HLA allele and haplotype diversity in the Croatian population: State of the art.

The aim of this report was to compare our data with data presented in the European Federation for Immunogenetics (EFI) catalogue (version 1.0) and to evaluate whether or not current (CUR) HLA alleles found in the Croatian population fall into the same categories of common (COM) or well-documented (WD) HLA alleles as those listed in the EFI catalogue. Among 237 HLA-A, -B, -DRB1 alleles observed in the Croatian population so far, 181 alleles were observed ≥3 times. According to our criteria, 36 alleles at HLA-A locus, 71 alleles at HLA-B locus and 51 alleles at HLA-DRB1 locus were characterised as CUR in Croatia (COM or WD in EFI catalogue), while 23 local HLA alleles are not listed at all among COM or WD alleles in EFI catalogue.

Clustering HLA class I superfamilies using structural interaction patterns.

Human leukocyte antigen (HLA) class I molecules are critical components of the cell-mediated immune system that bind and present intracellular antigenic peptides to CD8(+) T cell receptors. To understand the interaction mechanism underlying human leukocyte antigen (HLA) class I specificity in detail, we studied the structural interaction characteristics of 16,393 nonameric peptides binding to 58 HLA-A and -B molecules. Our analysis showed for the first time that HLA-peptide intermolecular bonding patterns vary among different alleles and may be grouped in a superfamily dependent manner. Through the use of these HLA class I 'fingerprints', a high resolution HLA class I superfamily classification schema was developed. This classification is capable of separating HLA alleles into well resolved, non-overlapping clusters, which is consistent with known HLA superfamily definitions. Such structural interaction approach serves as an excellent alternative to the traditional methods of HLA superfamily definitions that use peptide binding motifs or receptor information, and will help identify appropriate antigens suitable for broad-based subunit vaccine design.

Distributions of human leukocyte antigen-A, -B, and -DRB1 alleles and haplotypes based on 46,915 Taiwanese donors.

Human leukocyte antigen (HLA)-A, -B, and -DRB1 alleles were typed in 46,915 healthy Taiwanese volunteers recruited for Tzu Chi Taiwan Marrow Donor Registry (TCTMDR). The volunteers were separated into Taiwanese and Taiwanese aborigines. In this study, a total of 51 A, 121 B, and 53 DRB1 alleles were found in the Taiwanese group, and 17 A, 32 B, and 23 DRB1 alleles were identified in the Taiwanese aborigines. Some commonly shared alleles appeared more frequently in one group than in the other. The two haplotypes, among the 20 most frequently observed haplotypes in each group, shared in common by both groups were A*3303-B*5801-DRB1*0301 and A*0207-B*4601-DRB1* 0901. However, both haplotype frequencies in each group were extremely different, indicating the existence of genetic diversity between the two groups. In addition, principal component analysis and clustering results based on high-resolution HLA-A, -B, and -DRB1 alleles indicated that the Taiwanese group was closest to Southern Chinese and reiterated HLA diversity between the Taiwanese and the Taiwanese aborigines. We believe that our findings in this study may provide useful information in search for HLA-matched donors for patients.

Role of human leukocyte antigen class I alleles in progressive multifocal leukoencephalopathy.

Because human leukocyte antigen (HLA) associations with various infectious diseases have recently been reported, we examined the role of HLA class I alleles in the development of progressive multifocal leukoencephalopathy (PML) or its outcome in 152 patients, including 123 Caucasians and 29 African Americans. Compared to a human immunodeficiency virus positive (HIV+) control population, we observed decreased frequency of HLA-A3 (P = 0.03) in the Caucasian PML group, whereas B18 (P = 0.02), was more frequent. No such difference was found among African American PML patients. We then sought to characterize differences in HLA between PML progressors, whose survival doesn't exceed 1 year, and survivors. Caucasian survivors were less likely to harbor A68 (P = 0.01), whereas African American survivors less frequently displayed Cw4 (P = .01). However, none of these differences reached statistical significance after Bonferroni correction for multiple testing. Further investigations are needed to assess the role of genetics in the incidence of PML or its outcome. Physicians may exercise caution in the use of immunomodulatory medications in patients whose genetic background is associated with an increased risk of PML.

PEPVAC: a web server for multi-epitope vaccine development based on the prediction of supertypic MHC ligands.

