MAJOR HISTOCOMPATIBILITY COMPLEX (Aka Human

Leukocyte antigens (HLA)/ MHC molecules)

o Dausset
o First defined by discovering an antibody response to
circulating WBCs
o Determine whether transplanted tissue is histocompatible
and thus accepted or recognized as foreign and rejected
o Found on all nucleated cells in the body (Class I),
professional APC’s (dendritic cells, macrophages, B cells
Class II)
o Play a pivotal role in the development of both humoral and
cellular immunity
o MAIN FUNCTION:
 Bring antigen to the cell surface for recognition
by T cells, because T cell activation will occur
only when antigen is combined with only MHC
molecules
o Clinically, they are relevant because they may be involved
in transfusion reactions, graft rejection, and autoimmune
diseases
o Genes controlling expression of these molecules are
actually a system of genes known as the MAJOR
HISTOCOMPATIBILITY COMPLEX (MHC) CLASS I MHC MOLECULES
STRUCTURE:
o Polymorphic
o Consists of a structurally distinct α chain associated with a
o There are so many possible alleles at each location
second, shorter polypeptide called β2-microglobulin
 E.g., at least 580 alleles of HLA-A
o Class I α chain is organized in three folded domains (α1,
 921 alleles of HLA-B
α2, and α3) has a carboxy-terminal membrane anchor
 312 alleles of HLA-C
o Probability that any two individuals will express the same o The smaller β2-microglobulin, with one folded domain, is
MHC molecules is very low linked to the membrane only indirectly through its
association with the αchain
o GENES ARE CODOMINANT
o Critical for stabilizing the class I molecule and for
o E.g., all six (6) class I alleles are expressed together on the
facilitating its transport to the cell surface
surface of every nucleated cell
o The peptide-binding site in a class I protein is formed by
the α1 and α2 domains
GENES CODING FOR MHC MOLECULES o Can only accommodate peptides that are 8 – 10 amino
o Most polymorphic system found in humans acids long
o Found on the short arm of chromosome 6 (6p) o The α3 and β2 regions are similar to the constant regions
o Divided into three categories/ classes found in immunoglobulin molecules
1. Class I molecules o The α3 region reacts with CD8 on cytotoxic T cells
• HLA-A, HLA-B, HLA-C
2. Class II molecules
• D region → HLA-DR, HLA-DQ, HLA-DP
3. Class III molecules
• Code for complement proteins and cytokines
such as tumor necrosis factor
• Proteins are secreted proteins that have an
immune function but not expressed on the cell
surfaces

CLASS I MHC MOLECULES

o Expressed on all nucleated cells
o But differ in the level of expression
o Highest on lymphocytes and lowest on liver hepatocytes,
neural cells and muscle cells
 o HLA-C antigens are expressed at a lower level than HLA-A
and HLA-B antigens so the latter two are the most
important to match for transplantation
o HLA-E,HLA-F, HLA-G
 Another group of molecules called the
nonclassical class I antigens
 Except for HLA-G, are not expressed on cell
surfaces and do not function in antigen
recognition but may play other roles in the
immune response
o HLA-G
 Expressed on trophoblast cells during the first
trimester of pregnancy
 Help ensure tolerance for the fetus by protecting
placental tissue from the action of NK cells

CLASS II MHC MOLECULES:

o Found primarily on antigen-presenting cells (B cells,
dendritic cells, monocytes, macrophages)
o Consist of two (2) noncovalently bound polypeptide chains
that are both encoded by genes in the MHC complex
o DR is expressed at the highest level
 Accounts for ½ of the all the class II molecules
on a particular cell
o Both the α chain (MW = 33 kD) and the  chain (MW = 27
kD) are anchored to the cell membrane
o Each has two domains
o Peptide binding site →formed by the α1 and β1 domains
o Both ends of the peptide-binding cleft are open
 Allow the capture of longer peptides ( 9 – 20
amino acids) than is the case for class I molecules
o CD4 contacts sequences in the 2 domain
o HLA-DM, HLA-DN, HLA-DO
 Nonclassical class II genes
 Products of these genes play a regulatory role in
antigen processing

