Chapter 15

Major Histocompatibility Complex

Major Histocompatibility ComplexCluster of genes found in all mammalsIts products play role in discriminating

self/non-selfParticipant in both humoral and cell-mediated

immunityMHC Act As Antigen Presenting StructuresIn humans MHC is found on chromosome 6

Referred to as HLA complexIn mice, MHC is found on Chromosome 17

Referred to as H-2 complex

The Major Histocompatibility Complex (MHC)

The MHC is located on chromosome 6.

The MHC contains the human leukocyte antigen (HLA) and other genes.

TNF 1 Mb 2 Mb 3 Mb 4 Mb

HLA- DP DQ DR B C A

Class II Class III Class I

Classes of MHC GenesClass I MHC genes

Glycoproteins expressed on all nucleated cells Major function to present processed Ags to TC

Class II MHC genes Glycoproteins expressed on M, B-cells, DCs Major function to present processed Ags to TH

Class III MHC genes Products that include secreted proteins that have

immune functions. Ex. Complement system, inflammatory molecules

Genes of the Major Histocompatibility LocusMHC region Gene products Tissue location Function

Class I HLA-A, HLA-B, HLA-C All nucleated cells

Identification and destruction of abnormal or infected cells by cytotoxic T cells

Class II HLA-D

B lymphocytes, monocytes, macrophages, dendritic cells, activated T cells, activated endothelial cells, skin (Langerhans cells)

Identification of foreign antigen by helper T cells

Class III Complement C2, C4, B Plasma proteins Defense against extracellular pathogens

Cytokine genes TNFa, TNFb Plasma proteins Cell growth and

differentiation

Class I MHC Genes found in regions A, B and C in humans (K and D in mice)

Class II MHC Genes found in regions DR, DP and DQ (IA and IE In mice)

Class I and Class II MHC share structural featuresBoth involved in APC

Class III MHC have no structural similarity to Class I and IIEx. TNF, heat shock proteins, complement

components

MHC Genes Are PolymorphicMHC products are highly polymorphic

Vary considerably from person to person

However, crossover rate is low0.5% crossover rateInherited as 2 sets (one from father, one from

mother)Haplotype refers to set from mother or father

MHC alleles are co-dominantly expressedBoth mother and father alleles are expressed

Inheritance Of HLA Haplotypes

Class I MHC MoleculeComprised of 2 molecules

chain (45 kDa), transmembrane2-microglobulin (12 kDa) Non-covalently associated with each oth

Association of chain and 2 is required for surface expression

chain made up of 3 domains (1, 2 and 3) 2-microglobulin similar to 3 1 and 2 form peptide binding cleft

Fits peptide of about 8-10 a/a long3 highly conserved among MHC I molecules

Interacts with CD8 (TC) molecule

Class II MHC MoleculeComprised of and chains

chain and chain associate non-covalently and chains made up of domains

1 and 2 ( chain) 1 and 2 ( chain)

1and 1 form antigen binding cleft and heterodimer has been shown to

dimerizeCD4 molecule binds 2/2 domains

Class I And II SpecificitySeveral hundred allelic variants have been

identifiedHowever, up to 6 MHC I and 12 MHC II

Molecules are expressed in an individualEnormous number of peptides needs to be

presented using these MHC moleculesTo achieve this task MHC molecules are not

very specific for peptides (unlike TCR and BCR)Promiscuous binding occurs

A peptide can bind a number of MHCAn MHC molecule can bind numerous peptides

Class I And II Diversity And PolymorphismMHC is one of the most polymorphic

complexes knownAlleles can differ up to 20 a/aClass I Alleles: 240 A, 470 B, 110 CClass II Alleles: HLA-DR 350 , 2 !HLA-DR

genes vary from 2-9 in different individuals!!!,

1 gene ( can combine with all products increasing number of APC molecules)

DP (2 , 2 ) and DQ (2 , 3 )

Class I MHC PeptidesPeptides presented thru MHC I are

endogenous proteinsAs few as 100 Peptide/MHC complexes can

activate TC

Peptide Featuressize 8-10 a/a, preferably 9

Peptides bind MHC due to presence of specific a/a found at the ends of peptide. Ex. Glycine @ position 2

