1. The genetics and heterogeneity of MHC
topics, keywords:
Heterogeneity of MHC molecules
Mutations
Alleles
Allele frequency
MHC gene region and genes
The inheritance of MHC
Heterogeneity and expression of MHC class I
Heterogeneity and expression of MHC class II
Mechanisms of heterogeneity

2. Why are so many MHC variants?
Multiple MHC variants  Various peptide binding „pockets”   Multiple various peptide binding specificity
The replication rate of pathogenic microorganisms is faster than human reproduction
The genes of a pathogen can mutate frequently in a given time and can easily evade the efficient antigen presentation by MHC molecules
To counteract the flexibility of pathogens
The MHC has developed many variants
Some variants could not provide protection from a particular pathogen, but there should be an appropriate variant in the population which gives efficient protection

3. The beneficence of the MHC polymorphism
hypothetical model
The beneficence of the MHC polymorphism
The outcome of an infection in a population with or without polymorphic MHC
Example: If a single type of MHC (MHC X) was the only in the population
Pathogen that evades MHC X
MHC XX v
Multiple variety of MHC
– vulnerable individuals
Population threatened
with extinction
Heterogeneous population is protected

4. The diversity of the peptid presenting MHC molecules of the individual
Polygenic – encoded by multiple genes (evolutionary gene duplications) – ISOTYPES!
human MHC class I molecule isotypes: Human: HLA-A, HLA-B, HLA-C genes
MHC class II molecule isotypes: Human: HLA-DP, HLA-DQ, HLA-DR genes
Polymorphic – genes can have various alleles
MHC genes are the most polymorphic known! The genes of the peptide presenting ”classical” MHC molecules have several various alleles in the population. Every isotype can have two alleles in a given heterozygous individual.
one gene with different alleles
multiple genes, without alleles
multiple genes with different alleles
Molecular basis of MHC types and variants
Haplotípus: egy kromoszómán levő MHC gének összessége (kombinációja)
Az MHC régió viszonylagos rövidsége miatt ritka benne a rekombináció (ezért általában kapcsoltan öröklődik az egész)
ezért az eredeti haplotípusok valamelyike öröklődik tovább az utódokba is
Későbbi előadáson esik még szó róla részletesebben

5. HLA – Human Leukocyte Antigen
The gene variants is termed as „isotypes”. DO NOT MIX with the „allele” term.
The classical peptide presenting MHC molecules are indicated within the red frame. They are very polymorphic – they have lots of alleles.
The other non polymorphic proteins have different roles in immunity and antigen presentation
HLA – Human Leukocyte Antigen

6. Polymorphic residues of the MHC molecules are located in the peptide binding site of the molecules
Fundamental immunology, 6th ed, Philadelphia, 2008, Lippincott Williams & Wilkins
FIGURE 6-11 Polymorphic residues of MHC molecules. The polymorphic residues of class I and class II
MHC molecules are located in the peptide-binding clefts and the α helices around the clefts. The regions of
greatest variability among different HLA alleles are indicated in red, of intermediate variability in green, and
of the lowest variability in blue. (Reproduced with permission from Margulies DH, Natarajan K, Rossjohn J,
McCluskey J: Major histocompatibility complex [MHC] molecules: structure, function, and genetics. In Paul
WE [ed]: Fundamental immunology, 6th ed, Philadelphia, 2008, Lippincott Williams & Wilkins.)
The polymorphic residues (allelic variations) is clustered in the peptide binding site Allelic variants can reach 20 aa differences in the sequence (10% of the total sequence )
MHC polymorphism influences the peptide binding: Different allelic variants bind different peptides with different efficiency (motifs!)
This has influence on the T cell activation
It can explain why different persons mount immune responses with different efficiency

