Major Histocompatibility Complex Dept. of Laboratory Medicine Eun Young Song

Major Histocompatibility Complex, MHC a set of cell surface proteins essential for the acquired immune system to recognize foreign molecules in vertebrates, which in turn determines histocompatibility  Function: bind to antigens derived from pathogens and display them on the cell surface for recognition by the appropriate T- cells Human leukocyte antigen (HLA) : a gene complex encoding the MHC proteins in humans

Contents Structure and function of HLA molecule HLA inheritance and polymorphism HLA nomenclature HLA typing & HLA Ab tests

HLA (Human Leukocyte Antigen) HLA - structure and function Class I molecule (HLA-A, B, C) expressed on all nucleated cells Presents endogenous Ag to T-lymphocyte (CD8+) Class II molecule (HLA-DR, DQ, DP) expressed on B lymphocyte, activated T lymphocyte, monocyte, macrophage, dendritic cells, etc. Presents exogenous Ag to helper T-lymphocyte (CD4+)

Structure of HLA class I, class II 57 kd: heavy chain (45 kd), β2-microglobulin (12 kd) cleft by α1 and α2 : Ag-binding pocket α3 domain : interact with CD8 on T cells < Class II > 63 kd: α chain (34 kd), β chain (29 kd) cleft by α1 and β1 : Ag-binding pocket α2, 2: interact with CD4 on T cells

T cell recognition of MHC-peptide complex

HLA genes 6p21.3 ~4 Mb, > 250 genetic loci Classical HLA: HLA-A, B, C, DR, DQ, DP

HLA polymorphism More than two kinds of molecules from same gene locus Most polymorphic genes in humans Cause graft rejection due to variable HLA molecules in each persons         

HLA class I & class II gene polymorphism B C DRB1 DQB1 DPB1 Allele No.(2009) (Jul, 2017) 965 3,968 1,543 4,828 626 3,579 762 2,376 107 1,142 138 894 Protein No. 2,781 3,501 2,490 1,739 774 641 * HLA polymorphism (allele, protein): 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 A33 B44 DR13 A11 B62 DR4 A1 B27 DR1 A30 B13 DR7  a b c d Child1 Child2 Child3 Child4 A33 B44 DR13 A1 B27 DR1 A33 B44 DR13 A30 B13 DR7 A11 B62 DR4 A1 B27 DR1 A11 B62 DR4 A30 B13 DR7 a c a d b c b d The probability of agreement of HLA with one’s sibling: 1/4

HLA Nomenclature (by WHO Committee for HLA Nomenclature) HLA Antigen ex) HLA-A2, B7, Cw1, DR4 HLA allele ex) HLA-A*02:01, B*07:02, C*01:02, DRB1*04:05

Nomenclature for HLA Ag assigned according to international histocompatibility workshops during which typing reagents were exchanged among participating laboratories based on the order in which the serologically defined specificity was defined.  split Ag (subtypic or private specificity) Over time, older serologically defined specificities were “split,” as the definition became more discriminating. broad Ag (supertypic specificity) The broader, shared specificities before ‘split’. e.g.) A9 → A23, A24 A23 and A24 are subtypic specificities of the supertypic specificity A9. Thus, a cell that is either A23+ or A24+ must also be positive for A9.

Serological and cellular specificities (2010)

HLA-A*29:01:01:02N Nomenclature for HLA allele Differences outside the coding region (introns) Signifies DNA Subtype HLA-A*29:01:01:02N Locus Type Silent Substitution Expression Corresponds to the serologic antigen or family <2004 nomenclature> HLA-A*29010102N <2010 nomenclature> HLA-A*29:01:01:02N

Nomenclature for HLA allele HLA-A*0201, Cw*0102, DRB1*0405 <2004 nomenclature> HLA-A*02:01, C*01:02, DRB1*04:05 <2010 nomenclature> Major changes in 2010 nomenclature 1) Separation using colon(:) into the allele names to act as delimiters of the separate fields 2) HLA-C* : The ‘w’ well be removed from the HLA-C allele names (but will be retained in the HLA-C antigen names, to avoid confusion with the factors of the complement system) Usually reports second fields (e.g. A*02:01) in clinical tests

HLA typing Serologic test Molecular test PCR-SSO PCR-SSP PCR-SBT Microlymphocytotoxicity Test Molecular test PCR-SSO PCR-SSP PCR-SBT

HLA molecular test PCR-SSO 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 – Luminex SSO probes are attached on colored bead Hybridization with Streptavidin-PE-labeled PCR products

http://es.slideshare.net/

Luminex PCR-SSO BEAD 1 DRB1*01:01 BEAD 2 DRB1*03:01 Positive Negative PE-primer PCR product Complementary Oligonucleotide Negative Positive BEAD 1 DRB1*01:01 BEAD 2 DRB1*03:01

SSP (sequence specific primer) PCR with sequence specific primers, typing using the existence of PCR products http://es.slideshare.net/

SSP (sequence-specific primer) typing 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 Using Sanger sequencing method flurorescent tagged A, G, C, T base

HLA Ab assay DSA (donor HLA-specific Ab) HLA crossmatch CDC-XM (crossmatch) FCXM (Flowcytometric) PRA (panel reactive antibody) tests Luminex Sensitivity Luminex PRA > FCXM > CDC-XM

CDC-crossmatch Microlymphocytotoxicity test Principle: complement-dependent cytotoxicity, CDC Methods: microplate – mix patient serum + lymphocytes of donor (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% XM negative XM positive

FCXM (Flowcytometric crossmatch) Detector PE-anti-CD3 T cell Ag(CD3) HLA Ag anti-HLA Ab Laser FITC-goat F(ab’)2-anti-human IgG

Flowcytometry crossmatch

PRA (Panel Reactive Antibody) Ab screen & Identification in patient serum Types Screen Phenotype panel Single antigen assay

Luminex technology MFI (mean fluorescence intensity) Negative PE-anti-IgG Allo Antibody A3 HLA Antigen Negative Positive BEAD 1 A1,A2,B7,B8 BEAD 2 A3,A29,B13,B27 No Antibody

PRA (Panel Reactive Antibody) Screen Detects HLA Ab Use antigen pool Phenotype Panel Use purified Ag, several tens of panels (~50) Identify the specificity of HLA Ab Single Antigen assay Use recombinant Ag advantage: easy identification weakness: distortion of molecule – false positive

PRA - Screen – Luminex LIFECOSES LifeScreen Deluxe (Gen-probe) Bead Specificity 1 Class I (300 donors) 2 A1, A19 CREG enriched 3 A2 CREG enriched 4 B5 CREG enriched 5 B7 CREG enriched 6 B8 CREG enriched 7 B12 CREG enriched 8 Class II (30 cell lines) 9 DR51 enriched 10 DR52 enriched 11 DR53 enriched 12 DQ enriched

PRA – ID (phenotype panel) LIFECODES Class I ID (Immucor, USA)

PRA - Single Ag LIFECODES LSA Class II (Immucor, USA)

Compatibility tests in organ transplantation (1) ABO typing (2) HLA-A, B, DR (3) HLA crossmatch (4) PRA solid organ : ABO typing, HLA crossmatch – critical kidney, pancreas – consider HLA match bone marrow (HSC) : HLA matching – critical