1. Major Histocompatibility Complex
Dept. of Laboratory Medicine
Eun Young Song

2. 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

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

4. 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+)

5. 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

6. T cell recognition of MHC-peptide complex

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

8. 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         

9. HLA class I & class II gene polymorphismB 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

10. 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
Child Child Child 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

11. 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

12. 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.

13. Serological and cellular specificities (2010)

14. 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* N
<2010 nomenclature> HLA-A*29:01:01:02N

15. 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

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

17. HLA molecular test PCR-SSO Sequence Specific Oligonucleotide probes
PCR-SSP
Sequence Specific Primers
PCR-SBT
Sequence Based Typing

18. 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)

19. 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

20. Reverse SSOP reverse SSO – Luminex
SSO probes are attached on colored bead
Hybridization with Streptavidin-PE-labeled PCR products

22. 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

23. SSP (sequence specific primer)
PCR with sequence specific primers, typing using the existence of PCR products

24. SSP (sequence-specific primer) typing
B*44/B*55

25. 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

27. 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

28. 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

30. Viable cells ≥ 90% Viable cells ≤ 10%
XM negative XM positive

31. 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

32. Flowcytometry crossmatch

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

34. 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

35. 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

36. 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

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

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

39. 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