7. Most polymorphisms derive from point mutations
30 HLA-DPB1 allele sequences between nucleotides 204 and 290 (amino acids 35-68)
Y-F
A-V
Silent
A-D
A-E
E-A
I-L
DPB1*01011 TAC GCG CGC TTC GAC AGC GAC GTG GGG GAG TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GCT GCG GAG TAC TGG AAC AGC CAG AAG GAC ATC CTG GAG GAG DPB1* A DPB1* T- -T A- -A DPB1* T- -T AC -A DPB1*0202 CT- -T AG DPB1* T- -T A- -A- --C C DPB1* T DPB1* T- -T A- -A DPB1*0501 CT- -T AG DPB1* T- -T A- -A- --C C DPB1* T- -T A- -A DPB1* T- -T A- -A- --C DPB1* T- -T A- -A DPB1* A C DPB1* A C DPB1* DPB1* T- -T A- -A- --C C DPB1* A C DPB1* T- -T A- -A DPB1* T- -T A- -A- --C DPB1* T- -T A- -A DPB1* T- -T AG DPB1* T- -T A- -A- --C C DPB1* T- -T A- -A- --C C DPB1*2101 CT- -T AG DPB1*2201 CT- -T AG DPB1* T- -T DPB1* T AG DPB1* T- -T A- -A C DPB1* A DPB1*
missense mutation – results amino acid change
Some polymorphism doesn’t influence the peptide binding specificity of the molecules
Polymorphic sequences encode amino acids associated with the peptide binding site

8. Natural selection can change the allele frequency in different groups or populations in areas hosting endemic pathogens
Some MHC allele could provide more efficient protection against a specific pathogen than others This could be observed in the distribution of MHC alleles’ frequency in different human geographical populations:
The allele corresponding the HLA-B53 serotype is strongly associated with the recovery from the lethal form of malaria. HLA-B53 serotype is very common in some region where malaria (Plasmodium - parasitic protozoa) is endemic.
HLA-B27 and B57 serotypes have higher allele frequency in the group of ”HIV controllers”
Some HLA allele frequency is in correlation with the occurrence of a specific disease in a population. The correlation could be either positive or negative.
szerotípus fogalma: adott tipizáló szérummal reagál (ellenanyag tartalmú szérummal)
HLA-B53: HLA-B szerotipizálás alapján 53-as típusa
Ne keverjük össze a később említésre kerülő gén nevekkel! (pl. HLA-DRB1, HLA-DRB5)
Ne keverjük össze a későbbi ábrákon látható szekvenálási adatbáziosok transzkript számozásával! (-001, -002, -201….)
Ne keverjük össze a szekvencia alapon meghatározott HLA típusok számozásával se, bár ezekkel mutatnak összefüggést
strong association of the HLA-B53 allele with recovery from
a potentially lethal form of malaria. This allele is very common in people
from West Africa, where malaria is endemic, and rare elsewhere, where lethal
malaria is uncommon.
Patients exhibiting such control for long
periods are termed ‘elite controllers’ or ‘HIV controllers
(HIC)’ [6]. The HLA alleles B27 and B57 are highly enriched
in this population. However, the presence of these protective
HLA alleles is neither sufficient nor always necessary to
achieve control of infection.

9. The heterogeneity of human MHC alleles and proteins
HLA alleles
HLA proteins (2015)
The numbers of the identified alleles has been increasing year by year

10. 3 subregion – according the function of the genes
Leukocytes were used for the identification of the proteins  Human Leukocyte Antigen (HLA)
Janeway’s Immunobiology, 8th ed. (Garland Science 2012)
(Human Leukocyte Antigen)
(Histocompatibility-2)
chromosome 6
3 subregion – according the function of the genes
mouse chromosome 17
Human: chromosome 6
Mouse: chromosome 17

11. FROM THE HUMAN GENOME PROJECT
MAP OF THE HUMAN MHC
FROM THE HUMAN GENOME PROJECT
3.8Mbp
~225 genes (orf)
on chromosome 6
orf – open reading frame (possible gene)
The MHC sequencing consortium
Nature 401, 1999
various protein coding genes, non-protein coding genes (e.g. miRNA), pseudogenes

