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Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality in hematopoietic stem cell transplant recipients despite the introduction of posttransplantation viral monitoring and preemptive antiviral therapy. We evaluated the use of HLA class I tetramers in monitoring CMV-specific T-cell recovery to predict patients at risk for CMV-related complications. This prospective multicenter clinical trial obtained nearly 1400 tetramer/allele results in more than 800 biweekly blood samples from 83 patients monitored for 1 year after transplantation. Major HLA types were included (A*0101, A*0201, B*0702, B*0801, B*3501). iTAg MHC Tetramers (Beckman Coulter) were used to enumerate CMV-specific CD8(+) T cells by flow cytometry using a single-platform absolute counting method. Assay variability was 8% or less and results were available within 3 hours. Delayed recovery of CMV-specific T cells (< 7 cells/μL in all blood samples during the first 65 days after transplantation) was found to be a significant risk factor for CMV-related complications; these patients were more likely to develop recurrent or persistent CMV infection (relative risk 2.6, CI 1.2-5.8, P = .01) than patients showing rapid recovery, which was associated with protection from CMV-related complications (P = .004). CMV tetramer-based immune monitoring, in conjunction with virologic monitoring, can be an important new tool to assess risk of CMV-related complications and to guide preemptive therapeutic choices.

Immunotherapy of colorectal carcinoma (CRC) has great promise as the presence of T lymphocytes in CRC tissues in situ is correlated with reduced recurrence and increased survival. Thus, identification of the antigens recognized by T cells of CRC patients may permit development of vaccines with potential benefit for these patients. Using expression cloning, we identified the antigen, nucleophosmin (Npm), recognized by an HLA-A1 restricted cytotoxic T lymphocyte (CTL) line derived from the peripheral blood mononuclear cells (PBMC) of a rectal cancer patient. A decamer peptide derived from the Npm sequence sensitized peptide-pulsed HLA-A1 positive cells to lysis by the CTL line. The peptide also induced proliferative and cytotoxic T lymphocytes in the PBMC of 4 of 6 CRC patients, which lysed HLA-A1 positive peptide-pulsed target cells and CRC cells endogenously expressing Npm. Overexpression of Npm by tumors of various histological types, recognition of the antigen by T cells derived from different CRC patients and association of the antigen with poor prognostic outcome make it a promising target for immunotherapeutic intervention in cancer patients.

A novel human leukocyte antigen-A allele, officially named HLA-A*0131N allele, was found in a potential Chinese bone marrow donor when direct sequence-based typing was carried out. The novel A*0131N is identical to A*01010101 with an exception of one base substitution at position 178 (G>A) of exon 2 resulting in codon #60 changed from TGG (Trp) to TAG (stop codon).

The aim of this study was to uncover the membrane protein profile differences between colorectal carcinoma and neighboring normal mucosa from colorectal cancer patients. Information from cellular membrane proteomes can be used not only to study the roles of membrane proteins in fundamental biological processes, but also to discover novel targets for improving the management of colorectal cancer patients. We used solvent extraction and a gel-assisted digestion method, together with isobaric tags with related and absolute quantitation (iTRAQ) reagents to label tumoral and adjacent normal tissues in a pairwise manner (n = 8). For high-throughput quantification, these digested labeled peptides were combined and simultaneously analyzed using LC-MS/MS. Using the shotgun approach, we identified a total of 438 distinct proteins from membrane fractions of all eight patients. After comparing protein expression between cancerous and corresponding normal tissue, we identified 34 upregulated and eight downregulated proteins with expression changes greater than twofold (Student's t-test, P < 0.05). Among these, the overexpression of well-established biomarkers such as carcinoembryonic antigens (CEACAM5, CEACAM6), as well as claudin-3, HLA class I histocompatibility antigen A-1, tapasin and mitochondrial solute carrier family 25A4 were confirmed by western blotting. We conclude that gel-assisted digestion and iTRAQ labeling MS is a potential approach for uncovering and comparing membrane protein profiles of tissue samples that has the potential to identify novel biomarkers.

Most antigenic peptides presented by major histocompatibility complex (MHC) class I molecules are produced by the proteasome. Here we show that a proteasome-independent peptide derived from the human tumor protein MAGE-A3 is produced directly by insulin-degrading enzyme (IDE), a cytosolic metallopeptidase. Cytotoxic T lymphocyte recognition of tumor cells was reduced after metallopeptidase inhibition or IDE silencing. Separate inhibition of the metallopeptidase and the proteasome impaired degradation of MAGE-A3 proteins, and simultaneous inhibition of both further stabilized MAGE-A3 proteins. These results suggest that MAGE-A3 proteins are degraded along two parallel pathways that involve either the proteasome or IDE and produce different sets of antigenic peptides presented by MHC class I molecules.

Introduction: Near 10 to 20% of patients with myositis have another systemic, sometimes inaugural, disease., Case Report: A 48-year-old woman was admitted with progressive hypoesthesia in V2 and V3 areas on both sides, difficulties to chew and swallow and then, proximal and axial muscular deficiency, with weight loss. Brain MRI showed gadolinium-enhanced trigeminal nerves and biological tests revealed anti-SSA and anti-Pm/Scl antibodies and a grade IV in Chisholm scoring system on the labial salivary gland biopsy. Neurophysiological studies revealed a myogenic pattern on tibialis anterior muscles and a muscle biopsy confirmed the diagnosis of polymyositis., Conclusion: The diagnosis of primitive Sjogren's syndrome was suspected because of the association of bilateral trigeminal neuropathy and anti-SSA and anti-Pm/Scl antibodies., (Copyright 2009 Elsevier Masson SAS. All rights reserved.)

Introduction: Selective IgA deficiency (IgAD; serum IgA concentration of <0.07 g/l) is the most common primary immunodeficiency in Caucasians with an estimated prevalence of 1/600. The frequency of the extended major histocompatibility complex haplotype HLA A1, B8, DR3, DQ2 (the "8.1" haplotype) is increased among patients with IgAD., Materials and Methods: We carried out a direct measurement of the relative risk of homozygosity of the 8.1 haplotype for IgA deficiency in a population-based sample of 117 B8, DR3 homozygous individuals., Results and Discussion: IgA deficiency was found to be present in 2 of 117 (1.7%) of these subjects, a figure that is concordant with estimates of relative risk from large case-control studies in the Swedish population. These data are consistent with a multiplicative model for the 8.1 haplotype contribution to IgA deficiency and contrasts with prior studies, suggesting a much higher risk for 8.1 homozygosity. Using a dense single nucleotide polymorphism marker analysis of the MHC region in HLA B8, DR3, DQ2 homozygous individuals, we did not observe consistent differences between cases (n = 26) and controls (n = 24). Overall, our results do not support the hypothesis that IgA deficiency is associated with a distinct subgroup of 8.1 related haplotypes, but rather indicate that risk is conferred by the common 8.1 haplotype acting in multiplicative manner.