Prediction of peptide binding to major histocompatibility complex (MHC) molecules is a basis for anticipating T-cell epitopes, as well as epitope discovery-driven vaccine development. In the human, MHC molecules are known as human leukocyte antigens (HLAs) and are extremely polymorphic. HLA polymorphism is the basis of differential peptide binding, until now limiting the practical use of current epitope-prediction tools for vaccine development. Here, we describe a web server, PEPVAC (Promiscuous EPitope-based VACcine), optimized for the formulation of multi-epitope vaccines with broad population coverage. This optimization is accomplished through the prediction of peptides that bind to several HLA molecules with similar peptide-binding specificity (supertypes). Specifically, we offer the possibility of identifying promiscuous peptide binders to five distinct HLA class I supertypes (A2, A3, B7, A24 and B15). We estimated the phenotypic population frequency of these supertypes to be 95%, regardless of ethnicity. Targeting these supertypes for promiscuous peptide-binding predictions results in a limited number of potential epitopes without compromising the population coverage required for practical vaccine design considerations. PEPVAC can also identify conserved MHC ligands, as well as those with a C-terminus resulting from proteasomal cleavage. The combination of these features with the prediction of promiscuous HLA class I ligands further limits the number of potential epitopes. The PEPVAC server is hosted by the Dana-Farber Cancer Institute at the site http://immunax.dfci.harvard.edu/PEPVAC/.

Characterization of a novel allele HLA-B*3549.

A novel HLA-B allele, B*3549, was identified in a bone marrow transplantation candidate. B*3549 differs from B*3525 by two nucleotides at exon 2, position 142 (T to G) and 165 (G to C). The difference at position 142 resulted in an amino acid difference (serine to alanine). However, the difference at position 165 did not cause any amino acid change. This novel allele was found on a haplotype with A*3101, B*3549, Cw*0401, DRB1*0407, and DQB1*0302.

Is there a relationship between HLA type and prognostic factors in breast cancer?

No convincing association exists between HLA type and breast cancer development but certain HLA types have been suggested to be associated with poor risk disease. Here, the HLA type (class I and II) for 141 breast cancer patients was compared to a control population of 100 individuals and to the prognostic indicators for the patients. No association was found between HLA type and breast cancer development. Consideration of individual HLA/prognostic factor relationships previously reported confirmed that HLA-B7 was over-represented in premenopausal oestrogen-receptor (ER)-positive, grade 3 tumours (p = 0.04) and that HLA-A1 correlated positively with Nottingham Prognostic Index (p < 0.05) and with ER-negative disease (p < 0.05). These findings suggest that some previously identified associations between HLA type I and particular prognostic factors may be real, if weak but appear to conflict with the only other sizeable study investigating HLA type II in breast cancer (which negatively correlated HLA-DR 11 with early onset disease).

New HLA-B alleles identified and sequences extended in potential bone marrow donors: B*5804, B*4418, B*1558, and B*4805.

Nucleic acid-based methods for allele identification have revealed more than 470 polymorphic variants at the HLA-B locus. Screening of potential bone marrow donors with sequence specific primer polymerase chain reactions and sequence specific oligonucleotide probe hybridization assays revealed apparent variants within the B*58, *44, *15, and *48 allele groups. DNA sequencing of cloned DNA identified the new alleles B*5804, B*4418, and B*1558 within these groups and observed new sequence information for the previously reported allele B*4805. These findings further extend our knowledge of the substantial genetic variation present at the HLA-B locus within human populations.

The HLA Dictionary 2001: a summary of HLA-A, -B, -C, -DRB1/3/4/5 and -DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR and -DQ antigens.

This report presents the serological equivalents of 123 HLA-A, 272 HLA-B and 155 HLA-DRB1 alleles. The equivalents cover over 64% of the presently identified HLA-A, -B and -DRB1 alleles. The dictionary is an update of the one published in 1999 (<1>Schreuder et al., 1999, Tissue Antigens, 54, 409) and also includes equivalents for HLA-C, DRB3, DRB4, DRB5 and DQB1 alleles. The data summarize information obtained by the WHO Nomenclature Committee for Factors of the HLA System, the International Cell Exchange (UCLA), the National Marrow Donor Program (NMDP) and individual laboratories. In addition, a listing is provided of alleles that are expressed as antigens with serological reaction patterns that differ from the well-established HLA specificities. The equivalents provided will be useful in guiding searches for unrelated hematopoietic stem cell donors in which patients and/or potential donors are typed by either serology or DNA-based methods. These equivalents will also serve typing and matching procedures for organ transplant programmes where HLA typings from donors and from recipients on waiting lists represent mixtures of serological and molecular typings. The tables with HLA equivalents and a questionnaire for submission of serological reaction patterns for poorly identified allelic products will also be available on the WMDA web page: www.worldmarrow.org

HLA-B*4413 identified in a UK Caucasoid potential bone marrow donor.