12. The MHC gene region is quiet short
Journal of Human Genetics (2009) 54, 15–39

13. You can have detailed information about the genes from databasesp q

14. The inheritance of HLA

15. Example of a human MHC I haplotype pair
THE HAPLOTYPE
MHC I
gének:
(izotípusok)
MHC haplotype – the combination of the MHC alleles encoded by one of the diploid chromosome pair
Example of a human MHC I haplotype pair B: C: A:
HLA-
One MHC I haplotype of the person:
B14, Cw1, A3
The other MHC I haplotype:
B8, Cw4, A2
allélok (a populációban)
w – „workshop” (pl. a C gének alléljainak nevében)
„w” megjelölést kapnak az új vagy nehezen karakterizálható allélok. Utal arra, hogy még jelenleg is a nemzetközi workshopok segítségével próbálják véglegesíteni az azonosításukat. A HLA-C allélok gyengén immunogének, ezért nehézkes a vizsgálatuk.
The HLA allele names in the example are the so called ”serotypes”

16. possible combinations in the offsprings
Inheritence of MHC A B C DP DQ DR
possible combinations in the offsprings
The MHC region is rather short
Rare meiotic recombinations (linkage)
generally the haplotypes are inherited
parent 1 A C B DP DQ DR ×
parent 2
Because of the thight genetic linkage, one-one haplotype is inherited in the original form from parent to offspring. The progeny inherit one haplotype from each ancestor, and pass them down to their offsprings in the original forms. A B C DP DQ DR

17. The genetics and heterogeneity of MHC Ip q
The genetics and heterogeneity of MHC I 6:
Janeway’s Immunobiology, 8th ed. (Garland Science 2012)
(Human Leukocyte Antigen)
(Histocompatibility-2)

18. * * The genetics and heterogeneity of MHC I 6: exons: β2-microglobulinp q
The genetics and heterogeneity of MHC I 6: * *
exons: 1 2 3 4 5 6 7 8
leader,
signal
n.t.
β2-microglobulin
(chromosome 15)
α-chain tm c α1 α2 α3
NH2-
-COOH
protein
domains
Exon2 and exon3 encode the peptide binding specificity – heterogeneous parts – used in sequence based HLA typing
-COOH α3 α2 α1
β2m
-COOH α3 α2 α1
β2m
-COOH α3 α2 α1
β2m
HLA-B
HLA-C
HLA-A
cytosol
membrane

19. generally 6 kind of MHC I moleculep q
The heterogeneity of the human MHC I 6:
diploid individual
chromosome 6 MHC I region A B C
codominant expression
haplotype
(maternal origin)
haplotype
(paternal origin)
alleles
One individual:
generally 6 kind of MHC I molecule

20. The genetics and heterogeneity of MHC IIp q 6:
Janeway’s Immunobiology, 8th ed. (Garland Science 2012)
(Human Leukocyte Antigen)
(Histocompatibility-2)

21. Allelic variants of the classical polymorphic MHC II molecules
The alpha chains are less polymorphic than the beta chains
(Alpha chain should bind the non-polymorphic HLA-DM during the ”peptide editing”)
HLA-DRA is monomorphic in practice. The two proteins are almost identical.

22. Intrahaplotype or ”cis” combinationsq 6:
The genetics and heterogeneity of MHC II DR DQ DP A B
haplotype
(of maternal origin) A B
haplotype
(of paternal origin)
HLA-DRA practically monomorphic
Intrahaplotype or ”cis” combinations DR
intrahaplotype, intraisotype combinations DQ DP DR DQ DP β2 β1 α1 α2 β2 β1 α1 α2 β2 β1 α1 α2 β2 β1 α1 α2 β2 β1 α1 α2 β2 β1 α1 α2
The most favoured and frequent combinations on the cell

23. The genetics and heterogeneity of MHC IIp q 6:
The genetics and heterogeneity of MHC II DP DR A B DQ A B
haplotype
(of maternal origin)
haplotype
(of paternal origin)
The α and β chains of the maternal and paternal haplotype can be combined
All isotype (DP, DQ , DR) can produce cross haplotype combinations (”trans”) β2 β1 α1 α2
”cis”
HLA-DQ α1 α2 β2 β1
”trans”
HLA-DQ β2 β1 α1 α2
”trans”
HLA-DQ β2 β1 α1 α2
”cis”
HLA-DQ
cross haplotype, intraisotype combinations
α and β chains of different alleles can prefer or unfit each other

