Major Histocompatibility Complex Dept. of Lab Med Eun Young Song

Contents Sturcutre and function of HLA molecule HLA inheritance and polymorphism HLA nomenclature HLA tests

HLA (Human Leukocyte Antigen) HLA - structrue and function Class I molecule (HLA-A, B, C) Most nucleated cells Ag presentation to cytotoxic T-lymphocyte (CD8+) rejection, kill virus-infected cells Class II molecule (HLA-DR, DQ, DP) B lymphocyte, activated T lymphocyte, monocyte, macrophage, dendritic cells… Ag presentation to (helper) T-lymphocyte(CD4+)

HLA class I molecule cleft by α1 and α2 : Ag-binding pocket α3 domain : interact with CD8 on T cells

HLA class II molecule α2,  2: interact with CD4 on T cells

HLA genes 6p21.3

HLA polymorphism More than two kinds of molecules from same gene locus Several tens ~ hundreds of alleles Cause graft rejection due to variable HLA molecules in each persons         

HLA class I and class II polymorphism B C DRB1 DQB1 DPB1 Allele No. (Dec. 2009) (Mar, 2012) (Jul, 2014) 965 1,757 2,884 1,543 2,338 3,589 626 1,304 2,375 762 1,166 1,540 107 162 664 138 152 422 Antigen No. 26 53 10 18 9 6 * HLA polymorphism : class I : B > A > C class II : DR > DQ, DP

The probability of agreement of HLA with one’s sibling : 1/4 HLA inheritance HLA genes are located very closely each other and inherited as a haplotype. Father Mother A2 B44 DR13 A11 B54 DR9 A3 B27 DR8 A24 B40 DR7  Child A2 B44 DR13 A3 B27 DR8 The probability of agreement of HLA with one’s sibling : 1/4

HLA nomenclature (by WHO committee) HLA Ag ex) HLA-A2, B7, Cw1, DR4 HLA allele ex) HLA-A*02:01, B*07:02, C*01:02, DRB1*04:05

subtypic or private specificity HLA Ag nomenclature named after the order in which the serologic specificty was defiend in International histocompatibility workshop. subtypic or private specificity subdivided Ag, when the original Ag was split and named supertypic specificity Original Ag before split (e.g., B12 → B44, B45). B44 Ag-positive lymphocyte shows positive reaction also with B12 anti-sera.

Split antigens

HLA allele nomenclature HLA-A*0201, Cw*0102, DRB1*0405 <2004 nomenclature> HLA-A*01:01:01:02N <2010 nomenclature> First region agreement with serologic specificity e.g.) A*0201 shows A2 serologic specificity Disagreement with serologic specificity when named after nucleotide sequence similarity e.g.) B*15:18 didn’t show serologic B15 specificity, but shows B71 specificity. Second region The order in which alleles were defined. A*02:01 was defined first, A*02:03 was defined third. 68년 WHO nomenclature committee구성 87년 처음 DNA nomenclature 도입 serologic 정보없이 allele 급증하면서 96년부터는 첫 두자리는 단순히 염기서열의 유사성 2010년 4월

HLA allele nomenclature Third region Silent or synonymous substitution. Amino acids are same, though nucleotide sequence of coding region was different. e.g.) B*27:05:02, B*27:05:03 Fourth region nucleotide sequence of noncoding region was different. DRB4*01:03:01:01, DRB4*01:03:01:02 Suffix, N, L, S, Q N (null): No protein expression L (low): Very low protein expression

2010 HLA nomenclature For A*02 and B*15 alleles, ‘92’ and ‘95’ had been used because the number of alleles were over 99. Three digits are used in 2010 nomenclature.

The HLA Dictionary 2008 Tissue Antigens 2009:73:95-170 1997, 1999, 2001, and 2004 (5th update)

HLA typing Serologic test Molecular test Cellular detection method Microlymphocytotoxicity Test Molecular test PCR-SSOP, Reverse SSOP PCR-SSP PCR-SBT Cellular detection method Mixed Lymphocyte Culture (MLC) The polymorphism of the HLA molecules is determined by serological test using specific antibodies, cellular method such MLC and molecular analyses such as PCR-SSP (Sequence Specific Primers), PCR-SSOP (Sequence Specific Oligonucleotide Probes) and PCR-SBT (Sequence Base Typing). I am going to tell you more detail about PCR-SSOP.

Microlymphocytotoxicity test Principle: complement-dependent cytotoxicity, CDC Methods: microplate with various antisera (eg. anti-HLA-A2 Ab) + lymphocytes of patients (HLA Ag) → adds rabbit complement → penetration of Eosin Y due to cell lysis → reading with inverted phase contrast microscopy . + complement + Cell lysis Penetration of Eosin Y

Viable cells ≥ 90% Viable cells ≤ 10%

ASHI reading standard

HLA molecular test PCR-SSOP Sequence Specific Oligonucleotide Probes PCR-SSP Sequence Specific Primers PCR-SBT Sequence Based Typing

Classification of resolution of HLA tests Low (low resolution) serologic level (serologic/generic) Intermediate several allele groups (DRB1*0401/0421/0433/0434/0435/0438g) High (high resolution) allele level (allelic)

HLA molecular test Class I Class II Exon 2 Exon 3 Exon 1 Exon 4 Intron 1 Intron 2 Intron 3 Typing region Class II Exon 2 Exon 3 Exon 1 Exon 4 Intron 1 Intron 2 Intron 3 Typing region

Reverse SSOP reverse SSO – line blot SSO probes are attached on strip. biotinylated primer are used in PCR. Hybridization with probes on strip -> visualization -> reading Kits used in Korea Murex INNO-LIPA, Dynal RELI reverse SSO – Luminex SSO probes are attached on colored bead Hybridization with Streptavidin-PE-labed PCR products e.g.) LIFECODES HLA DNA (Tepnel Lifecodes)

Reverse-SSOP, line blot

Luminex corp. xMAP system www.rpciflow.org

SSP (sequence specific primer) PCR with sequence specific primers, typing using the existence of PCR products The main kits in Korea Biotest, One Lambda, Biosewoom etc., B*44/B*55

SBT (sequence-based typing) high resolution test class I (HLA-A, -B, -C): exon 2, 3, (4) class II (HLA-DRB1): exon 2 The main kits in Korea Abott AlleleSEQR SBT kit, Biosewoom, In-house etc.,

Ambiguity of HLA typing low resolution vs high resolution - low resolution: DRB1* 04 + 13 vs 04 + 14 - high resolution: B* 0705 + 4001 vs 0706 + 4001 Heterozygote vs Allelic - Heterozygote ambiguity: allele 1+2 = 3+4 A*0201+2402 = 0204+2452 = 0270+2429 - Allelic ambiguity: allele 1(=2) + 3 A*0201/04/70, *2402/29/52 * Heterozygote ambiguity > Allelic amibiguity

Heterozygote ambiguity International Histocompatibility Working Group (http://www.ihwg.org/protocols/sbt/ambiguities2.pdf)

HLA tests using cellular detection MLC (mixed lymphocyte culture) In vitro measure of class II disparity (mainly the response against HLA-DR molecule) Replaced with molecular tests due to difficulties in standardization

Compatibility tests in organ transplantation (1) ABO typing (2) HLA-A, B, DR (3) HLA crossmatch (4) PRA (5) MLC solid organ : ABO typing, HLA crossmatch – critical kidney, pancreas – consider HLA match bone marrow (HSC) : HLA matching – critical ABDR full match (1 antigen mismatch) related : serologic (≒allele) level match unrelated : serologic (≠allele) level match