The Major Histocompatibility Complex 3/28/2022
MAJOR HISTOCOMPATIBILITY
COMPLEX
MAJOR • Human Leukocyte antigens (HLA)
HISTOCOMPATIBILITY •
•
MHC molecules
Dausset
COMPLEX • First defined by discovering an antibody response to
circulating wbcs
• Determine whether transplanted tissue is
Maida Fatima E. Chan histocompatible and thus accepted or recognized as
foreign and rejected
1 2
MAJOR HISTOCOMPATIBILITY COMPLEX GENES CODING FOR MHC MOLECULES
• Found on all nucleated cells in the body (Class I), professional APC’s
• Most polymorphic system found in humans
(dendritic cells, macrophages, B cells →Class II)
• Found on the short arm of chromosome 6 (6p)
• Play a pivotal role in the development of both humoral and cellular
immunity • Divided into three categories/ classes
• MAIN FUNCTION: 1. Class I molecules
• Bring antigen to the cell surface for recognition by T cells, because T cell • HLA-A, HLA-B, HLA-C
activation will occur only when antigen is combined with only MHC 2. Class II molecules
molecules • D region → HLA-DR, HLA-DQ, HLA-DP
• Clinically, they are relevant because they may be involved in transfusion 3. Class III molecules
reactions, graft rejection, and autoimmune diseases • Code for complement proteins and cytokines such as tumor necrosis factor
• Genes controlling expression of these molecules are actually a system of • Proteins are secreted proteins that have an immune function but not
genes known as the MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) expressed on the cell surfaces
3 4
Maida Fatima E. Chan 1
The Major Histocompatibility Complex 3/28/2022
GENES CODING FOR MHC MOLECULES MAJOR HISTOCOMPATIBILITY COMPLEX
• Polymorphic
• There are so many possible alleles at each location
• E.g., at least 580 alleles of HLA-A
• 921 alleles of HLA-B
• 312 alleles of HLA-C
• Probability that any two individuals will express the same
MHC molecules is very low
• GENES ARE CODOMINANT
• E.g., all six (6) class I alleles are expressed together on the
surface of every nucleated cell
5 6
MAJOR HISTOCOMPATIBILITY COMPLEX CODOMINANT EXPRESSION OF MHC
7 8
Maida Fatima E. Chan 2
The Major Histocompatibility Complex 3/28/2022
Feature &
Significance of
CLASS I MHC MOLECULES
MHC STRUCTURE:
• Consists of a structurally distinct a chain associated with a
second, shorter polypeptide called b2-microglobulin
• Class I a chain is organized in three folded domains ( a1, a2,
and a3) has a carboxy-terminal membrane anchor
• The smaller b2-microglobulin, with one folded domain, is
linked to the membrane only indirectly through its
association with the a chain
• Critical for stabilizing the class I molecule and for
facilitating its transport to the cell surface
9 10
CLASS I MHC MOLECULES CLASS I MHC MOLECULES
STRUCTURE:
• The peptide-binding site in a class I protein is formed by the
a1 and a2 domains
• Can only accommodate peptides that are 8 – 10 amino
acids long
• The a3 and b2 regions are similar to the constant regions
found in immunoglobulin molecules
• The a3 region reacts with CD8 on cytotoxic T cells
11 12
Maida Fatima E. Chan 3
The Major Histocompatibility Complex 3/28/2022
CLASS I MHC MOLECULES CLASS I MHC MOLECULES
• Expressed on all nucleated cells • HLA-E,HLA-F, HLA-G
• But differ in the level of expression • Another group of molecules called the nonclassical class I
antigens
• Highest on lymphocytes and lowest on liver • Except for HLA-G, are not expressed on cell surfaces and do not
hepatocytes, neural cells and muscle cells function in antigen recognition but may play other roles in the
immune response
• HLA-C antigens are expressed at a lower level than • HLA-G
HLA-A and HLA-B antigens so the latter two are the • Expressed on trophoblast cells during the first trimester of
most important to match for transplantation pregnancy
• Help ensure tolerance for the fetus by protecting placental tissue
from the action of NK cells
13 14
CLASS II MHC MOLECULES CLASS II MHC MOLECULES
CHARACTERISTICS: STRUCTURE:
• Found primarily on antigen-presenting cells (B cells, dendritic • Both the a chain (MW = 33 kD) and the b chain (MW = 27 kD)
cells, monocytes, macrophages) are anchored to the cell membrane
• Consist of two (2) noncovalently bound polypeptide chains • Each has two domains
that are both encoded by genes in the MHC complex • Peptide binding site →formed by the a1 and b1 domains
• DR is expressed at the highest level • Both ends of the peptide-binding cleft are open
• Accounts for ½ of the all the class II molecules on a • Allow the capture of longer peptides ( 9 – 20 amino acids)
particular cell than is the case for class I molecules
• CD4 contacts sequences in the b2 domain
15 16
Maida Fatima E. Chan 4
The Major Histocompatibility Complex 3/28/2022
CLASS II MHC MOLECULES CLASS I & II MHC MOLECULES
17 18
CLASS II MHC MOLECULES THE ANTIGEN PROCESSING PATHWAYS
ENDOCYTIC PATHWAY CYTOSOLIC PATHWAY
• HLA-DM, HLA-DN, HLA-DO Major antigen Endocytosed extracellular Cytosolic proteins of host or
• Nonclassical class II genes sources proteins (host & foreign) intracellular pathogens (viral,
Membrane proteins (host & bacterial, parasitic)
• Products of these genes play a regulatory role in antigen foreign) Signal peptides (host & foreign)
processing
Processing Lysosomal enzymes Proteasomes (including low-
machinery molecular-weight protein (LMPs)
• **** READ MORE ABOUT CLASS I AND CLASS II MOLECULES, how Cell types where Professional APCs All nucleated cells
are they formed, mechanisms of antigen processing by each etc. active
(Books by Stanley, Stevens, Stites, Roitt etc. ) Site of antigen – Endocytic vesicles, Rough endoplasmic reticulum
• Know what are: TAPs (TAP1, TAP2), CLIP, Invariant chain, calnexin, MHC binding prelysosomes
calreticulin, tapasin, ERp57, DRiPs, ubiquitin, proteasomes ( and MHC utilized Class II Class I
their roles) Presents to CD4 (helper) T cells CD8 (cytotoxic) T cells
19 20
Maida Fatima E. Chan 5
The Major Histocompatibility Complex 3/28/2022
CYTOSOLIC PATHWAY CYTOSOLIC PATHWAY
21 22
ENDOCYTIC PATHWAY
CONCEPT MAP
23 24
Maida Fatima E. Chan 6
The Major Histocompatibility Complex 3/28/2022
CLINICAL SIGNIFICANCE OF MHC
Major • MHC molecules can induce a response that leads to graft
Histocompatibility rejection
• Play a role in development of autoimmune diseases
• Determine the type of peptides to which an individual can
Complex Part 2 mount an immune response
• Presence of a particular MHC protein may confer additional
protection (e.g., HLA B8 and increased resistance to HIV)
• Future developments to tailor vaccines to certain groups of
molecules
25 26
DISEASE ASSOCIATION WITH CLASS I
and CLASS II MHC EXPRESSION
Disease Examples of Associated HLA alleles
Ankylosing spondylitis HLA-B27
Birdshot retinopathy HLA-A29
Celiac disease HLA-DR3, - DR5, - DR7
Graves’ disease HLA-DR3
Narcolepsy HLA-DR2
Multiple sclerosis HLA-DR2
Rheumatoid arthritis HLA-DR4
Type 1 diabetes mellitus HLA-DQ8, - DQ2, - DR3, - DR4
27 28
Maida Fatima E. Chan 7
The Major Histocompatibility Complex 3/28/2022
HISTOCOMPATIBILITY TESTING:
HISTOCOMPATIBILITY TESTING
APPLICATIONS
1. Prevention of graft rejection / graft vs. host 1. Tissue typing
reaction • HLA Phenotyping: Serologic techniques
2. Paternity exclusion • HLA Genotyping: Molecular methods
3. Disease associations 2. Antibody screening
4. Prevent platelet refractoriness in Platelet transfusions
3. Tissue matching/ Crossmatching
5. Prevent Transfusion-related acute lung injury (TRALI)
6. Hematopoietic stem cell transplantation
29 30
Preparation of Samples
Histocompatibility
Testing Methods
• Purified Lymphocyte suspension for Antigen
detection
Serological
Cellular Molecular • Anticoagulated whole blood is overlaid into:
approaches
approaches approaches • Ficoll-Hypaque reagent
Antibody
Tissue
screening Tissue • Then centrifuge
typing Mixed Genotyping:
matching Lymphocyte platelets
Lymphocytotoxity PCR
Phenotyping: reaction
Flow Cytometry RFLP
Agglutination Lymphocytotoxicity
