Molecular Analysis Of HLA Class Research Articles

Identification of HLA ligands and T-cell epitopes for immunotherapy of lung cancer.

Lung cancer is the most common cancer worldwide. Every year, as many people die of lung cancer as of breast, colon and rectum cancers combined. Because most patients are being diagnosed in advanced, not resectable stages and therefore have a poor prognosis, there is an urgent need for alternative therapies. Since it has been demonstrated that a high number of tumor- and stromal-infiltrating cytotoxic T cells (CTLs) is associated with an increased disease-specific survival in lung cancer patients, it can be assumed that immunotherapy, e.g. peptide vaccines that are able to induce a CTL response against the tumor, might be a promising approach. We analyzed surgically resected lung cancer tissues with respect to HLA class I- and II-presented peptides and gene expression profiles, aiming at the identification of (novel) tumor antigens. In addition, we tested the ability of HLA ligands derived from such antigens to generate a CTL response in healthy donors. Among 170 HLA ligands characterized, we were able to identify several potential targets for specific CTL recognition and to generate CD8+ T cells which were specific for peptides derived from cyclin D1 or protein-kinase, DNA-activated, catalytic polypeptide and lysed tumor cells loaded with peptide. This is the first molecular analysis of HLA class I and II ligands ex vivo from human lung cancer tissues which reveals known and novel tumor antigens able to elicit a CTL response.

Molecular analysis of HLA class II associations with hepatitis B virus clearance and vaccine nonresponsiveness

Clearance of acute hepatitis B virus (HBV) infection is associated with a vigorous CD4+ T-cell response focusing on the core protein. HLA class II glycoproteins present viral peptides to CD4+ T cells and influence the immune responses. HLA-DRB1*1301/2 have been associated with viral clearance, and HLA-DRB1*0301 is associated with nonresponse to vaccination with envelope proteins. Binding affinities of overlapping peptides covering the core and envelope proteins of HBV were measured to HLA glycoproteins encoded by HLA-DRA1*0101,-DRB1*0101 (HLA-DR1), HLA-DRA1*0101,-DRB1*0301 (HLA-DR3), HLA-DRA1*0101,-DRB1*0701 (HLA-DR7) and HLA-DRA1*0101,-DRB1*1301 (HLA-DR13) molecules and compared with published peptide-specific CD4+ T-cell responses. There are more high-affinity ligands (IC50 < 1 micromol/L) derived from the core protein than the surface antigen (P < .04 for HLA-DR1/7/13), but there was no increase in the number or the affinity of ligands for HLA-DR13. Clusters of particular core peptides bound to multiple HLA types, explaining the immunodominance of these regions for T-cell responses. Within the envelope protein, the low-affinity ligands (IC50 < 10 micromol/L) are found mainly in the surface antigen, with a marked paucity of ligands for HLA-DR3 (HLA-DR3 vs. non-DR3; P < .05) consistent with the lower vaccination responses for this HLA type. Of all peptides tested, 8 to 10 bound mainly to one HLA type, allowing a substantially greater breadth of response in heterozygotes. In conclusion, these data offer a mechanistic explanation for the dominant response to the HBV core protein during infection and support the direct involvement of the HLA-DRB1 gene in vaccine nonresponsiveness but not altered susceptibility to viral persistence.

Molecular analysis of HLA class II region genes in ulcerative colitis in India

Molecular analysis of HLA class II region genes in ulcerative colitis in India

Molecular analysis of HLA class II region genes in ulcerative colitis in India

Molecular analysis of HLA class II region genes in ulcerative colitis in India

Molecular analysis of HLA class II polymorphisms among different ethnic groups in Israel

The Jewish population in Israel comprises of inhabitants of heterogeneous ethnic backgrounds. Genetic studies classify the Israeli Jewish population into two major groups: Ashkenazi from Central and Eastern Europe and Sephardic or non Ashkenazi, from the Mediterranean and North Africa. The present study was aimed at elucidating the differential influx of HLA class II alleles in Ashkenazi, in various non-Ashkenazi subgroups and in Israeli Moslem Arabs. Using the PCR-SSOP technique, a large number of alleles were detected at each of the loci examined (DRB1, DQA1 and DQB1). In addition, gene frequencies, characteristic DR/DQ linkage disequilibria, population distances and their corresponding dendogram, were used to study the relationship between Israelis as a group, non Jewish Caucasians and Blacks. These populations could be grouped into three main clusters: the first consists of all the Israeli groups with the exception of the Ethiopian Jews; the second consists of non Jewish Caucasians, with a clear distinction seen between Israelis and non Jewish Europeans and U.S. Caucasians; the third, composed of Blacks, is distinctly different from the other populations. Ethiopian Jews were found to be closer to the Blacks than to any of the Israeli Jewish groups. We have shown that Jews share common features, a fact that points to a common ancestry. A certain degree of admixture with their pre-immigration neighbors exists despite the cultural and religious constraints against intermarriage.