Immunological correlates of AIDS-free survival after human immunodeficiency virus type 1 (HIV-1) infection are largely unknown. Cytotoxic T lymphocyte (CTL) responses are generally believed to be a major component of protective immunity against viral infections. However, the relationship between HIV-1-specific CTL responses and disease progression rate is presently unclear. Here we show in twelve HIV-1-infected individuals that detection of Rev-specific CTL precursors (CTLp) early in the asymptomatic stage, as well as detection of Rev- and Tat-specific CTLp later during follow-up, inversely correlate with rapid disease progression. No such correlation was found for detection of CTLp against Gag, RT or Nef. Further studies are required to determine whether a protective mechanism is indeed the basis of the observed correlation. The data presented are in agreement with the hypothesis that CTL against proteins that are important for early viral transcription and translation are of particular importance in protection from rapid disease progression.

S100A4 (metastasin 1) belongs to the S100 family of Ca(2+) binding proteins. While not present in most differentiated adult tissues, S100A4 is upregulated in the micromilieu of tumors. It is primarily expressed by tumor-associated macrophages, fibroblasts, and tumor endothelial cells. Due to its strong induction in tumors S100A4 is a promising target for cancer immunotherapy. By reverse immunology, using epitope prediction programs, we identified 3 HLA-A1-restricted peptide epitopes (S100A4 A1-1, A1-2, and A1-3) which are subject to human T cell responses as detected in peripheral blood of melanoma patients by means of IFN-gamma ELISPOT and cytotoxicity assays. In addition, IFN-gamma responses to S100A4 A1-2 can not only be induced by stimulation of T cells with peptide-loaded DC but also by stimulation with S100A4 protein-loaded DC, indicating that this epitope is indeed generated by processing of the endogenously expressed protein. In addition, S100A4 A1-2 reactive T cells demonstrate lysis of HLA-A1(+) fibroblasts in comparison to HLA-A1(-) fibroblasts. In summary, this HLA-A1-restricted peptide epitope is a candidate for immunotherapeutical approaches targeting S100A4-expressing cells in the tumor stroma.

Objectives: As the HLA system is involved in recognition of self and non-self, an association with the development of ANCA-associated vasculitis (AAV) seems probable. In this study, the relation between HLA antigens and AAV and it's severity were investigated., Methods: Consecutive patients diagnosed with AAV at our centre, who were followed for at least 2 years, were included. The frequency of HLA antigens of AAV and WG patients was compared with 5872 healthy blood donors from the same region and with 4000 healthy Dutch controls originating from a Eurotransplant database., Results: From 304 AAV patients, sufficient data were available. We found DR13(6) to be less prevalent and both DR4 and the ancestral haplotype A1B8DR3 more prevalent in patients with AAV compared with controls, particularly in patients with WG. In addition, DR1 was less prevalent in patients with WG in comparison with controls. Further, DR8 was more prevalent in patients with CSS compared with other forms of vasculitis and controls. There were no associations between HLA antigens and disease characteristics or course of AAV or WG., Conclusions: AAV is associated with increased prevalence of DR4 and the ancestral haplotype A1B8DR3 and with decreased prevalence of DR13(6), particularly in patients with WG. In patients with WG, prevalence of DR1 was decreased, whereas in patients with CSS DR8 was increased. No associations between HLA antigens and disease characteristics or course of AAV were found.

An optimal antitumoral immune response requires the activation of both CD8(+) and CD4(+) T lymphocytes by the peptide antigen presentation via the human leukocyte antigen (HLA) class I and class II molecules, respectively. The frequency of A1, A26, DR11 alleles are significantly elevated and seem to be the predisposing alleles in RCC, while HLA A29 and DQ1 are the protective alleles and are found more frequently in the healthy group. To investigate the association between renal cell carcinoma (RCC) and the host's immune system, we immunohistochemically examined RCCs in 44 Turkish patients for the expression of class I and class II antigens. We found a significantly higher frequency of the alleles HLA-A1 (p= 0.001), HLA-A26 (p=0.047) and HLA-DR11 (p= 0.03) in RCC patients compared to the control group. The most frequent alleles in the control population were A29, DQ1 (p= 0.004 and 0.002 respectively). We observed no significant difference between patients and controls in HLA-B and HLA-C allele frequency. We conclude that our study found an association between HLA antigens and RCC in Turkish patients. We found a significantly higher frequency of the alleles HLA-A1, HLA-A26 and HLA-DR11in RCC patients compared to the control group. Larger studies are required to confirm these results.

Background: The ability to secrete blood group antigens into body fluids and secretions is controlled by a single gene on chromosome 19. By means of erythrocyte Lewis (Le) antigen phenotype secretor status can be inferred. An increase prevalence of non-secretors of blood group antigens among coeliac patients has recently been described., Methods: Blood was collected from 112 coeliac patients and 103 controls and tested for secretor status. Secretor status was correlated with human leucocyte antigens (HLA) in coeliac patients, thus evaluating a proposed interaction of susceptibility genes--that is, the secretor gene on chromosome 19 and HLA-linked genes on chromosome 6. Case notes for coeliacs were reviewed with regard to clinical outcome., Results: Of 112 coeliacs who had either Le(a) or Le(b) antigens, 36 (32%) were non-secretors Le(a+, b-), compared with 27% (28) of 103 disease-free controls (P = 0.313). Recessive Lewis phenotype Le(a-, b-) was found in 9% of coeliacs versus 2% of controls. Prevalence of HLA-A1, B8, DR3, and DQ2 was unrelated to secretor status in coeliac versus patients. An increased prevalence of complications and coeliac-associated abnormalities was found in the non-secreting and recessive coeliac groups., Conclusions: This study shows no firm relationship between the non-secretor state and coeliac disease, nor any difference in the distribution of HLA markers among secretor and non-secretor coeliacs. It is unlikely, therefore, that the secretor gene is the much sought-after second coeliac gene.

Smoking behavior has been associated in two independent European cohorts with the most common Caucasian human leukocyte antigen (HLA) haplotype (A1-B8-DR3). We aimed to test whether polymorphic members of the two odorant receptor (OR) clusters within the extended HLA complex might be responsible for the observed association, by genotyping a cohort of Hungarian women in which the mentioned association had been found. One hundred and eighty HLA haplotypes from Centre d'Etude du Polymorphisme Humain families were analyzed in silico to identify single-nucleotide polymorphisms (SNPs) within OR genes that are in linkage disequilibrium with the A1-B8-DR3 haplotype, as well as with two other haplotypes indirectly linked to smoking behavior. A nonsynonymous SNP within the OR12D3 gene (rs3749971(T)) was found to be linked to the A1-B8-DR3 haplotype. This polymorphism leads to a (97)Thr --> Ile exchange that affects a putative ligand binding region of the OR12D3 protein. Smoking was found to be associated in the Hungarian cohort with the rs3749971(T) allele (p = 1.05 x 10(-2)), with higher significance than with A1-B8-DR3 (p = 2.38 x 10(-2)). Our results link smoking to a distinct OR allele, and demonstrate that the rs3749971(T) polymorphism is associated with the HLA haplotype-dependent differential recognition of cigarette smoke components, at least among Caucasian women.

