statistical comparison of B*1402, B*1403, and B*2705-bound nonamers. Nevertheless, shared ligands between B*2705 and B*1403 were formally identified, although ligands common to B*2705 and B*1403, but absent from B*1402, were not found. Alloreactive T-cells were used as a tool to analyze epitope sharing among B*1402, B*1403, and B*2705. The percentage of cross-reactive T-cell clones closely paralleled peptide overlap, suggesting that shared ligands tend to maintain their antigenic features when bound to the different allotypes. Our results indicate that B*1403 and B*2705 can present common peptides. However, both the disparity of their peptide repertoires and the lack of binding features shared by these two allotypes, but not B*1402, argue against, although do not exclude, a mechanism of spondyloarthritis mediated by specific ligands of B*2705 and B*1403.

New evidence that the MHC influences odor perception in humans: a study with 58 Southern Brazilian students.

Increasing evidence suggests a correlation between mate choice, odor preference, and genetic similarity at the Major Histocompatibility Complex (MHC) in a variety of animals, including our species. The MHC is a highly polymorphic group of genes that play an important role in the immunological self/nonself recognition. Its products have been reported to take part on the variety of compounds and reactions that together build an individual's body odor. It has been suggested, therefore, that animals use body odor as a guide to identify possible mates as MHC-similar or MHC-dissimilar from their own genotype. Preference for a MHC-dissimilar partner enhances MHC heterozygosity of an individual's offspring. The possible adaptive advantages are clear: it is a mechanism of avoiding inbreeding and MHC-heterozygous offspring may have enhanced immunocompetence. The aim of this study was to search, in our species, new evidence on the correlation between specificities at HLA-A and HLA-B and assessments of pleasantness regarding specific body odors. HLA is the name for the human MHC. Four olfactory sessions were performed with 58 young Southern Brazilian students, in order to investigate whether assessments of pleasantness of body odors from individuals correlate to a person's HLA phenotype. Body odors were collected via sweat and urine from all participants. Women smelled and scored all male odor samples and men did the same with all female samples. We found a significant correlation only when female smellers evaluated male sweat odors.

Minor histocompatibility antigens--big in tumour therapy.

Technical advances combined with the deciphering of the human genome have facilitated the identification of the molecular nature of human minor histocompatibility (H) antigens. To date, it is believed that minor H antigens result from just any polymorphic protein, regardless of their functional properties. A closer look at the first series of autosomally encoded human minor H proteins reveals a striking functional relationship. Here, we propose that T cells generated after HLA-identical stem cell transplantation (SCT) for malignancies are likely to be directed towards peptides derived from minor H proteins involved in tumourigenesis. This novel insight has important consequences in the search for, and the use of, minor H antigens as immunotherapeutics in stem-cell-based immunotherapy of haematological malignancies and solid tumours.

A mutation in the HLA-B*2705-restricted NP383-391 epitope affects the human influenza A virus-specific cytotoxic T-lymphocyte response in vitro.

Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in or adjacent to CTL epitopes. Recently, an amino acid substitution (R384G) in an HLA-B*2705-restricted CTL epitope in the influenza A virus nucleoprotein (nucleoprotein containing residues 383 to 391 [NP(383-391)]; SRYWAIRTR, where R is the residue that was mutated) was associated with escape from CTL-mediated immunity. The effect of this mutation on the in vitro influenza A virus-specific CTL response was studied. To this end, two influenza A viruses, one with and one without the NP(383-391) epitope, were constructed by reverse genetics and designated influenza viruses A/NL/94-384R and A/NL/94-384G, respectively. The absence of the HLA-B*2705-restricted CTL epitope in influenza virus A/NL/94-384G was confirmed by using (51)Cr release assays with a T-cell clone specific for the NP(383-391) epitope. In addition, peripheral blood mononuclear cells (PBMC) stimulated with influenza virus A/NL/94-384G failed to recognize HLA-B*2705-positive target cells pulsed with the original NP(383-391) peptide. The proportion of virus-specific CD8+ gamma interferon (IFN-gamma)-positive T cells in in vitro-stimulated PBMC was determined by intracellular IFN-gamma staining after restimulation with virus-infected autologous B-lymphoblastoid cell lines and C1R cell lines expressing only HLA-B*2705. The proportion of virus-specific CD8+ T cells was lower in PBMC stimulated in vitro with influenza virus A/NL/94-384G obtained from several HLA-B*2705-positive donors than in PBMC stimulated with influenza virus A/NL/94-384R. This finding indicated that amino acid variations in CTL epitopes can affect the virus-specific CTL response and that the NP(383-391) epitope is the most important HLA-B*2705-restricted epitope in the nucleoprotein of influenza A viruses.