Class II MHC PeptidesPeptides presented thru MHC II are exogenous

Processed thru endocytic pathwayPeptides are presented to TH

Peptides are 13-18 a/a longBinding is due to central 13 a/a Longer peptides can still bind MHC II

Like a long hot dogMHC I peptides fit exactly, not the case with

MHC II peptides

MHC ExpressionExpression is regulated by many cytokines

IFN, IFN, IFN and TNF increase MHC expression

Transcription factors that increase MHC gene expressionCIITA (transactivator), RFX (transactivator)

Some viruses decrease MHC expressionCMV, HBV, Ad12

Reduction of MHC may allow for immune system evasion

Human Leukocyte Antigens (HLA)Human leukocyte antigens, the MHC gene

products, are membrane proteins that are responsible for rejection of transplanted organs and tissues.

2 microglobulin

1 1

2 2

2 1

3

HLA-D

Cell membrane

chain chain chain

Human Leukocyte Antigens (HLA)

HLA-gene sequences differ from one individual to another.

Also written as:

Each sequence is a different allele.

CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA

CGG GCC GCC GTG GAC ACC TAT TGC AGA CAC AAC TAC GGG GCT GTG GAG AGC TTC ACA

CGG GCC GCC GTG GAC ACC TAT TGC AGA CAC AAC TAC GGG GCT GTG GNN NNN NNN NNN

CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA

--- --- --- --- --- --- --T --- --- --- --- --- --- -C - -TG --- --- --- ---

--- --- --C --- --- --- --T --- --- --- --- --- --- -C- -TG -** *** *** ***

a.

b.

HLA Allele Nomenclature

A standard nomenclature has been established by the World Health Organization (WHO) Nomenclature Committee.

A small “w” is included in HLA-C, HLAB-4, and HLAB-6 allele nomenclature: HLA-Cw, HLABw-4, HLABw-6.

HLA-DRB1Gene region

Gene locus

Subregion

- or -chain polypeptide

A24

Cw1B14

DR14

A30

Cw3B7

DR15

A1

Cw1B12

DR5

A6

Cw7B44

DR14

A24

Cw1B14

DR14

A30

Cw3B7

DR15

A1

Cw1B12

DR5

A6

Cw7B44

DR14

A24

Cw1B14

DR14

A30

Cw3B7

DR15

A1

Cw1B12

DR5

A6

Cw7B44

DR14

haplotype

alleles

X

HLA TypingEvery person (except identical twins) has different sets of HLA alleles.

Transplanted organs are allografts, in which the donor organ and the recipient are genetically different.

Compatibility (matching) of the HLA of the donor and the recipient increases the chance for a successful engraftment.

Matching is determined by comparing alleles.

Resolution is the level of detail with which an allele is determined.

Serological TypingRecipient antihuman antibodies are assessed by crossmatching to donor lymphocytes.

Recipient serum

Lymphocytes from organ donor or lymphocytes of known HLA types

Positive reaction to antibodykills cells: dead cells pick up dye.

Negative reaction to antibody:cells survive and exclude dye.

Serological Typing Using Bead ArraysRecipient antihuman antibodies are assessed by

crossmatching to known lymphocyte antigens conjugated to microparticles. Results are assessed by flow cytometry.

(Wash)

Negative for antibody

Serum antibodies

Fluorescent reporter antibodies

Beads conjugated to different lymphocyte antigens

Positive for antibody

Other Serological Typing MethodsCytotoxic and noncytotoxic methods with

flow cytometry detection.Enzyme-linked immunosorbent assay

(ELISA) with solubilized HLA antigens.Mixed lymphocyte culture measuring growth

of lymphocytes activated by cross-reactivity.Measure of HLA-protein mobility differences

in one-dimensional gel isoelectric focusing or two-dimensional gel electrophoresis.

ELISAClass I and class II are solid phase enzyme

linked immuno sorbent assays (ELISA). Microtitre plates are coated with different highly purified human HLA class I and II glycoproteins. If the sample being tested contains specific antibodies against HLA class I or class II, they will bind to the antigens in the wells of the microtitre plate.

ELISAThe resulting antibody-antigen complex is

detected using a specific enzyme-labelled (alkaline phosphatase) antibody which is directed against human IgG (conjugate). The presence of bound antibodies is demonstrated by adding a chromogenic substrate (PNPP) which results in a coloured product. The reaction is interpreted by a photometric reader.