Molecular analysis of HLA class I alleles in the Mexican Seri Indians: implications for their origin.

The molecular analysis of HLA class I loci has demonstrated that, although, the genetic profile is restricted in Amerindians, several micropolymorphisms may be important in conferring a biological advantage. We analyzed the HLA-A and B genetic profile of Seris, a Mexican Indian tribe living in northwestern Mexico in the state of Sonora. There are presently only 619 individuals. Our study included 100 Seris belonging to nine families. HLA-A and -B loci typing was performed by polymerase chain reaction using an amplification refractory mutation system (PCR-ARMS) on a select group of samples; all of them were typed by polymerase chain reaction using sequence-specific oliogonuoleotide probes (PCR-SSOP) at a low-intermediate resolution level. The correlation between the techniques was 100%. Only five HLA-A alleles and seven HLA-B alleles were found. A*0201, A*68, A*31, A*24, B*3501, B*40, B*51, B*3512 and B*15 were present in over 5% of the individuals. B*27052 was detected in 2%. B27 is absent in any other Mexican Indian groups previously studied. The presence of B27 may be the result of a founder effect due to different waves of southward migrations. The B-locus is more diverse and the prevalent haplotypes were: A*0201-B*3501, A*0201-B*40, A*0201-B*3512, A*31-B*51, A*68-B*3501 and A*68-B*40. This genetic profile is different from the pattern of other Mexicans. The phylogenetic tree suggests that Seris are more closely related to the Warao Indians from Venezuela, who live in a similar ecosystem, and to some groups of Argentina, than they are to the Mexican Lacandones who live in the jungle. These data emphasize the relevance of the interaction between genes and environment.

Molecular analysis of HLA class I (HLA-A and -B) and HLA class II (HLA-DRB1) genes in Japanese patients with multiple sclerosis (Western type and Asian type).

The types of HLA-A, -B and -DRB1 genes were studied in 146 Japanese patients with multiple sclerosis (MS) using polymerase chain reaction-sequence-specific oligonucleotide probe analysis. Fifty-seven patients who displayed selective clinical involvement of the optic nerve and spinal cord were classified as having Asian type MS. The other 89 patients had disseminated central nervous system involvement and were classified as having Western type MS. The frequency of HLA-B*5101 was increased in both types of MS patients compared with controls. The frequency of HLA-DRB1*1501 was increased in Western type MS and the frequency of HLA-DRB1*0802 was increased in Asian type MS compared to controls. After correction of P values, the association of Western type MS patients with HLA-DRB1*1501 was statistically significant (Pc=0.0003) whereas other HLA alleles showed no significant association. These results suggest that HLA class I (HLA-A and -B) alleles may not contribute to a strong susceptibility to MS in Japanese compared to HLA class II (HLA-DRB1) alleles.

Serological and molecular analysis of HLA class I and II alleles in Thai patients with psoriasis vulgaris.

The HLA class I and class II alleles in 67 patients with type I psoriasis vulgaris, 23 patients with type II psoriasis vulgaris and 140 healthy individuals were analyzed. The frequencies of HLA-A2, -B46, -B57 and DQB1*0303 were significantly increased in type I psoriasis compared to the controls (Pc<0.05). Molecular analysis of HLA-A2 alleles showed an increase in HLA-A*0207 and a decrease in HLA-A*0203 in type I psoriasis. HLA-DQB1*0301 was significantly decreased in type I psoriasis compared to the normal controls (Pc<0.05). No association of any alleles with type II psoriasis was observed. This data demonstrated two susceptible haplotypes: HLA-A1-B57-DRB1*0701-DQA1*0201-DQB1*0303 (AH57.1) and HLA-A2-B46-DRB1*0901-DQA1*0301-DQB1*0303 (AH46.1) for type I psoriasis in the Thai population. Besides, the haplotype AH46.1 was also associated with type II psoriasis.