We describe a novel allele belonging to the B*44 allele group. HLA-B*4413 was identified in a Caucasoid potential bone marrow donor from the Anthony Nolan Bone Marrow Trust register. Initial HLA typing of this donor revealed unusual serological reactivity. Further investigation was carried out by reference strand conformation analysis (RSCA) and sequence-based typing (SBT) using DNA extracted from an Epstein-Barr virus (EBV)-transformed B-cell line made from this donor (AMI005AN). In this individual, two B*44 alleles were revealed upon initial investigation: B*4409 and a previously unseen allele which has been named B*4413. B*4413 is identical to B*44031 except for a unique nucleotide substitution resulting in an amino acid difference at residue 61 in the alpha1 helix.

HLA-B*15 diversity in the Korean population.

Alleles in the HLA-B*15 group encode molecules belonging to several serologic subgroups, B15 (B62, B63, B75, B76, B77) and B70 (B71, B72), representing many of the most problematic types to assign in routine clinical typing laboratories due to their serologic cross-reactivity resulting from structural similarity. More than 25% of Koreans express HLA-B molecules encoded by the HLA-B*15 alleles. To further characterize HLA-B*15 in this population, B*15-specific polymerase chain reaction (PCR) and sequence-specific oligonucleotide probe (SSOP) hybridization analysis using 39 digoxigenin-labeled probes were applied to DNA samples obtained from 237 B15/B70 serologically positive unrelated individuals. Nine B*15 alleles were identified. B*1501 was the most frequent allele (64.8%) followed by B*1511 (14.1%), B*1507 (8.6%), and B*1518 (5.5%) comprising more than 90% of B*15-positive samples. B62 molecules encoded by 4 of the identified alleles (B*1501, B*1507, B*1525, and B*1527) could not be discriminated by serologic reaction patterns. Among the fifteen B15/B70 apparent homozygotes, eight were heterozygotes carrying two different B*15 alleles. Several B*15 alleles exhibited strong associations with specific Cw, DRB1, and A allelic types (e.g., B*1507-Cw3 (22/22); B*1507-DRB1*04 (21/22), B*1507-A24 (17/22)). The data obtained in this study confirmed B*15 diversity in the study population and will be useful in hematopoietic stem cell donor searches as well as in determining the supplementary DNA typing strategy for B15/B70-positive samples in this population.

Characterization of the novel allele HLA-B*4417: implications for bone marrow transplantation.

The identification of the novel allele HLA-B*4417, which was found in a blood donor of Caucasian origin, is described. In the sequence analysis the new allele differs from B*4402 by three nucleotides in exon 3. At the protein level, the new allele has two residue differences compared to B*4402. Due to substantial differences with other B*44 variants a possible mismatch may impair clinical outcome of bone marrow transplantation.

Induction of human papillomavirus-specific CD4(+) and CD8(+) lymphocytes by E7-pulsed autologous dendritic cells in patients with human papillomavirus type 16- and 18-positive cervical cancer.

Human papillomavirus (HPV) type 16 (HPV 16) and HPV type 18 (HPV 18) are implicated in the induction and progression of the majority of cervical cancers. Since the E6 and E7 oncoproteins of these viruses are expressed in these lesions, such proteins might be potential tumor-specific targets for immunotherapy. In this report, we demonstrate that recombinant, full-length E7-pulsed autologous dendritic cells (DC) can elicit a specific CD8(+) cytotoxic T-lymphocyte (CTL) response against autologous tumor target cells in three patients with HPV 16- or HPV 18-positive cervical cancer. E7-specific CTL populations expressed strong cytolytic activity against autologous tumor cells, did not lyse autologous concanavalin A-treated lymphoblasts or autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL), and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. Cytotoxicity against autologous tumor cells could be significantly blocked by anti-HLA class I (W6/32) and anti-CD11a/LFA-1 antibodies. Phenotypically, all CTL populations were CD3(+)/CD8(+), with variable levels of CD56 expression. CTL induced by E7-pulsed DC were also highly cytotoxic against an allogeneic HLA-A2(+) HPV 16-positive matched cell line (CaSki). In addition, we show that specific lymphoproliferative responses by autologous CD4(+) T cells can also be induced by E7-pulsed autologus DC. E7-specific CD4(+) T cells proliferated in response to E7-pulsed LCL but not unpulsed LCL, and this response could be blocked by anti-HLA class II antibody. Finally, with two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, a marked Th1-like bias (as determined by the frequency of gamma interferon- and interleukin 4-expressing cells) was observable for both CD8(+) and CD4(+) E7-specific lymphocyte populations. Taken together, these data demonstrate that full-length E7-pulsed DC can induce both E7-specific CD4(+) T-cell proliferative responses and strong CD8(+) CTL responses capable of lysing autologous naturally HPV-infected cancer cells in patients with cervical cancer. These results may have important implications for the treatment of cervical cancer patients with active or adoptive immunotherapy.

