Download - DNA-Based Tissue Typing
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Chapter 15
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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
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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
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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
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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
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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
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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
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Inheritance Of HLA Haplotypes
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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
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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
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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
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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 )
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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
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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
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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
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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
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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.
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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
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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
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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
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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.
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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
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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.
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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
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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
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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
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Isolate DNA
PCR
clean amplicons
sequenceamplicon
Sequences are compared to reference sequences for previously assigned alleles.
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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.
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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
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Combining Typing ResultsSSP-PCR followed by PCR RFLPSSOP followed by SSP-PCRSBT results clarified by serology