Search

Genes encoding the human leukocyte antigens (HLA) are associated with diverse immunological disorders, including autoimmune diseases and infections. Recently, significant progresses have been made in the HLA typing technologies through the use of next generation sequencers. The reliable platforms for the SNP-based imputation of HLA genotypes have also been established. These technical advancements should enable further identification of HLA associations with diseases. One of the remaining questions is the mechanism through which HLA confer disease susceptibility. As a first step toward comprehensive understanding of functional variations among HLA allele products, we established a protocol to analyze the HLA-binding peptides through quantification of cell-surface HLA expression in an engineered cell line. In this article, we summarize the overview of the cell-surface HLA expression assay, which we plan to use for screening and collection of HLA-peptide interaction profiles for large sets of HLA alleles and peptides.

Clinical trial of organ transplantation was renal transplantation by Voronoy at 1936. The discovery of HLA in the 1950s was one of the most important new findings in the area of transplantation. Nowadays, developing HLA genotyping methods, the serum analysis does not use for donor and recipient HLA typing but for cross-matching test. Because each of HLA genotyping methods has its merits and demerits, it is important to choice right methods for avoiding type error. PCR-Luminex method using fluorescence microsphere was developed for high-resolution HLA-A, HLA-B and HLA-DRB1 genotyping in the Japanese population. This genotyping method allows to define all the possible combinations of alleles at each loci existing in Japanese at the four-digital level. In hematopoietic stem cell transplantation, to match high resolution level of HLA between donor and recipient lead an improvement of recipient's survival. In organ transplantation, removed organ has to be so immediately transplanted into recipient that no time is left for HLA genotyping. In order to have good survival of transplanted organ, HLA, cytokine promoter lesion and immunoglobulin like receptor genotyping might be helpful. We focused on this review at HLA genotyping, especially new SSO methods.

Micro-lymphocyte cytotoxicity test (LCT) have been developed by Dr. Terasaki at 1964 and usually used for serological typing up to date. HLA typing has been complicated by the extensive diversity of and the sequence homology among HLA alleles. However, it is need to match HLA types between donor and patient for hematopoietic stem/progenitor cell transplantation, therefore accurate HLA typing is required. Recently, it is able to perform accurate lymphocyte separation and HLA typing of much blood samples by using immunomagnetic beads and computer analysis, respectively.

HLA genes exhibit high degrees of polymorphism, and a total of about 400 alleles have been identified at HLA-A, B, C and DR loci up to now. A number of DNA-based typing methods using specific primers, specific probes or restriction enzymes have been developed. In HLA class II allele typing, a few methods which are simple in handling and able to determine the alleles automatically, have been available. We have recently developed PCR MPH (microtiter plate hybridization) method, and this method has been in use for routine typing of DRBI alleles. In HLA class I typing, however, DNA typing system suitable for routine typing laboratories has not been established yet, because the polymorphism spreads two exons intervened by an intron. Nevertheless, allele typing of some antigen groups such as A2 and B61 groups have been established using PCR MPH method.

The enzymatic amplification of specific DNA sequences by the polymerase chain reaction (PCR) has provided a new approach to genetic typing of HLA-class II region specificities. However, we found the existing PCR protocols to be adequate and/or time consuming, especially when large numbers of samples need to be processed. We have devised a simplified method which alleviates these problems. Genomic DNA prepared from peripheral blood nucleated cells was facilitated using the punched-out filter paper absorbed with the peripheral blood. Second exon HLA-DRB segments of the genomic DNA were amplified by the polymerase chain reaction (PCR) (30 cycles: denaturing at 94 C for 1 min; annealing at 55 for 1 min, extension at 72 C for 2 min) using HLA-DRB primer DRB 5'-1 (ACCGGTCGTTCITGTCCCCICAGCA) and DRB 3'-1 (CTCGCCICTGCACIGTIAAGC) designed in our laboratory. The presence of specific alleles in a PCR-amplified sample was determined by dot-blot hybridization with 32 P-labeled sequence-specific-oligonucleotides (SSOs). Quantitation of the producer is being evaluated using an automated scanner. These newly designed primers allowed amplification of all the known expressed alleles of DRB 1, B 3, B 4 and B 5 loci. This type of reproducible and precise assay is very useful in the HLA-class II typing of large number of patients.

DNA-HLA (human leukocyte antigen) typing was performed for ocular tissues in order to determine the usefulness of the method in corneal transplantation. Each type of ocular tissue was dissected from eye bank eyes (n = 3). DNA was extracted, and HLA-DRB1, DQB1, and DPB1 genes were amplified using PCR (polymerase chain reaction) method. DNA can be extracted and amplified from each ocular tissue except for the crystalline lens, but DNA from iris and choroid can be amplified only after diluting the samples 10 to 100 times. HLA class II antigens were successfully determined in these ocular tissues by the RFLP (restriction fragment length polymorphism) method. The method was applied to the corneal tissues of donor and recipient used for the corneal transplantation (n = 7). HLA class II antigens can be determined in both the donor and recipient corneal samples, and the results of recipients' HLA typing were the same as those determined using the blood samples. These results indicate that the DNA-HLA typing can be used for ocular tissues. Since DNA-HLA typing is more accurate than the conventional serological typing, the method is promising for the study of HLA class II typing in corneal transplantation.

Pretransplant tests necessary for kidney transplantation are HLA typing, mixed lymphocyte culture response (MLR), and direct crossmatch. HLA typing and MLR are closely related to graft survival rates. The significance of HLA matching is generally known. In our analysis of 25 living related kidney grafts, graft survival rate of two haplo identical donor transplants was 4/4 (100%), while that of one haplo identical donor transplant was 18/21 (85.7%). Acute rejection rate in the MLR low response group (S.I. less than or equal to 5) was 2/7 (28.6%), while that in the high response group (S.I. greater than 5) was 11/19 (57.9%). HLA typing and MLR are useful in selecting the most suitable recipient. In order to reduce the risk of hyperacute or accelerated graft rejection, T warm direct crossmatch is performed. Anti-T warm antibodies are mainly produced by blood transfusion. In our study of 239 hemodialysis patients, there were 31 patients with positive T warm (13.0%), the positive rate became higher in proportion to increases in blood transfusion. Recently, there have been reports of kidney transplants successfully performed across T warm-positive crossmatches due to IgM antibodies. We also investigated the immunoglobulin class. Not only pretransplant crossmatch but also posttransplant crossmatch is necessary. In the case of accelerated, acute or chronic rejection, anti-donor HLA antibodies are produced in patients' peripheral blood. Thus, the test of posttransplant anti-donor antibodies is useful for the early detection of rejection, its diagnosis, and index of the prognosis. The sensitivity of flow cytometry crossmatches was compared to standard cytotoxicity crossmatch. Titration studies indicate a 32-64 fold range of greater sensitivity than the cytotoxicity test.(ABSTRACT TRUNCATED AT 250 WORDS)