HLA-A, -B, -C genotyping and expression in human nonlymphoid tumor cell lines.

A combination of molecular genotyping and protein biochemistry methods was used to assess the HLA-A, -B, -C genotyping and expression of six tumor cell lines. Four cell lines had been previously HLA typed by conventional serologic methods. Two could not be typed by serology because deficient in the surface expression of HLA-A, -B, -C molecules. As shown herein, all the 25 alleles carried by the six tested cell lines were typed at the DNA level. In addition, discrepancies between the previous serologic and the present DNA typing results were detected in 9 of the 21 tested serologic specificities. Typing at the protein level by isoelectric focusing and allele-specific monoclonal antibodies confirmed the DNA typing data. Our results exemplify the limits of the serologic typing procedures and demonstrate that molecular methods are highly desirable to conduct functional experiments and identify HLA losses in neoplastic cells at single allele level.

HLA-B44 subtypes and the chance of finding HLA compatible donor/recipient pairs for bone marrow transplantation: a haplotype study of 303 Italian families.

A total of 1176 HLA-A,B,DR haplotypes were reconstructed by typing 303 unrelated families referred to our laboratory during the last seven years for the search of HLA identical sibs in view of bone marrow transplantation. A total of 614 different three-locus haplotypes were found. Most of them (83.6%) were present only once or twice, whereas 24/614 (3.9%) were found 6-28 times each. HLA-B44 was present in 4 of these most frequent haplotypes. HLA-B44 has been implicated as the molecular target for bone marrow allograft rejection. Therefore, a better knowledge of the HLA-B44 haplotype relationships might prove useful for the programming of registries of unrelated bone marrow donors. Eighty five serologically defined HLA-B44 unrelated subjects, either one or both parents from the above families, were subtyped by a high-resolution sequence-specific oligonucleotide probing approach. Moreover, 34 unrelated potential donors recruited for those patients that did not find a suitable donor among their siblings were subtyped also for HLA-B44. B*4403, which accounted for 47/85 (55.3%) serologically defined B44 alleles, appeared in strong, statistically significant, linkage disequilibrium with HLA-A29, -A23 and -DR7. On the other hand, B*4402, which covered virtually all other B44 alleles, showed prevalent gametic associations with HLA-A2 and HLA-A24. The linkage disequilibrium between HLA alleles is the key for the low frequency of HLA-B44 mismatches in donors selected as HLA-A,B,DRB1 identical to patients waiting for unrelated bone marrow transplantation. If a given patient presents unusual haplotypes, the chance of finding HLA-B44 mismatches may be higher because of the presence of different haplotype relationships in the donors.

The effect of TNF*B gene polymorphism on TNF-alpha and -beta secretion levels in patients with insulin-dependent diabetes mellitus and healthy controls.