Acute enteroviral infections ranging from meningitis, pancreatitis to myocarditis are common and normally well controlled by the host immune system comprising virus-specific CD8+ cytotoxic T lymphocytes (CTL). However, in some patients enteroviruses and especially coxsackieviruses of group B are capable of inducing severe chronic forms of diseases such as chronic myocarditis. Currently, it is not known whether divergences in the CTL-related immune response may contribute to the different outcome and course of enterovirus myocarditis. A pre-requisite for the study of CTL reactions in patients with acute and chronic myocarditis is the identification of CTL epitopes. In order to define dominant enterovirus CTL epitopes, we have screened, by using gamma interferon (IFN-gamma) ELISPOT, 62 HLA-A*01- and 59 HLA-A*02-positive healthy blood donors for pre-existing CTL reactions against 12 HLA-A*01 and 20 HLA-A*02 predicted CTL epitopes derived from coxsackieviruses of group B. Positive CTL reactions were verified by FACS analysis in a combined major histocompatibility complex-tetramer IFN-gamma staining. A total of 14.8% of all donors reacted against one of the three identified epitopes MLDGHLIAFDY, YGDDVIASY or GIIYIIYKL. The HLA-A*02-restricted epitope ILMNDQEVGV was recognized by 25% of all tested blood donors. For this peptide, we could demonstrate specific granzyme B secretion, a strong cytolytic potential and endogenous processing. All four epitopes were homologous in 36-92% of group B enteroviruses, providing a strong basis for monitoring the divergence of T-cell-based immune responses in enterovirus-induced acute and chronic diseases.

Adenovirus vectors have great potential in cancer gene therapy. Targeting of cancer-testis (CT) antigens, which are specifically presented at the surface of tumor cells by human leukocyte antigen (HLA) class I molecules, is an attractive option. In this study, a single-chain T-cell receptor (scTCR) directed against the CT antigen melanoma-associated antigen (MAGE)-A1 in complex with the HLA class I molecule of haplotype HLA-A1 is fused with the C terminus of the adenovirus minor capsid protein IX. Propagation of a protein-IX (pIX)-gene-deleted human adenovirus 5 (HAdV-5) vector on cells that constitutively express the pIXscTCR fusion protein yielded viral particles with the pIXscTCR fusion protein incorporated in their capsid. Generated particles specifically transduced melanoma cell lines expressing the HLA-A1/MAGE-A1 target complex with at least 10-fold higher efficiency than control viruses. Whereas loading of HLA-A1-positive cells with MAGE-A1 peptides leads to enhanced transduction of the cells, the efficiency of virus transduction is strongly reduced if the HLA-A1 molecules are not accessible at the target cell. Taken together, these data provide proof of principle that pIXscTCR fusions can be used to target HAdV-5 vectors to tumor cells expressing intracellular CT antigens.

Tumor regressions have been observed in a small proportion of melanoma patients vaccinated with a MAGE-A3 peptide presented by HLA-A1, administered as peptide, ALVAC canarypox virus containing a MAGE-A3 minigene, or peptide-pulsed dendritic cells (DC). There was a correlation between tumor regression and the detection of anti-MAGE-3.A1 CTL responses. These responses were monoclonal and often of a very low magnitude after vaccination with peptide or ALVAC, and usually polyclonal and of a higher magnitude after DC vaccination. These results suggested that, at least in some patients, surprisingly few anti-MAGE-3.A1 T-cells could initiate a tumor regression process. To understand the role of these T cells, we carried out a functional analysis of anti-MAGE-3.A1 CTL clones derived from vaccinated patients who displayed tumor regression. The functional avidities of these CTL clones, evaluated in lysis assays, were surprisingly low, suggesting that high avidity was not part of the putative capability of these CTL to trigger tumor rejection. Most anti-MAGE-3.A1 CTL clones obtained after DC vaccination, but not after peptide or ALVAC vaccination, produced interleukin 10. Transcript profiling confirmed these results and indicated that approximately 20 genes, including CD40L, prostaglandin D2 synthase, granzyme K, and granzyme H, were highly differentially expressed between the anti-MAGE-3.A1 CTL clones derived from patients vaccinated with either peptide-ALVAC or peptide-pulsed DC. These results indicate that the modality of vaccination with a tumor-specific antigen influences the differentiation pathway of the antivaccine CD8 T-cells, which may have an effect on their capacity to trigger a tumor rejection response.

Cytochrome P450 1B1 (CYP1B1) is widely expressed in human malignancies, but silent in most normal tissues. Importantly, the protein is believed to play an important role in the survival and growth of cancer cells in a stressed environment, e.g., as a result of hypoxia or chemotherapy. Thus, targeting of CYP1B1 represents a potentially successful strategy in the treatment of metastatic cancer, e.g., by therapeutic vaccination. Herein, we describe the characterization of a novel peptide from the CYP1B1 protein (CYP240), which is spontaneously recognized by CD8 T cells in cancer patients. Interestingly, the peptide binds to both human leukocyte antigen (HLA)-A1 and HLA-B35. Hence, peripheral blood lymphocytes from a total of 49 cancer patients (25 melanoma, 13 RCC, and 11 breast cancer; 41 HLA-A1 positive, 8 HLA-B35 positive) were analyzed for reactivity taking advantage of the EliSpot assay. Rare but strong responses were detected in HLA-A1-positive patients, and more frequent responses were detected in HLA-B35-positive patients. No reactivity against the peptide could be detected in healthy donors. Furthermore, we demonstrated that peptide-specific T cells were able to lyze target cells presenting the peptide on the surface. The characterized CYP240 peptide presented herein opens the avenue for more broader recruitment of patients in vaccination trials targeting CYB1B1.

Patients with high-risk human leukocyte antigen (HLA)-DR-DQ genotypes for type 1 diabetes (T1D) were compared with HLA-matched controls to evaluate T1D risk for other HLA loci, including HLA-A, -B, -Cw, and DPB1. Patients (n = 133) with high-risk genotypes (DR3/DR3, DR3/DR4, DR4/DR4) were selected from the Lazio (Rome) region of Italy. Screening of more than 9000 patients from the Lazio region and northern Italy yielded 162 controls with high-T1D-risk haplotypes. Although the overall distributions did not differ significantly, allele frequency differences were discovered between the controls from Lazio and controls from northern Italy for some alleles previously determined to affect T1D risk, such as A*3002, DPB1*0301, and DPB1*0402. Therefore, Lazio patient data were compared both with the Lazio subset of controls (n = 53) and with the entire group of controls for association analyses. Significant allele frequency differences between patients and DR-DQ-matched controls existed for specific alleles at all loci. Data for the DR3/DR3 subset of patients and controls demonstrated an increase of Cw*0702 in patients. Compared with controls, reduced patient frequencies were seen for several alleles, including A*0101, B*0801, and Cw*0701, all on the highly conserved, extended DR3 haplotype known as 8.1 in DR3/DR3, but not DR3/DR4, subgroup. DPB1*0101, often reported on 8.1 haplotypes, was also less frequent in DR3/DR3 patients than controls. Analysis of family-based data from the HBDI repository was consistent with the observed results from the Italian patients, indicating the presence of a T1D-protective locus at or near A*0101 and a second T1D-protective locus at or near DPB1*0101. These data indicate that T1D risk conferred by the 8.1 haplotype is genotype dependent.

