[DISCUSSION] MODULE 1 UNIT 5 Flashcards
1
Q
-MAJOR HISTOCOMPATIBILITY COMPLEX IS ALSO CALLED
A
Human Leukocyte antigens (HLA)
2
Q
-MAJOR HISTOCOMPATIBILITY COMPLEX PRPONENT
A
Dausset
3
Q
- First defined by discovering an antibody response to circulating wbcs
A
MAJOR HISTOCOMPATIBILITY COMPLEX
4
Q
– Determine whether transplanted tissue is histocompatible and thus accepted or recognized as foreign and rejected
A
MAJOR HISTOCOMPATIBILITY COMPLEX
5
Q
-all nucleated cells in the body
A
(Class I)
6
Q
-professional APC’s (dendritic cells, macrophages, B cells)
A
(Class II)
7
Q
-Play a pivotal role in the development of both humoral and cellular immunity
A
MAJOR HISTOCOMPATIBILITY COMPLEX
8
Q
MHC Main Function
A
- 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
9
Q
– Clinical relevance of MHC
A
transfusion reactions
graft rejection
autoimmune diseases
10
Q
– Genes controlling expression of these molecules are actually a system of genes known as the
A
MAJOR HISTOCOMPATIBILITY COMPLEX (MHC)
11
Q
-Most polymorphic system found in humans
A
Genes coding for MHC molecules
12
Q
– Found on the short arm of chromosome 6 (6p)
A
GENES CODING FOR MHC MOLECULES
13
Q
CATEGORIES OF GENES CODING FOR MHC MOLECULES
A
Class I molecules
Class II molecules
Class III molecules
14
Q
classical class I molecules
A
HLA-A, HLA-B and HLA-C antigens
15
Q
classical class II molecules
A
D region → HLA-DR, HLA-DQ, HLA-DP
16
Q
classical class Ill molecules
A
Code for complement proteins and cytokines such as tumor necrosis factor
17
Q
- Polymorphic
A
MAJOR HISTOCOMPATIBILITY COMPLEX
18
Q
- There are so many possible alleles at each location
- E.g., at least 580 alleles of HLA-A
A
- Polymorphic
19
Q
- alleles of HLA-B
A
921
20
Q
- alleles of HLA-C
A
312
21
Q
- Probability that any two individuals will express the
same MHC molecules is
A
very low
22
Q
GENES ARE
A
CODOMINANT
23
Q
Consists of a structurally distinct a chain associated with a second, shorter polypeptide called b2
-microglobulin
A
CLASS I MHC MOLECULES
24
Q
is organized in three folded domains ( a1
, a2
, and a3
) has a
carboxy-terminal membrane anchor
A
Class I a chain
25
(?), with one folded domain, is linked to the
membrane only indirectly through its association with the a chain
smaller b2
-microglobulin
26
Critical for stabilizing the class I molecule and for facilitating its transport to the cell surface
smaller b2
-microglobulin
27
The peptide-binding site in a class I protein is formed by the
a1 and a2 domains
28
Can only accommodate peptides that are (?) amino acids long
8 – 10
29
are similar to the constant regions found in immunoglobulin molecules
a3 and b2 regions
30
reacts with CD8 on cytotoxic T cells
a3 region
31
Expressed on all nucleated cells, but differ in the level of expression
CLASS I
32
Highest on lymphocytes and lowest on liver
hepatocytes, neural cells and muscle cells
CLASS I
33
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
HLA-C antigens
34
Another group of molecules called the nonclassical class I antigens
HLA-E, HLA-F, HLA-G
35
are not expressed on cell surfaces and do not
function in antigen recognition but may play other roles in the immune
response
HLA-E, HLA-F
36
Expressed on trophoblast cells during the first trimester of pregnancy
HLA-G
37
Found primarily on antigen-presenting cells (B cells, dendritic cells, monocytes, macrophages)
CLASS II
38
Consist of two (2) noncovalently bound polypeptide chains that are both encoded by genes in the MHC complex
CLASS II
39
DR is expressed at the highest level
CLASS II
40
Accounts for ½ of the all the class II molecules on a particular cell
DR
41