ENDOCYTIC CYTOSOLIC
PATHWAY PATHWAY
Major antigen Endocytosed Cytosolic proteins
sources extracellular proteins of host or
(host & foreign) intracellular
Membrane proteins pathogens (viral,
(host & bacterial, parasitic)
foreign) Signal peptides (host &
foreign)
Processing Lysosomal enzymes Proteasomes
machinery (including low-
molecular-weight
protein (LMPs) CLINICAL SIGNIFICANCE OF MHC
Cell types Professional APCs All nucleated cells o MHC molecules can induce a response that leads to graft
where active rejection
Site of antigen – Endocytic Rough endoplasmic o Play a role in development of autoimmune diseases
MHC binding vesicles, reticulum
o Determine the type of peptides to which an individual can
prelysosomes
MHC utilized Class II Class I mount an immune response
o Presence of a particular MHC protein may confer
Presents to CD4 (helper) T cells CD8 (cytotoxic) T cells
additional protection (e.g., HLA B8 and increased
resistance to HIV)
o Future developments to tailor vaccines to certain groups of
molecules
Disease Examples of Associated HLA alleles o Purified Lymphocyte suspension for Antigen detection
Ankylosing spondylitis HLA-B27 o Anticoagulated whole blood is overlaid into:
Birdshot retinopathy HLA-A29 o Ficoll-Hypaque reagent
Celiac disease HLA-DR3, - DR5, - DR7 o Then centrifuge
Graves’ disease HLA-DR3
Narcolepsy HLA-DR2
Multiple sclerosis HLA-DR2
Rheumatoid arthritis HLA-DR4
Type 1 diabetes mellitus HLA-DQ8, - DQ2, - DR3, - DR4

HISTOCOMPATIBILITY TESTING: APPLICATIONS

1. Prevention of graft rejection / graft vs. host reaction
2. Paternity exclusion
3. Disease associations
4. Prevent platelet refractoriness in Platelet transfusions o Purified Lymphocyte suspension for Antigen detection
5. Prevent Transfusion-related acute lung injury (TRALI)  Class I Antigens (HLA-A, HLA-B, HLA-C)
6. Hematopoietic stem cell transplantation • Use T lymphocytes
 Class II Antigens (HLA-DR, HLA-DP, HLA-
HISTOCOMPATIBILITY TESTING DQ)
1. Tissue typing • Use B lymphocytes
• HLA Phenotyping: Serologic techniques o B lymphocyte separation:
• HLA Genotyping: Molecular methods 1. Nylon wool separation
2. Antibody screening 2. Use immunomagnetic beads
3. Tissue matching/ Crossmatching 3. Fluorescent labeling (use FITC)

Sources of Antibodies
1. Multiparous women
2. Patients who received multiple transfusions (WBC and
platelets)
3. Volunteers who were sensitized by blood transfusion or
tissue grafts
4. Patients who have rejected a transplanted kidney

SEROLOGIC METHODS: TISSUE TYPING:

Lymphocytotoxicity Test (Complement- dependent
microlymphocytotoxicity)
o Expose unknown cell to a battery of antisera of known
HLA specificity
o Incubate at room temperature for 30 minutes
o Complement is added (rabbit serum)
o Incubated at room temperature for 60 minutes

Complement-Dependent Lymphocytotoxicity
o Then add trypan blue dye (eosin Y)
o Take an aliquot from well and examine under light
microscope using hemocytometer
o Dead cells take up the dye
o Flattened, appear large, dark and nonrefractile
o Unaffected cells appear small, bright and refractile

% Dead SCORE INTERPRETATI

Lymphocytes ON
0 – 10 1 Negative
11 – 20 2 Doubtful positive
21 – 50 4 Weak positive
51 – 80 6 Positive
81 – 100 8 Strong positive

Preparation of Samples
TISSUE TYPING: Complement-Dependent Lymphocytotoxicity
 HLA GENOTYPING: MOLECULAR METHODS
A. Restriction Fragment Length Polymorphism (RFLP)
B. PCR- based
1. Sequence-Specific oligonucleotides (SSO)
2. Sequence-specific primers (SSP)
3. Sequence-based typing (SBT)

