A preliminary analysis of the 11th International Histocompatibility Workshop monoclonal antibodies

In the 11th International Histocompatibility Workshop prescreening study, HLA Class I and Class II monoclonal antibodies (mAb) were tested on panels of peripheral T and B cells and homozygous typing cell (HTC) lymphoblastoid lines. The 102 Class I mAb, of which 64 were new, were screened on 63 HTC lines and 20 peripheral T cells. The 144 Class II mAb were tested on 63 HTC lines, 20 peripheral B and 10 peripheral T cells. For Class I, 45 of the 102 mAbs tested proved to have clear and identifiable reaction patterns on both PBL and HTC lines and included several interesting new HLA-A and -C locus antibodies. Of the 144 Class II mAb, of which 99 were new, 18 DR, 22 DQ and 7 DP antibodies were judged excellent. The DR antibodies included some which were antigen-specific but the majority were complex antibodies, while the DQ antibodies, as previously, identified the majority of DQ antigens both singly and in combinations. The encouraging number of DP antibodies was interesting in the limited range of sites they appear to detect.     

Molecular typing for HLA class I using ARMS-PCR: further developments following the 12th International Histocompatibility Workshop

Molecular typing for HLA class I was introduced in the 12th International Histocompatibility Workshop. Following a pilot study using three methods, sequence specific oligotyping (SSO), reverse dot blot and amplification refractory mutation system (ARMS)-PCR, the ARMS-PCR method was selected for use. A great advantage of an ARMS-PCR method is that, unlike the other two methods, it can determine whether sequence motifs are in cis or in trans, as ARMS-PCR detects two cis located motifs per reaction using forward and reverse sequence specific primers. Resolution was designed to be low to medium level for HLA-A, -B and -C alleles. Two hundred and fifty class I kits and 83 HLA-A2 subtyping kits were distributed. The A2 subtyping kit used a two round nested PCR system to identify all of the A2 alleles known at the time. Typing results on control DNA samples distributed with both the kits showed a very satisfactory performance. Since the 12th Workshop, the kits have been developed with the addition of new primers and primer mixes to increase the resolution of the test.     

Next-generation HLA typing of 382 International Histocompatibility Working Group reference B-lymphoblastoid cell lines: Report from the 17th International HLA and Immunogenetics Workshop

Extended molecular characterization of HLA genes in the IHWG reference B-lymphoblastoid cell lines (B-LCLs) was one of the major goals for the 17th International HLA and Immunogenetics Workshop (IHIW). Although reference B-LCLs have been examined extensively in previous workshops complete high-resolution typing was not completed for all the classical class I and class II HLA genes. To address this, we conducted a single-blind study where select panels of B-LCL genomic DNA samples were distributed to multiple laboratories for HLA genotyping by next-generation sequencing methods. Identical cell panels comprised of 24 and 346 samples were distributed and typed by at least four laboratories in order to derive accurate consensus HLA genotypes. Overall concordance rates calculated at both 2- and 4-field allele-level resolutions ranged from 90.4% to 100%. Concordance for the class I genes ranged from 91.7 to 100%, whereas concordance for class II genes was variable; the lowest observed at HLA-DRB3 (84.2%). At the maximum allele-resolution 78 B-LCLs were defined as homozygous for all 11 loci. We identified 11 novel exon polymorphisms in the entire cell panel. A comparison of the B-LCLs NGS HLA genotypes with the HLA genotypes catalogued in the IPD-IMGT/HLA Database Cell Repository, revealed an overall allele match at 68.4%. Typing discrepancies between the two datasets were mostly due to the lower-resolution historical typing methods resulting in incomplete HLA genotypes for some samples listed in the IPD-IMGT/HLA Database Cell Repository. Our approach of multiple-laboratory NGS HLA typing of the B-LCLs has provided accurate genotyping data. The data generated by the tremendous collaborative efforts of the 17th IHIW participants is useful for updating the current cell and sequence databases and will be a valuable resource for future studies.     

European Mathematical Genetics Meeting, Rotterdam, the Netherlands, 10th–11th April 2008

Bertram Müller-Myhsok
Max-Planck Institute of Psychiatry, Munich
Genome-wide association has become a major success story in the very recent past. It has altered the standing that association studies have in the field, and has delivered remarkably well replicating results. Contrasting the results with genome-wide linkage studies it is apparent that the overlap between the two approaches is very small. I will try to explore this question and come up with a rationale what to expect and what not to expect from a comparison of the results of the two approaches.     