Both the a chain (MW = 33 kD) and the b chain (MW = 27 kD) are anchored to the cell membrane
CLASS II
42
Each has two domains
CLASS II
43
Peptide binding site →formed by the a1 and b1 domains
CLASS II
44
Both ends of the peptide-binding cleft are open
CLASS II
45
Allow the capture of longer peptides ( 9 – 20 amino acids) than is the case for class I molecules
CLASS II
46
CD4 contacts sequences in the b2 domain
CLASS II
47
Nonclassical class II genes
HLA-DM, HLA-DN, HLA-DO
48
Products of these genes play a regulatory role in antigen processing
HLA-DM, HLA-DN, HLA-DO
49
ENDOCYTIC PATHWAY Major antigen
sources
Endocytosed extracellular
proteins (host & foreign)
Membrane proteins (host &
foreign)
50
CYTOSOLIC PATHWAY Major antigen
sources
Cytosolic proteins of host or
intracellular pathogens (viral,
bacterial, parasitic)
Signal peptides (host & foreign)
51
ENDOCYTIC PATHWAY Processing machinery
Lysosomal enzymes
52
CYTOSOLIC PATHWAY Processing
machinery
Proteasomes (including low-
molecular-weight protein (LMPs)
53
ENDOCYTIC PATHWAY Cell types where active
Professional APCs
54
CYTOSOLIC PATHWAY Cell types where active
All nucleated cells
55
ENDOCYTIC PATHWAY Site of antigen –
MHC binding
Endocytic vesicles,
prelysosomes
56
CYTOSOLIC PATHWAY Site of antigen –
MHC binding
Rough endoplasmic reticulum
57
ENDOCYTIC PATHWAY MHC utilized
Class II
58
CYTOSOLIC PATHWAYbMHC utilized
Class I
59
ENDOCYTIC PATHWAY Presents to
CD4 (helper) T cells
60
CYTOSOLIC PATHWAY Presents to
CD8 (cytotoxic) T cells
61
CLINICAL SIGNIFICANCE OF MHC
- MHC molecules can induce a response that leads to graft
rejection
- Play a role in development of autoimmune diseases
- Determine the type of peptides to which an individual can 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
62
Ankylosing spondylitis
HLA-B27
63
Birdshot retinopathy
HLA-A29
64
Celiac disease
HLA-DR3, - DR5, - DR7
65
Graves’ disease
HLA-DR3
66
Narcolepsy
HLA-DR2
67
Multiple sclerosis
HLA-DR2
68
Rheumatoid arthritis
HLA-DR4
69
Type 1 diabetes mellitus
HLA-DQ8, - DQ2, - DR3, - DR4
70
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
5. Prevent Transfusion-related acute lung injury (TRALI)
6. Hematopoietic stem cell transplantation
71
HISTOCOMPATIBILITY
TESTING
1. Tissue typing
2. Antibody screening
3. Tissue matching/ Crossmatching
72
1. Tissue typing
-HLA Phenotyping: Serologic techniques
-HLA Genotyping: Molecular methods
73
Purified Lymphocyte suspension for Antigen detection
- Anticoagulated whole blood is overlaid into:
Ficoll-Hypaque reagent
Then centrifuge
74
Purified Lymphocyte suspension for Antigen
Use T lymphocytes
Class I Antigens (HLA-A, HLA-B, HLA-C)
75
Use B lymphocytes
Class II Antigens (HLA-DR, HLA-DP, HLA-DQ)
76
B lymphocyte separation:
1. Nylon wool separation
2. Use immunomagnetic beads
3. Fluorescent labeling (use FITC)
77
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
78
SEROLOGIC METHODS: TISSUE
TYPING
Lymphocytotoxicity Test (Complement-dependent
microlymphocytotoxicity)
79
Expose unknown cell to a battery of antisera of known
HLA specificity
Lymphocytotoxicity Test (Complement-dependent
microlymphocytotoxicity)
80
Lymphocytotoxicity Test (Complement-dependent
microlymphocytotoxicity)
-Incubate at room temperature for 30 minutes
81
Lymphocytotoxicity Test (Complement-dependent
microlymphocytotoxicity)
- Complement is added (rabbit serum)
82
Lymphocytotoxicity Test (Complement-dependent
microlymphocytotoxicity)
-Incubated at room temperature for 60 minutes
83
SEROLOGIC METHODS: TISSUE
TYPING
Lymphocytotoxicity Test (Complement-dependent
microlymphocytotoxicity)
84
Lymphocytotoxicity Test
- Expose unknown cell to a battery of antisera of known (?)