ELISA
Lymphocytotoxicity Flow cytometry
ELISA
31 32
Maida Fatima E. Chan 8
The Major Histocompatibility Complex 3/28/2022
Preparation of Samples Sources of Antibodies
• Purified Lymphocyte suspension for Antigen
detection 1. Multiparous women
• Class I Antigens (HLA-A, HLA-B, HLA-C) 2. Patients who received multiple transfusions
• Use T lymphocytes (WBC and platelets)
• Class II Antigens (HLA-DR, HLA-DP, HLA-DQ) 3. Volunteers who were sensitized by blood
• Use B lymphocytes transfusion or tissue grafts
• B lymphocyte separation:
1. Nylon wool separation 4. Patients who have rejected a transplanted kidney
2. Use immunomagnetic beads
3. Fluorescent labeling (use FITC)
33 34
SEROLOGIC METHODS: TISSUE SEROLOGIC METHODS: TISSUE TYPING:
TYPING Complement-Dependent Lymphocytotoxicity
• Lymphocytotoxicity Test (Complement- • Then add trypan blue dye (eosin Y)
dependent microlymphocytotoxicity) • Take an aliquot from well and examine under
• Expose unknown cell to a battery of antisera of light microscope using hemocytometer
known HLA specificity
• Dead cells take up the dye
• Incubate at room temperature for 30 minutes
• Flattened, appear large, dark and nonrefractile
• Complement is added (rabbit serum)
• Incubated at room temperature for 60 minutes
• Unaffected cells appear small, bright and
refractile
35 36
Maida Fatima E. Chan 9
The Major Histocompatibility Complex 3/28/2022
SEROLOGIC METHODS: TISSUE TYPING: SEROLOGIC METHODS: TISSUE TYPING:
Complement-Dependent Lymphocytotoxicity Complement-Dependent Lymphocytotoxicity
• Reading of Results:
% Dead SCORE INTERPRETATION
Lymphocytes
0 – 10 1 Negative
11 – 20 2 Doubtful positive
21 – 50 4 Weak positive
51 – 80 6 Positive
81 – 100 8 Strong positive
37 38
TISSUE TYPING: Complement-Dependent Lymphocytotoxicity Disadvantages:
Lymphocytotoxicity 1. It requires having cells available for
testing.
• To increase sensitivity: 2. It is necessary to collect leukocytes and
1. Extended incubation perform cellular testing in a timely
2. The Amos wash step manner to enable transplantation
3. Antihuman globulin 3. it is necessary to maintain reliable and
consistent antigen panels that represent a
broad range of HLA antigens
39 40
Maida Fatima E. Chan 10
The Major Histocompatibility Complex 3/28/2022
SEROLOGIC METHODS: Antibody SEROLOGIC METHODS: Antibody Screening
Screening
• MICROLYMPHOCYTOTOX
• Detect antibodies in patients who are ICITY
candidates for transplant • PERCENT PANEL
REACTIVE ANTIBODY
1. Complement-dependent (%PRA)
lymphocytotoxicity/ • The proportion of
Microlymphocytotoxicity lymphocytes in the
panel that are killed by
2. ELISA the patient’s serum
3. Flow cytometry
41 42
SEROLOGIC METHODS: Antibody SEROLOGIC METHODS: Antibody
Screening: ELISA Screening: ELISA
• utilize purified HLA antigens bound to the wells • Serves as a qualitative screen for the
of microtiter plates. presence of HLA antibody in a serum
• Patient serum is added
• Increased specificity
• If HLA-specific antibody is present, it will bind
• Recognizes false-positive reactions
• Bound antibody is detected by addition of an
enzyme-labeled anti immunoglobulin reagent • Distinguishes Class I from Class II
• Differentiates IgM from IgG antibodies
43 44
Maida Fatima E. Chan 11
The Major Histocompatibility Complex 3/28/2022
SEROLOGIC METHODS: Antibody Screening: SEROLOGIC METHODS:
Flow Cytometry CROSSMATCHING
• Detects antibody binding directly • Lymphocytotoxicity
• Can distinguish between IgM and IgG • Flow cytometry
• Uses T or B cells; or purified HLA antigens coated
• ELISA being developed
with microparticles
• Bound antibody is detected by adding an FITC-
labeled anti-IgG reagent
• Percent PRA is determined
• Most sensitive, most specific
45 46
HLA GENOTYPING: MOLECULAR
METHODS
Molecular Techniques: RFLP
A. Restriction Fragment Length • Restriction enzymes (restriction endonucleases)
Polymorphism (RFLP) are used
• Cleaves genomic DNA
B. PCR- based
• Obtain a pattern of fragmentation