Failure of the adaptive immune response to control infection with the hepatitis C virus (HCV) can result from mutational escape in targeted T-cell epitopes. Recent studies suggest that T-cell immune pressure is an important factor in the evolution of the nonstructural proteins in HCV. The aim of this study was to characterize the forces that contribute to viral evolution in an HLA-A*01-restricted epitope in HCV NS3. This epitope represents a potentially attractive target for vaccination strategies since it is conserved across all genotypes. In our cohort of subjects with chronic HCV infection (genotype 1b or 3a), it is a frequently recognized CD8 epitope in HLA-A*01-positive subjects. Viral sequence data reveal that an escape variant is the dominant residue in both genotypes. The predominant Y1444F substitution seemingly impairs binding to the HLA-A*01 molecule, which may have an important impact on the ability to prime a functional CD8 response upon infection. Interestingly, a case of evolution toward the prototype sequence was observed during chronic infection, possibly because the helicase activity of the protein containing the Y1444F substitution is reduced compared to the prototype sequence. Comparison of HCV sequences from Asia and Europe suggests that the frequency of the HLA-A*01 allele in a population may influence the frequency of the escape variant in circulating strains. These data suggest a complex interaction of multiple forces shaping the evolution of HCV in which immune pressure both within the individual and also at the population level in addition to functional constraints are important contributing factors.

Purpose: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are acute-onset mucocutaneous diseases induced by infectious agents and/or inciting drugs. Although the pathobiological mechanisms underlying the onset of SJS/TEN have not been fully established, the extreme rarity of cutaneous and ocular surface reactions to drug therapies led us to suspect individual susceptibility. Our previous study of polymorphisms in the HLA-class I genes of 40 Japanese SJS/TEN patients with ocular surface complications showed that in the Japanese, HLA-A*0206 was strongly associated with SJS/TEN. In this study, we investigated the association between HLA class II antigens in addition to HLA class I antigens and SJS/TEN., Methods: We studied the histocompatibility antigen genes, HLA-A, B, C, DRB1, and DQB1, of 71 Japanese SJS/TEN patients with ocular complications. We also genotyped 113 healthy volunteers for HLA-A, B, C, DRB1, and DQB1. We performed polymerase chain reaction amplification followed by hybridization with sequence-specific oligonucleotide probes (PCR-SSO) using commercial bead-based typing kits., Results: HLA-A*0206 was strongly associated with SJS/TEN. HLA-A*1101 was inversely associated. HLA-B*5901 exhibited a high odds ratio for SJS/TEN with ocular complications. However, when we corrected the p-value for the number of alleles detected (n=29), the results ceased to be significant. There was no association between HLA-C and SJS/TEN. There was also no significant association between HLA-DRB1 and SJS/TEN. HLA-DQB1*0502 was negatively and weakly associated with SJS/TEN although correction of the p-value for the number of alleles detected rendered the result not significant., Conclusions: Because our findings are completely different from data reported on Caucasian patients, they suggest strong ethnic differences in the HLA-SJS associations.

Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8(+) T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.

In the previous studies, some human major histocompatibility complex (MHC) genes such as TNF, LTA and human leukocyte antigen (HLA)-DR2 genes and A1-B8-TNF(-308A) haplotype were implied in non-Hodgkin's lymphoma (NHL) outcome. In the current study, we have assigned most probable six-locus haplotypes determined by HLA-A, -Cw, -B and -DRB1 highly polymorphic genes and non-HLA LTA(+252) and TNF(-308) single nucleotide polymorphisms (SNPs) in 152 NHL Caucasian French patients. We have broadly mapped the MHC region by its component blocks and tagging alleles. Ten frequent (with haplotype frequency >1%) six-locus extended haplotypes (EHs) were revealed in NHL patients. The only two adjacent locus fragment of 8.1 EH associated with shortened freedom from progression (FFP) was B*08-LTA(+252G) (P= 0.0084, RR = 2.45). Interestingly, 305-kbp-long, four-locus fragment of 8.1 EH, Cw*07-B*08-LTA(+252G)-TNF(-308A) block was much strongly associated with shortened FFP (P= 0.00045, RR = 3.26). The analysis of further extended haploblocks comprising five or six loci showed weaker association with outcome measures, suggesting linkage disequilibrium to be the cause of DRB1*03 and A*01 allele associations. In contrast, all fragments of 7.1 EH influenced FFP favorably with top association of TNF(-308G) allele. In multivariate analysis, only Cw*07-B*08-LTA(+252G)-TNF(-308A) and TNF(-308G)-DRB1*01 haplotypes remained predictive for shortened FFP (P= 0.024 and 0.027, respectively) and independent of International Prognostic Index (P= 0.00044). This study reveals that the block composition of EHs may cause important functional differences for NHL outcomes. Further study will be required in NHL patients by fine mapping with dense microsatellite or SNP tags to define susceptibility genes in associating regions.

Human melanoma cells can process the MAGE-1 gene product and present the processed nonapeptide EADPTGHSY on their major histocompatibility complex class I molecules, HLA-A1, as a determinant for cytolytic T lymphocytes (CTLs). Considering that autologous antigen presenting cells (APCs) pulsed with the synthetic nonapeptide might, therefore, be immunogenic, melanoma patients whose tumor cells express the MAGE-1 gene and who are HLA-A1+ were immunized with a vaccine made of cultured autologous APCs pulsed with the synthetic nonapeptide. Analyses of the nature of the in vivo host immune response to the vaccine revealed that the peptide-pulsed APCs are capable of inducing autologous melanoma-reactive and the nonapeptide-specific CTLs in situ at the immunization site and at distant metastatic disease sites.

Previous studies showed that the HLA class I region is associated with Epstein-Barr virus (EBV)-positive Hodgkin lymphoma (HL) and that HLA-A is the most likely candidate gene in this region. This suggests that antigenic presentation of EBV-derived peptides in the context of HLA-A is involved in the pathogenesis of EBV+ HL by precluding efficient immune responses. We genotyped exons 2 and 3, encoding the peptide-binding groove of HLA-A, for 32 single nucleotide polymorphisms in 70 patients with EBV+ HL, 31 patients with EBV- HL, and 59 control participants. HLA-A*01 was significantly overrepresented and HLA-A*02 was significantly underrepresented in patients with EBV+ HL versus controls and patients with EBV- HL. In addition, HLA-A*02 status was determined by immunohistochemistry or HLA-A*02-specific polymerase chain reaction (PCR) on 152 patients with EBV+ HL and 322 patients with EBV- HL. The percentage of HLA-A*02+ patients in the EBV+ HL group (35.5%) was significantly lower than in 6107 general control participants (53.0%) and the EBV- HL group (50.9%). Our results indicate that individuals carrying the HLA-A*02 allele have a reduced risk of developing EBV+ HL, while individuals carrying the HLA-A*01 allele have an increased risk. It is known that HLA-A*02 can present EBV-derived peptides and can evoke an effective immune response, which may explain the protective phenotype.