-Incubate at room temperature for (?)
- Complement is added (?)
-Incubated at room temperature for
- Then add (?) (eosin Y)
- Take an aliquot from well and examine under light microscope using (?)
- (?) take up the dye
HLA specificity
30 minutes
rabbit serum
60 minutes
trypan blue dye
hemocytometer
Dead cells
85
Dead cells
Flattened, appear large, dark and nonrefractile
86
Unaffected cells
small, bright and refractile
87
1 Negative
2 Doubtful positive
4 Weak positive
6 Positive
8 Strong positive
0 – 10
11 – 20
21 – 50
51 – 80
81 – 100
88
1 Negative
2 Doubtful positive
4 Weak positive
6 Positive
8 Strong positive
0 – 10
11 – 20
21 – 50
51 – 80
81 – 100
89
SEROLOGIC METHODS: TISSUE TYPING:
Complement-Dependent Lymphocytotoxicity
90
TISSUE TYPING: Complement-
Dependent Lymphocytotoxicity
To increase sensitivity:
1. Extended incubation
2. The Amos wash step
3. Antihuman globulin
91
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
92
Detect antibodies in patients who are candidates for
transplant
SEROLOGIC METHODS: Antibody
Screening
93
Antibody
Screening
1. Complement-dependent lymphocytotoxicity/
Microlymphocytotoxicity
2. ELISA
3. Flow cytometry
94
MICROLYMPHOCYTOTOXICITY
PERCENT PANEL REACTIVE ANTIBODY (%PRA)
Antibody Screening
95
The proportion of lymphocytes in the panel that are killed by the patient’s serum
PERCENT PANEL REACTIVE ANTIBODY (%PRA)
96
Utilize purified HLA antigens bound to the wells of microtiter plates.
ELISA
97
-Patient serum is added
ELISA
98
-If HLA-specific antibody is present, it will bind
ELISA
99
-Bound antibody is detected by addition of an enzyme-labeled anti immunoglobulin reagent
ELISA
100
Serves as a qualitative screen for the presence of HLA antibody in a serum
ELISA
101
-Recognizes false-positive reactions
ELISA
102
-Distinguishes Class I from Class II
ELISA
103
-Differentiates IgM from IgG antibodies
ELISA
104
-Increased specificity
ELISA
105
Detects antibody binding directly
Flow Cytometry
106
- Can distinguish between IgM and IgG
Flow Cytometry
107
- Uses T or B cells; or purified HLA antigens coated with microparticles
Flow Cytometry
108
- Bound antibody is detected by adding an FITC-labeled anti-IgGreagent
Flow Cytometry
109
- Percent PRA is determined
Flow Cytometry
110
- Most sensitive, most specific
Flow Cytometry
111
CROSSMATCHING
- Lymphocytotoxicity
-Flow cytometry
-ELISA being developed
112
SEROLOGIC METHODS:
113
MOLECULAR METHODS:
114
HLA GENOTYPING:
A. Restriction Fragment Length Polymorphism (RFLP)
B. PCR- based
115
PCR- based
1. Sequence-Specific oligonucleotides (SSO)
2. Sequence- Specific Primer(SSP)
3. Sequence-based typing (SBT)
116
Restriction enzymes (restriction endonucleases) are used
Restriction Fragment Length Polymorphism
(RFLP)
117
- Cleaves genomic DNA
Restriction Fragment Length Polymorphism
(RFLP)
118
- Obtain a pattern of fragmentation
Restriction Fragment Length Polymorphism
(RFLP)
119
- Degree of disparity between donor and recipient can be assessed by COMPARING patterns of fragmentation
Restriction Fragment Length Polymorphism
(RFLP)
120
Automated, rapid and in vitro technique
PCR
121
-Allows direct amplification of a particular
DNA sequence
PCR
122
-Selected by the use of primers that border
the genes of interest
PCR
123
PCR-amplification of a chosen sequence using primers flanking the sequence
