Chapter 15
Major Histocompatibility Complex
 Major Histocompatibility Complex
 Cluster of genes found in all mammals
 Its products play role in discriminating self/non-self
 Participant in both humoral and cell-mediated
immunity
 MHC Act As Antigen Presenting Structures
 In humans MHC is found on chromosome 6
 Referred to as HLA complex
 In 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.
1 Mb
2 Mb
baba ba bbbba
HLA-
DP
3 Mb
4 Mb
a b
DQ DR
Class II
TNF
B C
Class III
A
Class I
 The MHC contains the human leukocyte antigen
(HLA) and other genes.
Classes of MHC Genes
 Class 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 Locus
MHC region
Gene products
Tissue location
Function
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
 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 features
 Both involved in APC
 Class III MHC have no structural similarity to Class I
and II
 Ex. TNF, heat shock proteins, complement components
MHC Genes Are Polymorphic
 MHC products are highly polymorphic
 Vary considerably from person to person
 However, crossover rate is low
 0.5% crossover rate
 Inherited as 2 sets (one from father, one from mother)
 Haplotype refers to set from mother or father
 MHC alleles are co-dominantly expressed
 Both mother and father alleles are expressed
Inheritance Of HLA Haplotypes
Class I MHC Molecule
 Comprised of 2 molecules
 a chain (45 kDa), transmembrane
 b2-microglobulin (12 kDa)
 Non-covalently associated with each oth
Association of a chain and b2 is required for surface expression
a chain made up of 3 domains (a1, a2 and a3)
b2-microglobulin similar to a3
a1 and a2 form peptide binding cleft
 Fits peptide of about 8-10 a/a long
 a3 highly conserved among MHC I molecules
 Interacts with CD8 (TC) molecule




Class II MHC Molecule
 Comprised of a and b chains
 a chain and b chain associate non-covalently
 a and b chains made up of domains
 a1 and a2 (a chain)
 b1 and b2 (b chain)
 a1and b1 form antigen binding cleft
 a and b heterodimer has been shown to dimerize
 CD4 molecule binds a2/b2 domains
Class I And II Specificity
 Several hundred allelic variants have been
identified
 However, up to 6 MHC I and 12 MHC II Molecules
are expressed in an individual
 Enormous number of peptides needs to be
presented using these MHC molecules
 To 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 MHC
 An MHC molecule can bind numerous peptides
Class I And II Diversity And
Polymorphism
 MHC is one of the most polymorphic complexes




known
Alleles can differ up to 20 a/a
Class I Alleles: 240 A, 470 B, 110 C
Class II Alleles: HLA-DR 350 b, 2 a!
HLA-DR
 b genes vary from 2-9 in different individuals!!!,
 1 a gene (a can combine with all b products increasing
number of APC molecules)
 DP (2 a, 2 b) and DQ (2 a, 3 b)
Class I MHC Peptides
 Peptides presented thru MHC I are endogenous
proteins
 As few as 100 Peptide/MHC complexes can activate TC
 Peptide Features
 size 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 Peptides
 Peptides presented thru MHC II are exogenous
 Processed thru endocytic pathway
 Peptides are presented to TH
 Peptides are 13-18 a/a long
 Binding is due to central 13 a/a
 Longer peptides can still bind MHC II
 Like a long hot dog
 MHC I peptides fit exactly, not the case with MHC II
peptides
MHC Expression
 Expression is regulated by many cytokines
 IFNa, IFNb, IFN and TNF increase MHC expression
 Transcription factors that increase MHC gene expression
 CIITA (transactivator), RFX (transactivator)
 Some viruses decrease MHC expression
 CMV, 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.
a 1 b1
HLA-D
a 2 a1
a 2 b2
a3
Cell membrane
a chain
b chain
a chain
b 2 microglobulin
Human Leukocyte Antigens (HLA)
 HLA-gene sequences differ from one individual to
another.
a.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
b.Also written as:
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 -**
 Each sequence is a different allele.
***
*** ***
HLA Allele Nomenclature
 A standard nomenclature has been established by the
World Health Organization (WHO) Nomenclature
Committee.
Gene region
Subregion
HLA-DRB1
Gene locus
a- or b-chain polypeptide
 A small “w” is included in HLA-C, HLAB-4, and HLAB-
6 allele nomenclature: HLA-Cw, HLABw-4, HLABw-6.
HLA alleles are inherited in blocks as
haplotypes.
A24
A30
A1
A6
alleles
haplotype
Cw1
B14
Cw3
B7
DR14
DR15
X
Cw1
B12
Cw7
B44
DR5
DR14
A24
A6
A1
A30
A6
A30
A24
A1
Cw1
B14
Cw7
B44
Cw1
B12
Cw3
B7
Cw7
B44
Cw3
B7
Cw1
B14
Cw1
B12
DR14
DR14
DR5
DR15
DR14
DR15
DR14
DR5
HLA Typing
 Every 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
Typing
 Recipient antihuman antibodies are assessed by
crossmatching to donor lymphocytes.
Lymphocytes from organ
donor or lymphocytes of
known HLA types
Recipient serum
Positive reaction to antibody
kills cells: dead cells pick up dye.
Negative reaction to antibody:
cells survive and exclude dye.
Serological Typing Using Bead Arrays
 Recipient antihuman antibodies are assessed by
crossmatching to known lymphocyte antigens conjugated
to microparticles. Results are assessed by flow cytometry.
Beads
conjugated to
different
lymphocyte
antigens
Serum
antibodies
Positive for antibody
(Wash)
Fluorescent
reporter
antibodies
Negative for antibody
Other Serological Typing Methods
 Cytotoxic 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 twodimensional gel electrophoresis.
ELISA
 Class 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.
ELISA
 The 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.
MLC
 Measure 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.
MLC
 In 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 Methods
 DNA 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: SSOP
 Sequence-specific oligonucleotide probe
hybridization (SSOP, SSOPH)
Specimen 1 (Type A*0203)
Specimen 2 (Type A*0501)
TAG C GAT
ATC G CTA
TAG A GAT
ATC T CTA
Amplify, denature, and
spot onto membranes
Specimen 1
Specimen 2
Probe with allele-specific probes
...TAGCGAT..(A*02)
Specimen 1
Specimen 2
...TAGAGAT…(A*05)
Specimen 1
Specimen 2
PCR-SSO
 Reverse 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 allelespecific primers.
Amplification
controls
SSP= Sequence-specific primer
Allele-specific
product
SSP matches allele
Amplification
SSP
No
amplification
SSP
SSP does not match allele
PCR-SSP
 PCR-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.
Reagent blank
Amplification control
Allele-specific product
Agarose gel
DNA-Based Typing Methods: SequenceBased Typing
 Sequence-based typing (SBT) is high resolution.
 Polymorphic regions are amplified by PCR and
then sequenced.
Reverse PCR primer
Forward PCR primer
Exon 2
Exon 3
HLA-B
Sequencing primers
SBT
 SBT 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
Sequence-Based Typing
Isolate DNA
PCR
clean amplicons
sequence
amplicon
Sequences are
compared to reference
sequences for
previously assigned
alleles.
Typing Discrepancies
 DNA 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
IntermediateResolution 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 Results
 SSP-PCR followed by PCR RFLP
 SSOP followed by SSP-PCR
 SBT results clarified by serology