P775 - Molecular analysis of HLA class II polymorphism in Ethiopian Jews

P775 - Molecular analysis of HLA class II polymorphism in Ethiopian Jews

Molecular analysis of HLA class II polymorphism in Croatians

HLA-class II polymorphisms have been studied in a population of 141 unrelated healthy Croatians using PCR amplification, followed by non-radioactive oligonucleotide hybridization. Thirty one DRB1, 8 DQA1, 13 DQB1 and 16 DPB1 alleles were found in the tested population. DRB1*1601, 0701, 1501, 0101 and 1104 are the most frequent alleles at the DRB1 locus. At the DQA1 locus two alleles predominate: DQA1*0501 and 0102, while the most frequent DQB1 allele is *0301. Analysis of HLA-DPB1 polymorphism showed that, as in other Europeans, DPB1*0401 is the most frequent allele. Four different two locus haplotypic associations (DRB1-DRB3, DRB1-DRB5, DRB1-DQB1 and DQA1-DQB1) as well as three locus DRB1-DQA1-DQB1 haplotypic associations were assigned on the basis of known linkage disequilibria. Several unusual two-locus associations have been observed: DRB1*0301-DRB3*0202, DRB1*1501-DRB5*02, DRB1*1601-DRB5*0101, DRB1*1502-DRB5*0101, DQA1*0103-DQB1*0503 and DQA1*0501-DQB1*0302. Among 236 examined DRB1-DQA1-DQB1 haplotypic combinations, the most frequent was DRB1*1601-DQA1*0102-DQB1*0502 that was found with statistically significant higher frequency than in other Europeans. Twenty-eight distinct probable haplotypes were observed just once, suggesting that the main characteristic of Croatian population is great heterogeneity of haplotypes. This study will serve as a reference for further anthropology studies, HLA and disease associations studies and for donor/recipient matching in organ and bone marrow transplantation.

Molecular analysis of HLA class II genes in Goodpasture's disease

A molecular analysis of HLA class II genes was undertaken in order to characterize the previously reported association between HLA-DR2 and glomerulonephritis caused by antibodies to glomerular basement membrane (Goodpasture's disease). Genomic DNA was prepared from 53 patients with Goodpasture's disease and analysed by: (i) Southern blotting using cDNA probes to DRB, DQA and DQB genes, after digestion with TaqI endonuclease; (ii) allele-specific oligonucleotide probing of specifically amplified DNA; and (iii) nucleotide sequencing of relevant alleles. The patients had a greatly increased frequency of DRw15 (a subspecificity of DR2) which was present in 75.5% of patients and 31% of controls (p < 0.0001). The frequency of DR4 was also increased, especially in patients without DRw15. Overall, 90.5% of the patients had either DRw15 or DR4. In contrast, the frequency of DR1 was significantly reduced (patients 5.6%, controls 20.7%, p < 0.01). Differences in the frequencies of DQA and DQB alleles could all be explained by linkage disequilibrium. Nucleotide sequences of relevant alleles were identical to those previously published. Comparison of derived amino acid sequences of expressed DR beta chains showed that the DR beta chains of DRw15 and DR4 shared a six-amino-acid motif from positions 26-31, that included four polymorphic amino acids none of which are shared with DR1. A sequence-specific oligonucleotide detected this amino-acid motif in 45/49 (91.8%) patients tested. Thus, this particular motif, which lies on the floor of the antigen binding groove, has a stronger association with Goodpasture's disease than any individual allele, and may be of pathogenic significance.

Molecular analysis of HLA class II genes in primary sjo¨gren's syndrome: A study of Israeli Jewish and Greek Non-Jewish patients

Abstract In an attempt to define the role of HLA class II genes in predisposition to primary Sjo¨rgen's syndrome, patients of two different ethnic groups (Israeli Jews and Greeks of non-Jewish origin) suffering from this disorder were studied. Oligonucleotide genotyping revealed the majority in both groups to carry either DRB1 *1101 or DRB1*1104, alleles tha re in linkage disequilibrium with DQB1*0301 and DQA1*0501. The high frequency of the two alleles in these SS patients is in contrast with the accepted association of primary SS with HLA-DR3 in Italian and American individuals. Molecular analysis of DQB1 and DQA1 alleles found in American Caucasian and American black SS (or SLE) patients demonstrated high frequencies of DQB1*0201 and DQA1*0501. The fact that the majority of SS patients, across racial and ethnic boundaries, carry a common allele, DQA1*0501, implies its involvement in the predisposition t to primary SS. Based on sequence analysis and the computer imaging of the HLA class II molecule structure, a hypothetical model for the role of the DQ molecule in promoting primary SS us proposed.