Sequence-
Specific Oligonucleotides (SSO)
124
- The amplified DNA is immobilized on a membrane
Sequence-
Specific Oligonucleotides (SSO)
125
- Then hybridized with selected, labeled
oligonucleotide probes
Sequence-
Specific Oligonucleotides (SSO)
126
Oligonucleotide primers are designed to obtain amplification of specific alleles or groups of alleles
Sequence-
Specific Primers (SSP)
127
- Assignment of allele is based on the presence or absence of amplified product
Sequence-
Specific Primers (SSP)
128
- Detected by agarose gel electrophoresis (AGE) and transillumination
Sequence-
Specific Primers (SSP)
129
SBT Two Methods:
- Sanger-based DNA sequencing
- Next-generation DNA sequencing (NGS)
130
Allows amplification of the most polymorphic regions of the HLA genes
Sequence
Based Typing (SBT)
131
-Preferred method for hematopoietic stem cell transplantation
Sequence
Based Typing (SBT)
132
Performed by terminal-end incorporation of fluorescently labeled nucleotides during PCR reactions
Sequence
Based Typing (SBT)
133
-Allows amplification of the most polymorphic regions of the HLA genes
Sequence
Based Typing (SBT)
134
-Preferred method for hematopoietic stem cell transplantation
Sequence
Based Typing (SBT)
135
Donor and recipient cells are cultured together for several days
Mixed
Lymphocyte Reaction
136
- Allow CD4+ T cells to be activated and proliferate
Mixed
Lymphocyte Reaction
137
- In response to disparate Class II antigens
Mixed
Lymphocyte Reaction
138
- Amount of proliferation is measured and used to predict the magnitude of rejection
Mixed
Lymphocyte Reaction
139
- Can be done in a One-way MLR or a Two-way MLR
One-Way MLR → used to test for recipient’s
response to donor cells
Mixed
Lymphocyte Reaction
140
-Donor cells are irradiated (using Cobalt or
Cesium) or treated with mitomycin C
Mixed
Lymphocyte Reaction
141
-Most useful for bone marrow grafts and in
cases of living related donors
Mixed
Lymphocyte Reaction
142
Radioactive label is added on day 5.
Mixed
Lymphocyte Reaction
143
-Beta ray emissions are measured using a liquid scintillation counter (counts per minute)
Mixed
Lymphocyte Reaction
144
-CPM correlates with the amount of
proliferation
Mixed
Lymphocyte Reaction
145
-Depends on the degree of disparity between the recipient cells and potential donor cells
Mixed
Lymphocyte Reaction
146
Types of Grafts
1. Autograft
2. Syngraft
3. Allograft (Homograft)
4. Xenograft (Heterograft)
147
Most Common Tissues used for
Transplantation
1. Kidney
2. Heart
3. Cornea
4. Lung
5. Skin
6. Bone marrow
148
HOST RESPONSE TO TRANSPLANTATION
1. Hyperacute Rejection
2. Acute or Accelerated rejection
3. Chronic rejection
4. Graft-versus-Host Disease
149
Graft-versus-Host Disease
- Acute GVHD
- Chronic GVHD
150
Induce intense immunosuppression in the
initial days post transplantation
Immunosuppressive Therapy
151
-Maintenance of the graft
Immunosuppressive Therapy
152
-Reversal of established rejection
Immunosuppressive Therapy
153
Types of Immunosuppressive Therapy
1. Corticosteroids
2. Cyclosporine (Cyclosporin A)
3. Tacrolimus
4. Cytotoxic drugs
5. Antilymphocyte (Antithymocyte) globulin
6. Monoclonal antibodies
154
Complications of Transplantation
- Cancer
-Osteoporosis
-Diabetes
-Hypertension
-Hypercholesterolemia