TNF-alpha and -beta have been implicated in the development of HLA-associated autoimmune diseases. It has been suggested that inter-individual differences in the secretion levels of these cytokines may contribute to the predisposition of certain individuals to the development of diseases such as insulin-dependent diabetes mellitus (IDDM). We have investigated whether a diallelic TNF*B polymorphism detected using the enzyme Ncol influences the TNF-alpha and/or -beta secretory capacity of peripheral blood mononuclear cells (PBMC) from PHA stimulated healthy individuals and IDDM patients. We have shown that the level of TNF-beta secreted correlates with the TNF*B genotype in healthy individuals: those with the TNF B*2 allele secreted significantly higher levels of TNF-beta (P = 0.025) than those with the TNF*B1 allele. In IDDM patients, the reverse situation was observed, with those patients with the TNF*B1 allele secreting higher levels of TNF-beta than those with the TNF*B2 allele. No correlation was found between TNF-alpha levels and TNF*B genotype. Furthermore, when IDDM patients and controls were matched for TNF*B genotype, the IDDM patients with the TNF*B2 allele secreted significantly lower levels of TNF-beta than controls with this allele. On analysis of IDDM-susceptible extended HLA haplotypes in the homozygous groups, 4/7 IDDM patients with the TNF*B2 allele were Bw62-DR4 compared with 0/16 matched controls. Thus, the extended haplotype Bw62-DR4-TNF*B2/2 rather than IDDM per se is almost certainly responsible for the depressed TNF-beta secretion found in the IDDM-TNF*B2 homozygous cohort.

Combined PCR-heteroduplex and PCR-SSCP analysis for matching of HLA-A, -B and -C allotypes in marrow transplantation.

We describe a method for rapid matching of HLA-A, -B and -C allotypes using simultaneous polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and heteroduplex analysis. Electrophoresis is performed at ambient temperature without requirements for buffer cooling. SSCP and heteroduplexes are revealed as discrete spatially separated band clusters. Using HLA-A, -B and -C locus-specific PCR primers, matching for alleles at these loci can be performed in 5 h. We tested 17 serologically matched patient-unrelated donor pairs and found considerable microheterogeneity at the DNA level. We propose that this technology has several advantages over conventional low-resolution typing methods and represents a potentially valuable screening method in unrelated donor selection.

The HLA-A,B,C genotype of the class I negative cell line Daudi reveals novel HLA-A and -B alleles.

Daudi, a lymphoblastoid B cell line derived from an African Burkitt lymphoma does not express HLA-A,B,C antigens at the cell surface. Although HLA-A,B,C heavy chains are made normally they do not assemble into functional molecules because beta 2-microglobulin is absent. Previous serological analysis of somatic cell hybrids indicated that the HLA haplotypes of Daudi encoded HLA-A1, A10(A26), B17, and B16(38) antigens. Here we describe the application of molecular methods: ARMS-PCR, cDNA cloning and sequencing, immunoprecipitation and gel electrophoresis, to define the class I genotype of the Daudi cell line which is HLA-A*0102, A*6601, B*5801, B*5802, Cw*0302 and Cw*0602. With the exception of the B38 antigen, which is not a product of the alleles defined, the genotype is consistent with the serological description. Two previously undiscovered alleles emerged from this analysis: A*0102 and B*5802. The A*0102 allele differs from A*0101 by 5 nucleotide substitutions within exon 2 where it has a motif shared with A*30 alleles; the B*5802 allele differs from B*5801 by 3 substitutions in exon 3 where it has a motif shared with B*14 alleles. Subtyping HLA-A1 alleles showed A*0102 was well represented amongst individuals typed serologically as A1 in an African population but was absent from caucasoids. B*5802 has been found in a second individual. Thus the novel A and B alleles are not specific to the Daudi tumor. Overall, this analysis of a single East African cell illustrates the power of molecular methods to define new class I HLA alleles in non-caucasoid populations.

A molecular analysis of the telomeric end of the major histocompatibility complex. DNA typing of HLA-A3 subtypes and -B7.

A molecular investigation of the HLA-A, -B, -C, I82, D6S128, and D6S265 loci was carried out using RFLP and PCR analyses in 331 healthy individuals, 21 HLA-A3B7 homozygous subjects, and 17 HLA homozygous cell lines. New polymorphic RFLP patterns were noted at the HLA-A and -B loci which were useful for distinguishing subtypes of HLA-A3 and -B44. Strictly specific bands were observed for HLA-A3, -A11, -B7, -B57. and -Cw4. Molecular identification of two HLA-A3 subtypes was possible by HLA-A RFLP and D6S265 PCR analyses. The two alleles of the I82 locus were associated with different HLA-A3 subtypes. It was shown that the serologically HLA-A3 homozygous cell line EHM was heterozygous for the two subtypes. The common subtype formed 91% of HLA-A3 antigens. Some serologically HLA-A3 homozygous healthy individuals were heterozygous for the subtypes. Using these new polymorphisms, a thorough molecular analysis of the haplotype HLA-A3B7DR15 at the three regions of the MHC was performed. The results have implications on HLA matching in transplantation, population genetics of the MHC, and disease association studies.