SSOP typing of the Tenth International Histocompatibility Workshop reference cell lines for HLA-C alleles

Abstract: HLA-C gene products are the most poorly understood of the HLA class I molecules because they express at low level on the cell surface compared to HLA-A and -B. However, recent evidence shows that HLA-C molecules are functionally competent in eliciting T-cell responses and in controlling NK-cell recognition. Approximately 20 to 50% of HLA-C alleles type "blank" in most populations. To provide a better definition of the HLA-C alleles, we analyzed 98 extensively characterized B-cell lines from the 10th International Histocompatibility Workshop. Selective HLA-C-specific DNA amplification of exons 2 and 3 from DNA prepared from the cell panel was achieved with the use of two sets of locus-specific primers. We used 64 sequence-specific oligonucleotide probes (SSOPs) complementary to variable sites in exons 2 and 3 to generate hybridization patterns. Twenty-five alleles were found among these patterns, including seven new alleles in the homozygous cell lines and seven potential new alleles in heterozygous cell lines. Differences between the new alleles and known alleles were generally small. Five major groups were identified in the Cw "blank" cells by the SSOP patterns. In addition, linkage between HLA-B specificities and HLA-C alleles was similar to previous observations. The present study demonstrated that SSOP typing was effective in identifying new alleles in homozygous typing cells but not in the heterozygous cells. Also, DNA typing can facilitate the identification of all HLA-C alleles, including those that serologically type as blanks. The HLA-C locus may be more polymorphic than was previously recognized.     

A multi-laboratory characterization of the KIR genotypes of 10th International Histocompatibility Workshop cell lines

Killer immunoglobulinlike receptors (KIRs) are expressed on natural killer and T cells. Both inhibitory and noninhibitory forms have been described, leading to inhibition or continuation of cellular killing activity. The natural ligands identified so far of KIRs are class I human leukocyte antigens (HLA). In particular, the interaction of some KIRs with HLA-Cw has been well characterized. Recent work has implicated KIRs in affecting the outcome of hematopoietic stem-cell transplant (HSCT). This may well lead to a requirement for prospective KIR typing of donor and recipient. We have utilized different typing systems (two using polymerase chain reaction-sequence-specific primers, and one using polymerase chain reaction-sequence-specific oligonucleotide probes) in three separate laboratories to characterize the KIR gene complement of 25 cell lines from the 10th International Histocompatibility Workshop. There were consistent results in 22, and minor differences in 3. When compared with previous results for some of these cell lines, no further differences were found. The differences are due to typing of KIRs KIR2DL1 and KIR2DS5, and may be explained by technical differences or the inability to type new variants. Further improvements in typing may be required if population and clinical studies are to produce accurate results.     

Rga (Rodgers) and the HLA Region: Linkage and Associations

In 19 families with 97 children the segregation of Rg^a (Rodgers) was found to be compatible with Mendelian inheritance and five backcross and 14 intercross families were found among HLA and Bf typed families. Close linkage (lods + 17.82) without recombination was found between Rg and the HLA region, with a direct count of 96 nonrecombinant meioses for Rg-HLA-B. Kg- was strongly associated with HLA-B8 (29 of 30 haplotypes) and probably associated with Bw40, but did occur on other HLA-B haplotypes. By inference .Rg^- is negatively associated with Ch^- (Chido). The Rg^-Ch^- haplotype has not been observed. Rg³ and Ch^a may or may not be coded for by different sites of the same cistron closely linked to HLA-B:C and cannot as yet be excluded from being parts of B or C.     

Rga (Rodgers) and the HLA Region: Linkage and Associations

In 19 families with 97 children the segregation of Rg^a (Rodgers) was found to be compatible with Mendelian inheritance and five backcross and 14 intercross families were found among HLA and Bf typed families. Close linkage (lods + 17.82) without recombination was found between Rg and the HLA region, with a direct count of 96 nonrecombinant meioses for Rg-HLA-B. Kg- was strongly associated with HLA-B8 (29 of 30 haplotypes) and probably associated with Bw40, but did occur on other HLA-B haplotypes. By inference.Rg^- is negatively associated with Ch^- (Chido). The Rg^-Ch^- haplotype has not been observed. Rg³ and Ch^a may or may not be coded for by different sites of the same cistron closely linked to HLA-B:C and cannot as yet be excluded from being parts of B or C.     

Oligotyping of Italian celiac patients with the 11th International Histocompatibility Workshop reagents.