Background: Hemochromatosis is a recessively inherited disorder caused by a point mutation, C282Y of the HFE gene on chromosome 6p21.3 near the human leukocyte antigen (HLA) locus. It is unknown why some homozygotes develop a severe iron loading, while others do not. A recent study suggested that the A1-B8 haplotype may be associated with higher iron storage., Methods: We studied HLA haplotypes of 85 probands, 31 females and 54 males, and their family members from a rural population where A1-B8 was common. We tested the hypothesis of a modifying effect of the A1-B8 haplotype., Results: Most homozygotes had a mild phenotypic expression, and were often detected accidentally because of a laboratory routine including transferrin saturation. A disease-related morbidity [serum alanine aminotransferase (S-ALT) > 43 U] was present in 40%. Three had porphyria cutanea tarda. Two brothers with A1-B8 died of bronze diabetes, probably caused by co-inheritance of congenital spherocytosis. In females there were no significant differences in phenotypic expression between groups with regard to the presence or absence of A1-B8. Two females, <50 yr of age, with this haplotype had iron deficiency. Males with two copies of A1-B8 had significantly lower serum ferritin (P = 0.02) values than those without. Those with one A1-B8 haplotype were not different from those without. In men without A1-B8, those carrying HLA-A3 were not phenotypically different from those without this ancestral haplotype., Conclusion: The A1-B8 haplotype hitchhiking with the C282Y mutation was not associated with a more efficient iron absorption. On the contrary, males with double copies of this haplotype expressed a milder phenotype, possibly an effect of local (environmental and/or genetic) factors.

Significant associations between human leucocyte antigen (HLA)-A and -B alleles and CD8+ T-lymphocyte numbers have been reported in the literature in both healthy populations and in HFE-haemochromatosis patients. In order to address whether HLA alleles themselves or alleles at linked genes are responsible for these associations, several genetic markers at the MHC class I region were typed on a population of 147 apparently healthy unrelated subjects phenotypically characterized for their CD8+ and CD4+ T-lymphocyte numbers. By using a machine learning approach, a set of rules was generated that predict the number of CD8+ T-lymphocyte numbers on the basis of the information of the D6S105 microsatellite alleles only. We demonstrate that the previously reported associations with HLA-A and -B alleles are due to the presence of common long (up to 4 megabases long) haplotypes that increased in frequency recently due to positive selection and that encompass a region where a putative gene contributing to the setting of CD8+ T lymphocytes is located, in the neighbourhood of microsatellite locus D6S105, in the 6p21.3 region.

Previous reports have implicated the tumor necrosis factor (TNF-308) locus to non-Hodgkin's lymphoma (NHL) outcome. The purpose of the study was to examine other chromosome components of the HLA 8.1 ancestral haplotype (AH) and their relation to the clinical course of NHL. HLA class I, II, TNF-308, and lymphotoxin alpha (LTA+252) alleles were analyzed in 154 newly diagnosed NHL patients. Three locus haplotypes were inferred from the unphased genotypes by a Bayesian implementation of the expectation maximization (EM) algorithm using the PHASE 2.1 program. TNF-308A was the only allele associated with fever, poor performance status, elevated beta2-microglobulin, TNF and its p75 receptor plasma levels. Although TNF-308A was in strong linkage disequilibrium with the remaining alleles of 8.1 AH, only HLA-A*01 and HLA-B*08 showed association with prognostic variables. A part of 8.1 AH (A*01-B*08-TNF-308A) was predictive for shorter freedom from progression and overall survival (RR=2.47, P=0.041; RR=3.15; P=0.0049), an association that was stronger than TNF-308A alone and independent from International Prognostic Index (RR=1.55, P<0.001; RR=2.36; P<0.0001). A*01-B*08-TNF-308A fragment of 8.1 AH remained an independent predictive factor in a multivariate model. We conclude that 8.1 AH is an important contributor to NHL outcome. In contrast to A*01-B*08-TNF(-308A, the remaining alleles (Cw*07, DRB1*03, LTA+252G) associated with the 8.1 AH seem to be its passive components., ((c) 2007 Wiley-Liss, Inc.)

Treatment of chondrosarcomas is limited to resection because these tumors are unresponsive to standard adjuvant treatments, such as chemotherapy and radiation. We have previously shown that high-grade chondrosarcomas express unspecified members of the Melanoma Antigen (MAGE) gene family. We show here that FS human chondrosarcoma (FS) cells express MAGE-A3 gene and HLA-A1 molecules. In vitro assays show that a cytolytic T-lymphocyte clone (CTL) specific for a MAGE-A3 peptide presented by HLA-A1 specifically lysed FS chondrosarcoma cells. Addition of antigenic peptide did not increase the susceptibility of FS cells to CTL mediated lysis, suggesting that HLA-A1 expression by the chondrosarcoma cells limited their susceptibility to lysis by the anti-MAGE-A3 CTL clone. Incubation of FS cells with 50 U/mL interferon-gamma increased surface expression of HLA class-I molecules, increased their susceptibility to lysis, and had no effect on MAGE-A3 gene expression. These results suggest that immunotherapy targeted against chondrosarcoma cells is possible., ((c) 2007 Orthopaedic Research Society.)

Cancer cells' growth in three-dimensional (3D) architectures promotes resistance to drugs, cytokines, or irradiation. We investigated effects of 3D culture as compared to monolayers (2D) on melanoma cells' recognition by tumour-associated antigen (TAA)-specific HLA-A(*)0201-restricted cytotoxic T-lymphocytes (CTL). Culture of HBL, D10 (both HLA-A(*)0201+, TAA+) and NA8 (HLA-A(*)0201+, TAA-) melanoma cells on polyHEMA-coated plates, resulted in generation of 3D multicellular tumour spheroids (MCTS). Interferon-gamma (IFN-gamma) production by HLA-A(*)0201-restricted Melan-A/MART-1(27-35) or gp 100(280-288)-specific CTL clones served as immunorecognition marker. Co-culture with melanoma MCTS, resulted in defective TAA recognition by CTL as compared to 2D as witnessed by decreased IFN-gamma production and decreased Fas Ligand, perforin and granzyme B gene expression. A multiplicity of mechanisms were potentially involved. First, MCTS per se limit CTL capacity of recognising HLA class I restricted antigens by reducing exposed cell surfaces. Second, expression of melanoma differentiation antigens is downregulated in MCTS. Third, expression of HLA class I molecules can be downregulated in melanoma MCTS, possibly due to decreased interferon-regulating factor-1 gene expression. Fourth, lactic acid production is increased in MCTS, as compared to 2D. These data suggest that melanoma cells growing in 3D, even in the absence of immune selection, feature characteristics capable of dramatically inhibiting TAA recognition by specific CTL.