MLCMeasure of histocompatibility at the hl-a

locus. Peripheral blood lymphocytes from two individuals are mixed together in tissue culture for several days. Lymphocytes from incompatible individuals will stimulate each other to proliferate significantly (measured by tritiated thymidine uptake) whereas those from compatible individuals will not.

MLCIn the one-way MLC test, the lymphocytes

from one of the individuals are inactivated (usually by treatment with mitomycin c or radiation) thereby allowing only the untreated remaining population of cells to proliferate in response to foreign histocompatibility antigens.

DNA-Based Typing MethodsDNA typing focuses on the most polymorphic

loci in the MHC, HLA-B, and HLA-DRB. Whole-blood patient specimens collected in

anticoagulant are used for DNA typing.Cell lines of known HLA type are used for

reference samples.

DNA-Based Typing Methods: SSOPSequence-specific oligonucleotide probe

hybridization (SSOP, SSOPH)

TAG CGATATC GCTA

TAG AGATATC TCTA

Specimen 1 (Type A*0203) Specimen 2 (Type A*0501)

Amplify, denature, and spot onto membranes

Specimen 1 Specimen 2

Probe with allele-specific probes...TAGCGAT..(A*02) ...TAGAGAT…(A*05)

Specimen 1 Specimen 2 Specimen 1 Specimen 2

PCR-SSOReverse SSO hybrodization is used to determine HLA-A, -B,

-C, -DR, -DQ and -DP locus types at an intermediate level of resolution, somewhat higher than serological testing. Tests of this type are used when low or intermediate resolution typing is required or as a screening test to identify potential donors or individuals who may later require higher resolution testing.

This technology is used for high volume testing and allows for relatively low-cost typing for bone marrow donor drives or other applications involving large sample numbers. The laboratory can process as many as 25,000 samples per drive. Special volume pricing and terms may apply.

DNA-Based Typing Methods: SSP-PCR

Sequence-specific PCR is performed with allele-specific primers.

product

Amplificationcontrols

Allele-specificSSP= Sequence-specific primer

Noamplification

SSP

SSP

Amplification

SSP matches allele

SSP does not match allele

PCR-SSPPCR-SSP is also used to determine HLA-A, -B,

-C, -DR and DQ locus types at a resolution similar to serological testing. PCR-SSP is a very rapid test that can be performed in 3-4 hours from the time a sample is received. PCR-SSP is used for typing deceased organ donors when speed is an important consideration. PCR-SSP can also be used to provide higher resolution testing and may be employed to resolve alleles.

DNA-Based Typing Methods: SSP-PCR

Primers recognizing different alleles are supplied in a 96-well plate format.

Amplification control

Allele-specific product

Agarose gel

Reagent blank

DNA-Based Typing Methods: Sequence-Based TypingSequence-based typing (SBT) is high

resolution.Polymorphic regions are amplified by PCR

and then sequenced.

Exon 2 Exon 3

HLA-B

Forward PCR primer

Sequencing primers

Reverse PCR primer

SBTSBT provides the highest resolution HLA

typing for HLA-A, -B, -C, -DR, -DQ and -DP locus alleles. SBT is used when the highest resolution typing is important as in donors and recipients of stem cell transplants or in examining disease associations

Isolate DNA

PCR

clean amplicons

sequenceamplicon

Sequences are compared to reference sequences for previously assigned alleles.

Typing DiscrepanciesDNA sequence changes do not always affect

epitopes.Serology does not recognize every allele detectable

by DNA.New antigens recognized by serology may be

assigned to a previously identified parent allele by SBT.

Serology antibodies may be cross-reactive for multiple alleles.

Due to new allele discovery, retyping results may differ from typing performed before the new allele was known.

Resolution Levels of HLA Typing Methods

Low-Resolution Methods

Intermediate-Resolution Methods

High-Resolution Methods

CDC (serology) PCR-SSP PCR-SSP

PCR-SSP PCR-SSOP PCR-SSOP

PCR-SSOP PCR-RFLP SSP-PCR + PCR-RFLP

SSOP-PCR + SSP-PCR

SBT

Combining Typing ResultsSSP-PCR followed by PCR RFLPSSOP followed by SSP-PCRSBT results clarified by serology