Oligotyping for HLA-DRB1, DQA1 and DQB1 specificities has been performed on PCR-amplified DNA from 55 Italian celiac children, living in Rome, and 50 blood donors. 52.6% of CD patients were DR3;DQA1*0501;DQB1*0201-positive versus 14% of controls (RR = 6.85) and 34.5% were DR5,7;DQA1*DQB1*0201-positive versus 2% of controls (RR = 25.86). 7 patients (12.7%) were negative for the DQA1*0501/B1*0201 dimer: 3 of them were DR4 (5.4%) and the others typed as DR1,5; 1,7; 5,7 and w6,7. No patient was negative for both DQA1*0501 and DQB1*0201 alleles.     

Oligotyping of celiac multiplex families with the 11th International Histocompatibility Workshop reagents.

Using PCR and SSO probes from the 11th International Histocompatibility Workshop, we oligotyped for HLA-DRB1 gene and DQA1*0501, DQB1*0201 alleles 10 celiac families each with 2 affected children. All families belong to the Italian population except for one, whose mother is originally from Cape Verde island. 8/10 sibling pairs share the DQA1*0501/B1*0201 heterodimer, inherited in cis or in trans arrangement. All the dimer-negative patients were DR4-positive.     

Mixed Lymphocyte Culture Technique: Standardization of a Test-System with 105 Responding and 105 Stimulating Lymphocytes per 1 ml

An MLC test system is described utilizing 10⁵ responding and 10⁵ stimulating lymphocytes in a total of 1 ml of culture medium. Some of the variables of the test have been evaluated and the reproducibility of the test within a given day and between days investigated according to a Workshop protocol. This system was first applied to three groups: HL-A identical siblings, one haplotype different family members and two haplotype different individuals. Within each group, the log converted stimulation ratios are normally distributed, allowing the use of simple statistical analysis in repeated testings. In addition, a group of weakly positive MLC reactions can be detected. This group includes the LD identical but SD different sibling combinations. Within the same group we find some unrelated HL-A sero-identical combinations and the so-called LD typing response, used to identify LD determinants by means of LD and SD homozygous stimulating test cells.
It is concluded that this MLC test system allows identification of identical combinations, weakly positive combinations and one and two haplotype responses even within unrelated combinations. This test system may have advantages over the microtiter test system because the large culture volume gives a low concentration of blastogenic factors in typing responses.     

DNA typing of human platelet antigen systems 1, 2, 3 and 5 in B-lymphoblastoid cell lines of the International Histocompatibility Workshop

Alloimmunization against human platelet antigens (HPA) can cause thrombocytopenia in different clinical settings, including neonatal alloimmune thrombocytopenia, post transfusion purpura, and refractoriness to platelet transfusion. Recently, DNA based methods have been described for analysis of the polymorphism of the human platelet antigens. However, until now, no reference material for standardization purposes is available. We thus determined the DNA polymorphism of the human platelet antigen (HPA) systems 1, 2, 3 and 5 in B-lymphoblastoid cell lines collected for the International Histocompatibility Workshops. DNA typing of the HPA systems was done by PCR amplification with sequence-specific primers (PCR-SSP). A new enzyme (AmpliTaq Gold) was introduced to enhance the specificity of the PCR. We present a panel of five B-lymphoblastoid cell lines, representing all heterozygous and homozygous genotypes of the tested HPA systems. This work thus provides the reference material required for DNA based diagnosis of human platelet antigens.     

Association of TNF-α genetic polymorphism with HLA DPB1*0301

BACKGROUND: We speculated TNF-alpha can be one of candidate gene for aspirin-intolerant asthma (AIA) because TNF-alpha is pro-inflammatory cytokine and known to be increased level in asthmatic airways. In addition, genetic interaction between TNF-alpha and human antigen leucocyte (HLA) DPB1*0301, which is a strong genetic marker for AIA, was examined for its close location within chromosome 6. METHOD: To investigate genetic association of TNF-alpha with an AIA phenotype, three study groups (163 patients with AIA, 197 patients with aspirin-tolerant asthma (ATA), 257 normal control subjects) were enrolled. Single nucleotide polymorphisms (SNPs) were genotyped using a single-base extension method and HLA DPB1 genotyping was determined by high-throughput sequencing method. RESULTS: All five SNPs of TNF-alpha were tested; there were no significant differences in allele and genotype frequencies among the three groups. However, significant association between TNF-alpha-308G>A polymorphism and atopy status was noted (P<0.05). Gene to gene interaction between TNF-alpha-1031T>C (or -863C>A or -857C>A) and HLA DPB1*0301could synergistically increase the susceptibility to AIA with odds ratio (OR) to 7.738 (or OR=8.184 for -863C>A, OR=7.500 for -857C>T, P<0.001, respectively). CONCLUSION: TNF-alpha promoter polymorphism may significantly increase susceptibility to AIA by gene-to-gene interaction with HLA DPB1*0301.