The FLT3 receptor tyrosine kinase is expressed in more than 90% of acute myelogeneous leukemias (AMLs), up to 30% of which carry an internal tandem duplication (ITD) within the FLT3 gene. Although varying duplication sites exist, most FLT3-ITDs affect a single protein domain. We analyzed the FLT3-ITD of an AML patient for encoding HLA class I-restricted immunogenic peptides. One of the tested peptides (YVDFREYEYY) induced in vitro autologous T-cell responses restricted by HLA-A*0101 that were also detectable ex vivo. These peptide-reactive T cells recognized targets transfected with the patient's FLT3-ITD, but not wild-type FLT3, and recognized the patient's AML cells. Our results demonstrate that AML leukemic blasts can in principle process and present immunogenic FLT3-ITD neoepitopes. Therefore, FLT3-ITD represents a potential candidate target antigen for the immunotherapy of AML.

We identified an A*0114 allele in an Irish patient with an apparent A*0236, A*3601 type.

Non small cell lung cancers (NSCLC) express cancer/testis antigens (CTA) genes and MAGE-A expression correlates with poor prognosis in squamous cell carcinomas. We addressed cytotoxic T lymphocytes (CTL) responses to HLA class I restricted CTA epitopes in TIL from NSCLC in an unselected group of 33 patients consecutively undergoing surgery. Expression of MAGE-A1, -A2, -A3, -A4, -A10, -A12 and NY-ESO-1 CTA genes was tested by quantitative RT-PCR. Monoclonal antibodies (MAb) recognizing MAGE-A and NY-ESO-1 CTA were used to detect CTA by immunohistochemistry. CD8(+) TIL obtained from tumors upon culture with anti CD3 and anti CD28 mAb and IL-2 were stimulated with autologous mature DC (mDC) and HLA-A*0101 restricted MAGE-A1(161-169) or MAGE-A3(168-176) peptides or HLA-A*0201 restricted MAGE-A4(230-239), MAGE-A10(254-262), NY-ESO-1(157-165) or multi-MAGE-A (YLEYRQVPV) peptides or a recombinant vaccinia virus (rVV) encoding MAGE-A and NY-ESO-1 HLA-A*0201 restricted epitopes and CD80 co-stimulatory molecule. Specificity was assessed by (51)Cr release and multimer staining. At least one CTA gene was expressed in tumors from 15/33 patients. In 10 specimens, at least 4 CTA genes were concomitantly expressed. These data were largely confirmed by immunohistochemistry. TIL were expanded from 26/33 specimens and CTA-specific CTL activity was detectable in 7/26 TIL. In 6, however, specific cytotoxicity was weak, (<40% lysis at a 50:1 E:T ratio) and multimer staining was undetectable. In one case, high (>60% lysis at 50:1 E:T ratio) MAGE-A10(254-262) specific, HLA-A*0201 restricted response was observed. Supportive evidence was provided by corresponding multimer staining. Although CTA genes are frequently expressed in NSCLC, detection of CTL reactivity against CTA epitopes in TIL from nonimmunized NSCLC patients represents a rare event., ((c) 2006 Wiley-Liss, Inc.)

Despite numerous attempts to increase the neutrophil count of infants with alloimmune neonatal neutropenia, no therapy has been consistently effective. We describe two infants with alloimmune neutropenia who had a rapid and prolonged increase in neutrophil number after treatment with granulocyte colony-stimulating factor (G-CSF). Patient 1 had antibody directed against the neutrophil antigen NA2. He received three daily doses of G-CSF, and within 2 days his neutrophil count increased from 0.350 x 10(9) to 3.584 x 10(9)/L (350 to 3584/mm3). Despite cessation of treatment the neutrophil count remained in the normal range. Patient 2 had antibody to the neutrophil antigen NA1, and received six daily doses of G-CSF. Within 4 days his neutrophil count increased from 0.477 x 10(9) to 4.320 x 10(9)/L (477 to 4320/mm3) and remained in the normal range for 11 days after the last dose of G-CSF. We recommend that treatment with G-CSF be considered for selected infants with alloimmune neutropenia.

Purpose: The transcription factor Wilms tumor protein 1 (WT1) belongs to a new generation of tumor antigens, as it is essential for tumor cell proliferation and is highly expressed in various hematologic and solid malignancies. The aim of this study was to apply a modified reverse immunology strategy to identify immunogenic epitopes of WT1 which could be useful for immunotherapy., Experimental Design: Potential HLA-A*01 epitopes predicted by a MHC binding algorithm were screened for recognition by peripheral blood mononuclear cells (PBMC) from patients with spontaneous T cell responses using intracellular cytokine cytometry. Epitope processing was shown by proteasomal cleavage. Epitope-specific T cells were generated from CD4+CD25+ regulatory T cell-depleted PBMC., Results: One of five predicted HLA-A*01-binding candidate epitopes showed high immunogenicity as 5 of 14 patients with hematologic malignancies had WT1.317-327-reactive T cells ranging from 0.4% to 1.5% of CD3+CD8+ T cells. Proteasomal degradation assays indicated the cleavage of WT1.317-327. The depletion of regulatory T cells from PBMCs enabled the rapid expansion of WT1.317-327-specific CTL, whereas no CTL could be generated from unfractionated PBMC. WT1.317-327-specific CTL efficiently lysed an autologous WT1-expressing tumor cell line but not HLA-A*01-negative WT1-expressing tumor cells. Immunogenicity of the epitope across histologies was verified by the demonstration of spontaneous ex vivo WT1.317-327-specific T cell responses in two of six patients with HLA-A*01-positive melanoma or lung cancer., Conclusion: In this study, a modified reverse immunology strategy was employed to identify a first immunogenic HLA-A*01-restricted T cell epitope of the tumor antigen WT1, which is of considerable interest for use in vaccination trials.

Background: Minor histocompatibility (H) antigens are peptides of allelic intracellular proteins that play an important role in human leukocyte antigen (HLA) matched transplantations. In an animal model of keratoplasty, minor H antigens have even been reported to exceed the immunogenicity of major H antigens (MHC). This investigation is to assess any benefit of matching the broadly expressed gender (H-Y) and HA-3 antigens in HLA-A1 donor positive human keratoplasty., Methods: A total of 229 HLA-A1 donor positive keratoplasties were analyzed. A Cox proportional hazards model and Kaplan-Meier analysis were applied to estimate the effect of H-Y or HA-3 mismatches on rejection-free graft survival., Results: Eighty-one cases were mismatched for H-Y (male donor to female recipient). A mean follow up of two years showed graft survival as high as 88% in the H-Y compatible group compared to only 77% in the H-Y mismatched group (P = 0.02). Eight out of 62 cases were mismatched for HA-3. No statistically significant influence of HA-3 matching on rejection-free graft survival was observed (85% vs. 73%, P=0.52)., Conclusion: HLA-A1/H-Y matching and matching for other broadly expressed minor H antigens may further improve prognosis in keratoplasty.