Molecular analysis of HLA DR4‐/β1 gene in malaria vaccinees. Typing and subtyping by PCR technique and oligonucleotides

The combination of the PCR technique and the synthetic oligonucleotides has proved to be a useful tool in the molecular analysis of HLA class II genes, allowing recognition of as little as a single nucleotide modification in the sequence of the gene. The molecules encoded by these genes have been associated with genetic control of the immune response and with susceptibility to certain diseases. Studies carried out in our laboratory have shown three patterns of humoral immune response in the human volunteers vaccinated with the synthetic protein SPf 66; high, intermediate and low responders. Approximately 73.3% of the low responders were serologically typed as HLA DR4 and 42% as DQw6. These results moved us to look for a subtype (Dw) correlation between the DR4 positive individuals and the different humoral immune response patterns. Using oligo-typing methods after previous amplification of the DR4 B1 exon, we subtyped 20 DR4 volunteers, classified as high, intermediate and low responders. We did not find any direct association between the HLA DR4 Dw special subtype in the high or low responders immunized with the SPf 66 vaccine.

A new HLA‐DRB1 allele within the DRw52 supertypic specificity (DRw13‐DwHAG): Sequencing and direct identification by oligonucleotide typing

Molecular analysis of HLA class II polymorphism represents a crucial parameter for HLA matching in transplantation immunology, for the study of HLA-disease association and for the understanding of major histocompatibility complex (MHC)-restricted antigen presentation. We report here the DNA sequence and the deduced amino acid sequence of the polymorphic first domain exon of the DRB1 and DRB3 alleles of the homozygous cell line HAG (DRw13-DwHAG-DQw7). The DRB1 sequence represents a new DRB allele, which clearly shows a close relationship to other DRB1 genes from the DRw52 group and is now officially named DRB1* 1303. The DRB1* 1303 allele is very similar to the two DRw13 alleles we have described earlier, with only five amino acid differences at positions 32, 37, 47, 57 and 71. Furthermore, its sequence in the third hypervariable region is unique among all known DRB1 and DRB3 alleles. The sequence of the DRB3 gene of HAG shows that it corresponds to the previously described DRB3* 0101 (DRw52a) allele. In addition we present analyses of a panel of healthy blood donors and leukemic patients by oligonucleotide typing showing that this new HLA-DR specificity can now be unequivocally identified in routine oligotyping with an allele-specific oligonucleotide probe.

Molecular analysis of HLA class I and class II antigen loss mutants reveals a homozygous deletion of the DR, DQ, and part of the DP region: Implications for class II gene order

The mutant human B-lymphoblastoid cell lines, 721.174 and 721.180, previously reported to exhibit greatly reduced expression of human HLA class I and II antigens (DeMars et al., Hum Immunol 11:77, 1984), were analyzed by Southern blotting using class II cDNA and genomic clones as hybridization probes. All genomic sequences complementary to DRα, DRß, DQα, and DQß probes were absent from these mutants. DZα genomic sequences were deleted as were the DPα1 and DPß2 loci but the DPß2 and most, if not all, of the DPα2 locus were retained. However, no RNA transcripts for either DPα2 or DPß2 could be detected. The mapping of the deletion breakpoint within the DP cluster allows the orientation of the loci in the DP region with respect to the centromere as follows: centromere, DPß2, DPα2, DPß1, DPα1, (DQ, DR). In addition, the analysis of a set of DR −, DQ −, DP + homozygous deletion mutants (721.82, 721.84, and 721.101) reveals a deletion breakpoint between the DQα1/DQß1 loci and the DQα2/DQß2 loci. These mutants retain DZα genomic sequences, tentatively mapping the DZα locus between the DQ and the DP region. The residual ability of the DR −, DQ −, DP − mutants (174 and 180) to stimulate allogeneic and autologous lymphoproliferative responses must be attributed to expression of as yet unidentified class II antigens, or to non-class II antigens.