Molecular Techniques: RFLP

To increase sensitivity:
1. Extended incubation
2. The Amos wash step
3. Antihuman globulin
Lymphocytotoxicity Disadvantages:
1. It requires having cells available for testing.
2. It is necessary to collect leukocytes and perform cellular
testing in a timely manner to enable transplantation
3. it is necessary to maintain reliable and consistent antigen
panels that represent a broad range of HLA antigens
SEROLOGIC METHODS: Antibody Screening
o Detect antibodies in patients who are candidates for transplant
1. Complement-dependent lymphocytotoxicity/
Microlymphocytotoxicity
2. ELISA
3. Flow cytometry
o Restriction enzymes (restriction endonucleases) are used
o Cleaves genomic DNA
o Obtain a pattern of fragmentation
o Degree of disparity between donor and recipient can be
assessed by COMPARING patterns of fragmentation
Molecular Techniques: PCR
o Automated, rapid and in vitro technique
o Allows direct amplification of a particular DNA sequence
o Selected by the use of primers that border the genes of
interest
PCR-Based Techniques: Sequence- Specific Oligonucleotides (SSO)
o PCR-amplification of a chosen sequence using primers
flanking the sequence
o The amplified DNA is immobilized on a membrane
o Then hybridized with selected, labeled oligonucleotide
probes

SEROLOGIC METHODS: Antibody Screening

o MICROLYMPHOCYTOTOX ICITY
o PERCENT PANEL REACTIVE ANTIBODY (%PRA)
o The proportion of lymphocytes in the panel that are killed
by the patient’s serum

SEROLOGIC METHODS: Antibody Screening: ELISA

o utilize purified HLA antigens bound to the wells of
microtiter plates.
o Patient serum is added
o If HLA-specific antibody is present, it will bind
o Bound antibody is detected by addition of an enzyme-
labeled anti immunoglobulin reagent

SEROLOGIC METHODS: Antibody Screening: ELISA

o Serves as a qualitative screen for the presence of HLA
antibody in a serum
o Increased specificity
o Recognizes false-positive reactions
o Distinguishes Class I from Class II
o Differentiates IgM from IgG antibodies

SEROLOGIC METHODS: Antibody Screening: Flow Cytometry

o Detects antibody binding directly
o Can distinguish between IgM and IgG
o Uses T or B cells; or purified HLA antigens coated with
microparticles
o Bound antibody is detected by adding an FITC- labeled PCR-Based Techniques: Sequence- Specific Primers (SSP)
anti-IgG reagent o Oligonucleotide primers are designed to obtain
o Percent PRA is determined amplification of specific alleles or groups of alleles
o Most sensitive, most specific
 o Assignment of allele is based on the presence or absence of
amplified product
o Detected by agarose gel electrophoresis (AGE) and
transillumination
o Beta ray emissions are measured using a liquid scintillation
counter (counts per minute)
o CPM correlates with the amount of proliferation
o Depends on the degree of disparity between the recipient
cells and potential donor cells

Types of Grafts
1. Autograft
2. Syngraft
3. Allograft (Homograft)
4. Xenograft (Heterograft)

Most Common Tissues used for Transplantation

1. Kidney
2. Heart
3. Cornea
4. Lung
5. Skin
PCR-Based Techniques: Sequence Based Typing (SBT)
6. Bone marrow
o Two Methods:
 Sanger-based DNA sequencing
 Next-generation DNA sequencing (NGS) HOST RESPONSE TO TRANSPLANTATION
o Allows amplification of the most polymorphic regions of 1. Hyperacute Rejection
the HLA genes 2. Acute or Accelerated rejection
o Preferred method for hematopoietic stem cell 3. Chronic rejection
4. Graft-versus-Host Disease
transplantation
o Acute GVHD
o Performed by terminal-end incorporation of fluorescently
o Chronic GVHD
labeled nucleotides during PCR reactions
o Allows amplification of the most polymorphic regions of
the HLA genes Immunosuppressive Therapy
o Preferred method for hematopoietic stem cell o Induce intense immunosuppression in the initial days post
transplantation transplantation
o Maintenance of the graft
o Reversal of established rejection

Types of Immunosuppressive Therapy

1. Corticosteroids
2. Cyclosporine (Cyclosporin A)
3. Tacrolimus
4. Cytotoxic drugs
5. Antilymphocyte (Antithymocyte) globulin
6. Monoclonal antibodies

Complications of Transplantation
o Cancer
o Osteoporosis
o Diabetes
Cellular Methods: Mixed Lymphocyte Reaction o Hypertension
o Donor and recipient cells are cultured together for several o Hypercholesterolemia
days
o Allow CD4+ T cells to be activated and proliferate
o In response to disparate Class II antigens
o Amount of proliferation is measured and used to predict the
magnitude of rejection
o Can be done in a One-way MLR or a Two-wayMLR
o One-Way MLR → used to test for recipient’s response to
donor cells
o Donor cells are irradiated (using Cobalt or Cesium) or
treated with mitomycin C
o Most useful for bone marrow grafts and in cases of living
related donors
o Radioactive label is added on day 5.