Human telomerase reverse transcriptase (hTERT) represents an attractive target for cancer immunotherapy because hTERT is reactivated in most human tumors. In an attempt to develop an effective vaccine against most human cancers, we constructed chemotactic-hTERT vaccine. Two hTERT fragments encoding multiple cytotoxic T lymphocyte and T helper cell epitopes were fused as a tumor antigen (named Te). The plasmid based DNA vaccine (pCCL21-Te-Fc) was constructed by linking human CCL21 and IgG Fc gene sequences to each end of Te. In poorly immunogenic B16F10 mouse melanoma model, DNA (pCCL21-Te-Fc) vaccination significantly inhibited tumor growth and all of the mice were dead by day 52. The immunization with pCCL21-Te-Fc-modified tumor cells (B16/CCL21-Te-Fc) resulted in a higher antitumor effect than DNA vaccination and 25% of tumor-bearing mice achieved long-term survival (> 120 days). The combined therapy of B16/CCL21-Te-Fc plus anti-4-1BB MAbs further enhanced the immune response, resulting in 75% of tumor-bearing mice achieved long-term survival (> 120 days) in subcutaneous model and few lung nodules in pulmonary metastasis model. Rechallenge experiment showed that a persistent memory response was successfully induced by the combined therapy. In vivo depletion of lymphocytes indicated that CD8+ T cells were essential in the antitumor activity induced by B16/CCL21-Te-Fc plus anti-4-1BB MAbs, whereas NK cells and CD4+ T cells played substantial roles. The CTL activity induced by pCCL21-Te-Fc-transfected PBMCs specifically lysed a variety of human leukocyte antigen-matched and hTERT-positive human tumor cells, suggesting pCCL21-Te-Fc could serve as a vaccine against most human cancers., (Copyright 2006 Wiley-Liss, Inc.)

Technology has become available to cost-effectively analyze thousands of single nucleotide polymorphisms (SNPs). We recently confirmed by genotyping a small series of class I alleles and microsatellite markers that the extended haplotype HLA-A1-B8-DR3 (8.1 AH) at the major histocompatibility complex (MHC) is a common and conserved haplotype. To further evaluate the region of conservation of the DR3 haplotypes, we genotyped 31 8.1 AHs and 29 other DR3 haplotypes with a panel of 656 SNPs spanning 4.8 Mb in the MHC region. This multi-SNP evaluation revealed a 2.9-Mb region that was essentially invariable for all 31 8.1 AHs. The 31 8.1 AHs were >99.9% identical for 384 consecutive SNPs of the 656 SNPs analyzed. Future association studies of MHC-linked susceptibility to type 1 diabetes will need to account for the extensive conservation of the 8.1 AH, since individuals who carry this haplotype provide no information about the differential effects of the alleles that are present on this haplotype.

We carried out a resequencing project that examined 552 kb of sequence from each of 46 individual HLA haplotypes representing a diversity of HLA allele types, generating nearly 27 Mb of fully phased genomic sequence. Haplotype blocks were defined extending from telomeric of HLA-F to centromeric of HLA-DP including in total 5186 MHC SNPs. To investigate basic questions about the evolutionary origin of common HLA haplotypes, and to obtain an estimate of rare variation in the MHC, we similarly examined two additional sets of samples. In 19 independent HLA-A1, B8, DR3 chromosomes, the most common HLA haplotype in Northern European Caucasians, variation was found at 11 SNP positions in the 3600-kb region from HLA-A to DR. Partial resequencing of 282 individuals in the gene-dense class III region identified significant variability beyond what could have been detected by linkage to common SNPs.

Emerging in vitro evidence points to an immunomodulatory activity of DNA hypomethylating drugs in human malignancies. We investigated the potential of 5-aza-2'-deoxycytidine (5-AZA-CdR) to modulate the expression of cancer testis antigens (CTA) and of HLA class I antigens by melanoma xenografts, and the resulting modifications in immunogenicity of neoplastic cells. Three primary cultures of melanoma cells, selected for immune phenotype and growth rate, were grafted into BALB/c nu/nu mice that were injected intraperitoneally with different dose- and time-schedules of 5-AZA-CdR. Molecular analyses demonstrated a de novo long-lasting expression of the CTA MAGE-1, -2, -3, -4, -10, GAGE 1-6, NY-ESO-1, and the upregulation of MAGE-1, MAGE-3, and NY-ESO-1 levels in melanoma xenografts from 5-AZA-CdR-treated mice. Serological and biochemical analyses identified a de novo expression of NY-ESO-1 protein and a concomitant and persistent upregulation of HLA class I antigens and of HLA-A1 and -A2 alleles. Immunization of BALB/c mice with 5-AZA-CdR-treated melanoma cells generated high titer circulating anti-NY-ESO-1 antibodies. Altogether, the data obtained identify an immunomodulatory activity of 5-AZA-CdR in vivo and strongly suggest for its clinical use to design novel strategies of CTA-based chemo-immunotherapy for melanoma patients.

We describe an HLA-A1 melanoma patient who has mounted a spontaneous cytolytic T cell (CTL) response against an antigenic peptide encoded by gene MAGE-A3 and presented by HLA-A1. The frequency of anti-MAGE-3.A1 CTLp was 5 x 10(-7) of the blood CD8 cells, with a dominant clonotype which was present in six out of seven independent anti-MAGE-3.A1 CTL clones. After vaccination with a recombinant poxvirus coding for the MAGE-3.A1 antigen, the blood frequency of anti-MAGE-3.A1 CTLp increased tenfold. Twenty-two independent CTL clones were derived. Surprisingly, only one of them corresponded to the dominant clonotype present before vaccination. Two new clonotypes were repeated 12 and 7 times, respectively. Our interpretation of these results is that the spontaneous anti-MAGE-3.A1 CTL response pre-existing to vaccination was polyclonal, and that the vaccine restimulated only some of these clones. To estimate the incidence of spontaneous anti-MAGE-3.A1 CTL responses in melanoma patients with a tumor expressing gene MAGE-A3, we measured the blood frequency of anti-MAGE-3.A1 T cells in 45 patients, and found only two clear responses.

Signals transduced by inhibitory receptors that recognize self-MHC class I molecules prevent NK cells from being activated by autologous healthy target cells. In order for NK cells to be activated upon contact with an infected cell, the balance between the activating and inhibitory signals that regulate NK cell function must be altered in favor of activation. By studying liver-derived NK cells, we show that only a subpopulation of NK cells expressing high levels of the inhibitory receptor NKG2A are able to lyse autologous vaccinia-infected targets, and that this is due to selective down-regulation of HLA-E. These data demonstrate that release from an inhibitory receptor:ligand interaction is one mechanism that permits NK cell recognition of a virally infected target, and that the variegated expression of inhibitory receptors in humans generates a repertoire of NK cells with different antiviral potentials.