24. The genetics and heterogeneity of MHC IIDR DQ
The α and β chains of different MHC II gene isotypes (HLA-DR, HLA-DQ, HLA-DP) can be combined if they prefer each other. A B A B
Mixed isotype MHC II heterodimers
It is also possible between (maternal and paternal) haplotypes
Lots of combinations are unfit (few percent can appear on the cells)
The expression of mixed isotype dimers can increase if some isotype express only one chain of the pair (e.g. the other is a null-allele)
DRα
DQβ
DQα
DRβ
interisotypic combinations or mixed isotype heterodimers
intrahaplotype, iterisotype and interhaplotype, interisotype combinations
Karp DR, Teletski CL, Jaraquemada D, Maloy WL, Coligan JE, Long EO.: J Exp Med Mar 1;171(3): Structural requirements for pairing of alpha and beta chains in HLA-DR and HLA-DP molecules.
Lotteau V, Teyton L, Burroughs D, Charron D.: A novel HLA class II molecule (DR alpha-DQ beta) created by mismatched isotype pairing. Nature Sep 24-30;329(6137):
Kaushansky N, Eisenstein M, Boura-Halfon S, Hansen BE, Nielsen CH, Milo R, Zeilig G, Lassmann H, Altmann DM Ben-Nun A.: Role of a Novel Human Leukocyte Antigen-DQA1*01:02;DRB1*15:01 Mixed Isotype Heterodimer in the Pathogenesis of "Humanized" Multiple Sclerosis-like Disease. J Biol Chem Jun 12;290(24): doi: /jbc.M Epub 2015 Apr 24.
Spencer JS, McCormack JE, Kubo RT.: Characterization of defective I-A surface expression in a mixed isotype expressing murine B cell lymphoma: continued expression of E alpha d A beta d despite competition from restored A alpha d A beta d pairs. Int Immunol Aug;4(8): ( β2 β1 α1 α2 α1 α2 β2 β1

25. The extreme heterogeneity of the HLA-DR locus (1.)
The genetics and heterogeneity of MHC II
The extreme heterogeneity of the HLA-DR locus (1.)
The structure of the HLA-DR locus can have large differences between individuals (Even the two haplotypes of an individual can greatly differ from each other.)
The HLA-DR locus encode the monomorphic alpha chain (HLA-DRA)
The HLA-DR locus encode at least one beta chain (HLA-DRB1)
a HLA-DRαβ molecule α1 α2 β2 β1
HLA-DRA gene
α-chain
HLA-DRB1 gene
β-chain
This two gene are expressed generally in everyone

26. The extreme heterogeneity of the HLA-DR locus (2.)
The genetics and heterogeneity of MHC II
The extreme heterogeneity of the HLA-DR locus (2.)
The structure of the HLA-DR locus can have large differences between individuals (Even the two haplotypes of an individual can greatly differ from each other.)
The HLA-DR locus can contain multiple HLA-DRB gene isotypes
The HLA-DRB isotypes can encode additional β protein chains (HLA-DRB3, -DRB4, -DRB5)
Some HLA-DRB isotypes are pseudogenes (Ψ) (HLA-DRB2, -DRB6, -DRB7, -DRB9)
The additional β chains can be combined with the α chain to form other functional HLA- DR isotypes
DRA
DRB1
DRB3
DRB5
DRB4
HLA-DR molecules
The isotypes are numbered acoording the sequence homology and the relative position in the HLA-DR locus
Ψ (psi) – symbol means pseudogene in the genetics
The HLA-DR locus of different individuals or haplotypes contains generally the HLA-DRA, HLA-DRB1, plus
one additional protein coding HLA-DRB isotype
and 2-3 HLA-DRB pseudogenes
in various combinations
See the examples:

27. HLA-DR locus can encode multiple beta chainsq 6:
HLA-DR locus can encode multiple beta chains
DRA
DRB1
DRB5
An example of a haplotype:
HLA-DRA
HLA-DRB1
HLA-DRB5
ΨDRB9
ΨDRB6
Ψ (psi) – symbol means pseudogenes
(DRB1 can use the last exon of ΨDRB6 in a transcript (ic part of the molecule))
alternative splicing
Possible DR β encoding isotypes: DRB1, DRB5