1. Sequence-Specific oligonucleotides (SSO)
• Degree of disparity between donor and recipient
2. Sequence-specific primers (SSP) can be assessed by COMPARING patterns of
3. Sequence-based typing (SBT) fragmentation
47 48
Maida Fatima E. Chan 12
The Major Histocompatibility Complex 3/28/2022
PCR-Based Techniques: Sequence-
Molecular Techniques: PCR Specific Oligonucleotides (SSO)
• Automated, rapid and in vitro technique • PCR-amplification of a chosen sequence using
• Allows direct amplification of a particular DNA primers flanking the sequence
sequence • The amplified DNA is immobilized on a
• Selected by the use of primers that border the membrane
genes of interest • Then hybridized with selected, labeled
oligonucleotide probes
49 50
Sequence-Specific Sequence-Specific Oligonucleotide Typing
Oligonucleotide Typing
51 52
Maida Fatima E. Chan 13
The Major Histocompatibility Complex 3/28/2022
PCR-Based Techniques: Sequence-
Specific Primers (SSP)
• Oligonucleotide primers are designed to obtain
amplification of specific alleles or groups of
alleles
• Assignment of allele is based on the presence or
absence of amplified product
• Detected by agarose gel electrophoresis (AGE)
and transillumination
53 54
PCR-Based Techniques: Sequence PCR-Based Techniques: Sequence
Based Typing (SBT) Based Typing (SBT)
• Two Methods:
• Performed by terminal-end incorporation of
• Sanger-based DNA sequencing
fluorescently labeled nucleotides during PCR
• Next-generation DNA sequencing (NGS) reactions
• Allows amplification of the most polymorphic • Allows amplification of the most polymorphic
regions of the HLA genes regions of the HLA genes
• Preferred method for hematopoietic stem cell • Preferred method for hematopoietic stem cell
transplantation transplantation
55 56
Maida Fatima E. Chan 14
The Major Histocompatibility Complex 3/28/2022
Cellular Methods: Mixed
Lymphocyte Reaction
• Donor and recipient cells are cultured together
for several days
• Allow CD4+ T cells to be activated and proliferate
• In response to disparate Class II antigens
• Amount of proliferation is measured and used to
predict the magnitude of rejection
• Can be done in a One-way MLR or a Two-way
MLR
57 58
Cellular Methods: Mixed Cellular Methods: Mixed
Lymphocyte Reaction Lymphocyte Reaction
• One-Way MLR → used to test for recipient’s • Radioactive label is added on day 5.
response to donor cells • Beta ray emissions are measured using a liquid
• Donor cells are irradiated (using Cobalt or scintillation counter (counts per minute)
Cesium) or treated with mitomycin C • CPM correlates with the amount of proliferation
• Most useful for bone marrow grafts and in cases • Depends on the degree of disparity between the
of living related donors recipient cells and potential donor cells
59 60
Maida Fatima E. Chan 15
The Major Histocompatibility Complex 3/28/2022
Most Common Tissues used
Types of Grafts
for Transplantation
1. Autograft 1. Kidney
2. Syngraft 2. Heart
3. Allograft (Homograft) 3. Cornea
4. Xenograft (Heterograft) 4. Lung
5. Skin
6. Bone marrow
61 62
HOST RESPONSE TO
Immunosuppressive Therapy
TRANSPLANTATION
1. Hyperacute Rejection • Induce intense immunosuppression in the initial
2. Acute or Accelerated rejection days post transplantation
3. Chronic rejection • Maintenance of the graft
4. Graft-versus-Host Disease • Reversal of established rejection
• Acute GVHD
• Chronic GVHD
63 64
Maida Fatima E. Chan 16
The Major Histocompatibility Complex 3/28/2022
Types of Immunosuppressive
Complications of Transplantation
Therapy
1. Corticosteroids • Cancer
2. Cyclosporine (Cyclosporin A) • Osteoporosis
3. Tacrolimus • Diabetes
4. Cytotoxic drugs • Hypertension
5. Antilymphocyte (Antithymocyte) globulin • Hypercholesterolemia
6. Monoclonal antibodies
65 66
References
• Abbas and Lichtman
• Elgert
• Harmening
• Mak, Tak
• McPherson & Pincus
• Roitt
• Stanley
• Stevens
• Stites
• Turgeon
67
Protect pdf from copying with [Link]
Maida Fatima E. Chan 17