Esophageal squamous cell carcinoma is the 6th most commonly occurring cancer worldwide. A relationship between HLA A1 and B40 and esophageal cancer was described in patients examined in China. The aim of this study was to investigate the relation of HLA class 1 and esophageal carcinoma in the northwestern region of Iran. Using specific monoclonal antibodies, different human leukocyte antigens (HLA) were quantified in 100 patients suffering esophageal carcinoma in Tabriz, a major city located in the Northwestern region of Iran. These data were compared to those of 100 healthy matched individuals as a control group from the same region. HLA B14 and A24 were increased and showed statistically significant correlation in squamous cell carcinoma. These findings may also indicate the association between genetic factors and esophageal carcinoma. Further studies are suggested for detecting correlation of HLA and esophageal carcinoma in other regions.

Expression of markers, collagens, and HLA-1 by human skin fibroblasts and fibroblast-like cells isolated from the umbilical Wharton's jelly was compared. Skin fibroblasts express collagens (proteins characteristic of differentiated cells of this histogenetic series) and HLA-1, while umbilical cells express, in addition to collagens, juvenile surface markers and almost no HLA-1. This indicates that fibroblast-like cells isolated from different sources are different and can serve as sources for the creation of cell preparations with different characteristics in future.

We report the identification of a new HLA-A null allele, HLA-A*0115N. This null allele has been identified within the A*01 group by a combination of serological and molecular typing [Polymerase chain reaction (PCR) sequence-specific primers, PCR sequence-specific oligoprobes and sequence-based typing (SBT)] in a potential intrafamilial bone marrow donor from Martinique (French West Indies). To characterize this A*01 null allele, we performed DNA typing by PCR-SBT on genomic DNA from the beginning of exon 2 (position 84) through the end of the exon 4 (position 895) and revealed a nucleotide deletion at the end of the exon 3. This sole difference between the new allele and the HLA-A*0101 generates a premature stop codon (TGA) in the beginning of exon 4. This deletion most likely explains the lack of cell surface expression of the encoded protein despite the presence of A*01 allele. The absence of correct expression of the antigen on the cell surface was confirmed by one-dimensional isoelectric focusing (1D-IEF). To date, this is the fourth null allele described within the A*01 group.

To determine if the hepatitis viruses are important etiologic factors in autoimmune hepatitis, the clinical, immunoserologic, virologic and HLA phenotypes of 105 of the latter patients were assessed prospectively and compared to 45 patients with chronic viral hepatitis. Patients with autoimmune hepatitis were more often women with higher serum aspartate aminotransferase and immunoglobulin levels than patients with viral disease. Only eight patients (8%) were seropositive for anti-HBc and anti-HBs (four patients) or anti-HCV (four patients) and none with anti-HCV were reactive by second generation immunoassay or recombinant immunoblot assay. Smooth muscle (90 vs. 22%, P < 0.001) and antinuclear (70 vs. 22%, P < 0.001) antibodies were more common in patients with autoimmune hepatitis and the titers more frequently exceeded 1:80 (84 vs. 11%, P < 0.0001). Patients with autoimmune hepatitis were more often positive for HLA B8 (48 vs. 20%, P < 0.01) and DR3 (49 vs. 20%, P < 0.003) and they more frequently had the HLA A1-B8-DR3 phenotype (38 vs. 10%, P < 0.003). Only one of the 120 patients tested for anti-LKM1 was seropositive. We conclude that in an American referral population autoimmune hepatitis usually lacks virologic markers and has a distinctive clinical, immunoserologic and HLA phenotype. Hepatitis viruses are not important immunogenic stimuli for non-organ specific antibodies and they are unlikely to be important causes of this form of autoimmune hepatitis.

Tyrosinase-related protein-2 (TRP-2) is a known target antigen of spontaneous cytotoxic T cell responses in melanoma patients. Its frequent expression in metastatic tumors suggests that it might be an ideal candidate antigen for T cell-based immunotherapy. To provide knowledge about TRP-2-derived T cell epitopes useful for immunotherapy we applied a "reverse immunology strategy" based on repeated in vitro peptide stimulation of peripheral blood lymphocytes (PBL) from normal donors with predicted HLA-A*01 ligands. This led to the identification of TRP-2(181-190) as the first HLA-A*01-presented TRP-2-derived epitope. T-cell lines specific for peptide TRP-2(181-190) could be established from PBL of 50% of the normal HLA-A*01(+) donors tested. Such T cells responded specifically to autologous dendritic cells transduced virally with TRP-2, as well as to HLA-A*01(+), TRP-2(+) melanoma cells, although tumor cells had to be pretreated with IFN-gamma to become susceptible to T cell recognition. Interestingly, short-term in vitro peptide stimulation of PBL from HLA-A*01(+) melanoma patients showed the presence of TRP-2(181-190)-reactive CD8(+) T cells in some donors, suggesting their in vivo sensitization. Because TRP-2(181-190) overlaps with the known HLA-A*0201-presented epitope TRP-2(180-188), an 11mer peptide encompassing both epitopes might be of specific value for vaccination of a broad population of melanoma patients.

The ability of influenza A virus-specific cytotoxic T lymphocytes (CTL) to degranulate and produce cytokines upon antigenic restimulation was studied in four HLA-A*0101 and HLA-A*0201 positive subjects. Peripheral blood mononuclear cells of these subjects were stimulated with influenza A virus in the presence of high or low interleukin (IL)-2 concentrations. CD8(+) T cell populations specific for the HLA-A*0101 restricted epitope NP(44-52) and the HLA-A*0201 restricted epitope M1(58-66) were identified by positive staining with tetramers of peptide major histocompatibility complexes (MHC) (NP-Tm and M1-Tm, respectively). Within these populations, the proportion of cells mobilizing CD107a, or expressing interferon (IFN)-gamma and tumour necrosis factor-(TNF)-alpha upon short-term peptide restimulation was determined by flow cytometry. Independent of IL-2 concentrations, large subject-dependent differences in the mobilization of CD107a and expression of IFN-gamma and TNF-alpha by both NP- and M1-specific T cells were observed. In two of the four subjects, the functional profile of NP-Tm(+) and M1-Tm(+) cells differed considerably. Overall, no difference in the proportion of NP-Tm(+) or M1-Tm(+) cells expressing CD107a was observed. The proportion of M1-Tm(+) cells that produced IFN-gamma (P < 0.05) was larger than for NP-Tm(+) cells, independent of IL-2 concentration. When cultured under IL-2(hi) concentrations higher TNF-alpha expression was also observed in M1-Tm(+) cells (P < 0.05). The IL-2 concentration during expansion of virus-specific cells had a profound effect on the functionality of both M1-Tm(+) and NP-Tm(+) cells.