28. HLA-DR locus can encode multiple beta chainsq 6:
HLA-DR locus can encode multiple beta chains
DRA
DRB1
DRB3
Other haplotype
HLA-DRA
HLA-DRB3
ΨDRB9
HLA-DRB1
ΨDRB2
alternative splicings
DRB1 and DRB3 can ”purchase” exons from each other
DRB3-202 transcript gets the second exon (beta 1 domain) of the DRB1  peptide binding similar to DRB1
DRB1-202 transcript gets the trailet exon of the ΨDRB2
Possible DR β encoding isotypes: DRB1, DRB3

29. HLA-DR locus can encode multiple beta chainsq 6:
HLA-DR locus can encode multiple beta chains
DRA
DRB1
DRB3
A third haplotype
(similar to the previous one)
HLA-DRA
HLA-DRB3
HLA-DQB1
ΨDRB9
One transcript of the DRB1 (205) gets the first two exons of the DQB1 (leader-signal sequence and beta1 domain of the protein)
ΨDRB2
HLA-DRB1
Possible DR β encoding isotypes: DRB1, DRB3

30. HLA-DR locus can encode multiple beta chainsq 6:
HLA-DR locus can encode multiple beta chains
DRA
DRB1
DRB4
A fourth haplotype
HLA-DRA
HLA-DRB4
ΨDRB8
ΨDRB9
HLA-DRB1
ΨDRB7
An alternative transcript of the DRB4 (202) gets the second exon of the ΨDRB7 pseudogene (beta1 domain) – the peptide binding specificity of a pseudogene can appear in the hybride molecule
Possible DR β encoding isotypes: DRB1, DRB4

31. A simplified example: individuals with multiple and single HLA-DRB isotypes
4 isotype in one person
a person with only one isotype
DRB3
DRB1
ΨDRB6
DRB1
maternal
haplotype
maternal
haplotype
identical
haplotypes
(with identical
alleles)
DRB5
DRB1
ΨDRB6
DRB1
paternal
haplotype
paternal
haplotype
DRα
DRβ
DRα
DRβ
4 different peptide binding
specificity
a single peptide binding
HLA-DR molecules

32. The HLA-DQ has two alpha and two beta coding isotype6:
The HLA-DQ has two alpha and two beta coding isotype
HLA-DQA1
These are the α and β chains
of the ”classical” HLA-DQ molecule
HLA-DQA2
These isotypes have shown expressed only by Langerhans cells
ΨHLA-DQB3
HLA-DQB2
HLA-DQB1
J Immunol Apr 15;188(8): doi: /jimmunol Epub 2012 Mar 9.
HLA-DQA2 and HLA-DQB2 genes are specifically expressed in human Langerhans cells and encode a new HLA class II molecule.
Lenormand C1, Bausinger H, Gross F, Signorino-Gelo F, Koch S, Peressin M, Fricker D, Cazenave JP, Bieber T, Hanau D, de la Salle H, Tourne S.
Generally the HLA-DQA1/HLA-DQB1 is expressed in the professional APC

33. Curiosities
It seems the neighbouring genes (isotypes) which are in the same orientation can use each other’s exons by alternative splicing
The exons of some pseudogenes can be used this way also
In the case of MHC I (HLA-A, -B, -C), such an exon exchange involving the second and third exons (alpha1 and alpha2 domains of the MHC I proteins) would increase the variability of the peptide binding site.
There is a hint in the database that suggests the existence of such a mechanism:

34. transmembrane domain is missing
A transcript of the HLA-C could use the first two exons of the HLA-B in the case of this haplotype
HLA-C
HLA-B
transcribed region L α1
HLA-B
HLA-C α2 α3
ex1
ex2
ex3
ex4
transmembrane domain is missing
-COOH α3 α2 α1
β2m
„HLA-B”
„HLA-C”
The protein could be soluble
instead of a membrane bound one:

35. Mechanisms of the MHC polymorphism
summary
Mechanisms of the MHC polymorphism
allelic variances of the population principally: combinations of several thousand alleles, practically: a pair of inherited haplotype combinations of the individual which change rather infrequently by recombinations
MHC gene/molecule isotypes 2x3 MHC I, 2x3-4 MHC II from the two inherited haplotype
α- and β-chain combinations of MHC II 6-8 to (principally up to 40) MHC II αβ dimer. The intra isotype, intra haplotype α β chains like to combine with each other the best.
alternative splicing (currently the evidences are based on sequence database data) Alternative splicing could combine peptide binding domains between isotypes (possibly involving the peptide binding domains of the pseudogene isotypes)
40 MHC II αβ combinations:
1x DRA combined with 2x2 DRB… or 2x1 DQB1 or 2x1 DPB1  8
2x DQA1 combined with 2x2 DRB… or 2x1 DQB1 or 2x1 DPB1 16
2x DPA1 combined with 2x2 DRB… or 2x1 DQB1 or 2x1 DPB1 16
And this is not involving the DQA2 DQB2 possibilities!!!

36. Other genes of the MHC regions
Class I region (Classical polymorphic MHC I molecules) Lots of MHC class Ib molecules: 2 microglobulin-associated, non-polymorphic MHC I–like molecules, with diverse tissue expression, influencing NK cell functions: e.g. HLA-E, HLA-F, HLA-G, MICA, MICB
Class II region (Classical polymorphic MHC II molecules) Genes involved in the antigen processing, chaperones: HLA-DM/, HLA-DO/, proteasome components (LMP-2 and 7), peptide transporters (TAP-1 and 2)
Class III region Genes of some complement proteins: C4 (multiple isotypes), C2 and factor B cytokines: Tumour Necrosis Factor (TNF) and Lymphotoxin (LT) genes
All three regions contain genes which could be irrelevant in the immunity and contain pseudogenes also:
e.g. cytochrome P450 monooxygenase (CYP21A2), RNA helicase (DDX39B), subunit of casein kinase (CSNK2B), heat shock protein HSP-70 (HSPA1A), sialidase (NEU1), etc. etc.

37. Functions of MHC encoded and MHC-like molecules
MHC function summary:
Functions of MHC encoded and MHC-like molecules
- T cell and NK cell selection and differentiation in the primary lymphoid organs - T cell survival on the periphery/memory
Classical polymorphic MHC molecules endogenic self and non-self peptide antigen presentation: HLA-A, -B, -C exogenic self and non-self peptide antigen presentation: HLA-DP, -DQ, -DR inhibiting NK cell function: HLA-C, (-A, -B) allogenic immune response against non-self MHC molecules (transplantation)
Non-classical, non-polymorphic MHC encoded molecules presentation of non-polymorphic peptide antigens: HLA-E (HLA-F?) participation in the antigen processing: TAP1, -2, HLA-DM, -DO, LMP-2, -7 influencing the NK cell function: HLA-E, -F, -G( ), MICA, -B( ) other immunological functions: C4, TNF… non immunological functions: HSP-70 (HSPA1A)…
MHC-like molecules encoded outside the MHC region presentation of non-polymorphic, non-peptide antigens: CD1, MR1 other immunological functions: FcRn (FCGRT) non immunological function: HFE

38. The genetics and heterogeneity of MHC
Themes and topics (to know):
Heterogeneity of MHC molecules (reasons and consequences)
Mutations, alleles, allele frequency
MHC gene region, haplotype
The inheritance of MHC
Heterogeneity and expression of MHC class I
Heterogeneity and expression of MHC class II
Mechanisms of heterogeneity
Other proteins of the MHC
various terms (you should know):
locus
gene
allele
haplotype
isotype (of MHC genes)
alternative splicing
polymorphism
polygeny
homozygote, heterozygote
pseudogene
null allele
allele frequency
exon, domain
MHC, HLA
The Immune System (Parham P): chapter 5-18 – 5-23 (4th ed: p )
Basic Immunology (Abbas AK): chapter 3 (4th ed: p55-61)

39. Lots of information about HLA: