Chapter 10: Immune Response Against Transplants

Front 1

Autograft

Back 1

Self tissue transferred from one body site to another
Skin grafts in burn victims
No immune response – graft accepted

Front 2

Isograft or syngeneic graft

Back 2

Grafts between genetically identical individuals
No immune response – graft accepted

Front 3

Blood transfusion

Back 3

First started in 1812
Still the most common transplant
Used for treating trauma/surgery or disease
Immediate replacement of
-Fluid
-Serum proteins
-Red blood cells
-Platelets

Front 4

Transplant rejection

Back 4

when a kidney is transplanted the recipients T cells attack the transplant

Front 5

Graft vs host dz

Back 5

when bone marrow is transplanted the T cells in the transplant attack the recipients tissues

Front 6

Donated blood

Back 6

Separated into component cells and plasma
Patient is given component that is needed
Short-term remedy
Within weeks patient’s bone marrow makes up the loss

Front 7

Paul Ehrlich

Back 7

Recognized isoantibodies (alloantibodies)
Ab produced in one individual against the RBC Ags of another individual

Front 8

Karl Landsteiner (1901)

Back 8

Established existence of ABO human blood groups by verifying the presence of hemagglutinins in normal human sera
4 blood groups: A, B, AB and O

Front 9

Hemagglutinins

Back 9

Cause agglutination of RBCs
Always present in normal, healthy individuals
Naturally occurring
Require no prior exposure to antigen for antibody to occur

Front 10

Inheritance

Back 10

Inheritance is determined by a polymorphic locus on chromosome 9
Three alleles: A, B, O
Inherit one ABO gene from each parent

Front 11

Phenotypes

Back 11

A and B express dominant phenotypes over O and are codominant over each other
O is recessive - amorphic
A is dominant – AA AO
B is dominant – BB BO
AB is codominant

Front 12

glycosyl-transferases
H substance

Back 12

Alleles code for specific enzymes (glycosyl-
transferases) that add a carbohydrate Ag to
a glycoprotein (H substance)

Front 13

H substance

Back 13

Nearly 100% of population inherits H gene
Formation of H substance (L-fucose)
Exception: Bombay type

Front 14

Formation of A antigen

Back 14

A gene codes for N-acetylgalactosamine

Front 15

Formation of B antige

Back 15

B gene codes for D-galactose

Front 16

O gene is inactive

Back 16

Individuals still produce H substance

Front 17

4 phenotypesbased on agglutinating antibodies

Back 17

Individuals are separated into 4 phenotypes
based on agglutinating antibodies normally
present in their serum
Type A: Anti-B
Type B: Anti-A
Type AB: Neither A or B
Type O: Both
A and B blood group Ags are also found on
other cells
Epithelial cells
Endothelial cells

Front 18

Development of ABO antibodies

Back 18

Result of cross reaction to A, or B, or neither
Develop during the 1st year of life, when gut becomes colonized with normal flora
Generally IgM
Other blood groups do not possess Abs to Ags they lack
They require exposure to the Ag to become sensitized

Front 19

A, B and H substances

Back 19

A, B and H substances are soluble and may appear in other body fluids
Saliva, sweat, gastric juices

Front 20

Secretion of A, B and H substance is controlled by

Back 20

Secretion of A, B and H substance is controlled by the alleles Se/se
Se is dominant
If individual is Se/Se or Se/se
-- Secretor
If individual is se/se
-- non-secretor

Front 21

Rh blood groups

Back 21

Landsteiner and Weiner (1930s)
Rabbit antisera against rhesus monkey RBCs agglutinated about 85% of human RBCs
Antisera was not ABO reactive
These individuals had an antigen (called Rh antigen) on their RBCs

Front 22

Rh blood group system

Back 22

Encoded by a group of alleles at a single locus on chromosome 1
single locus on chromosome 1 CDE/cde

Front 23

D antigen is the most important of the Rh Ags

Back 23

85% of population is D positive (Rh pos)
Antibodies against D cause >90% of all cases of hemolytic disease of the newborn (HDN)

Front 24

Rh genes are codominant

Back 24

Genes from both parents are demonstrated in offspring

Front 25

D, C, c, E, and e

Back 25

Represent actual Ag on RBCs that can be recognized by specific Abs
There is no “d” Ag

Front 26

Rh antibodies are immune antibodies

Back 26

IgG antibodies produced by exposure of an individual’s immune system to foreign Ag → sensitization

Front 27

Occurs if Rh neg patient

Back 27

Transfused with Rh pos blood → hemolytic transfusion reaction
Pregnant with Rh pos fetus → hemolytic disease of the newborn (HDN)

Front 28

HDN

Back 28

Only occurs to an Rh pos fetus of a previously sensitized Rh neg mother
Maternal sensitization may occur
-Transfusion with Rh pos blood
-Previous pregnancy with Rh pos fetus
Result is production of anti-D by the mother
Anti-D (IgG Ab) crosses the placenta and reacts with fetal RBCs
-Hemolysis and RBC destruction

Front 29

Symptoms of HDN

Back 29

First 24 hours of life:
Severe hemolysis
Can lead to death
If occurring after 1st day of life:
Increased blood bilirubin levels
Due to hemoglobin destruction
Result: kernicterus

Front 30

Prevention of HDN

Back 30

IM injection within 72 hours of delivery/ abortion of Rh immune globulin (RhoGam)
Given to all Rh neg mothers, regardless of fetal Rh status

Front 31

Mechanism of prevention IM injection

Back 31

Ig attaches to Rh Ag on fetal blood in maternal circulation
Ab-coated RBCs are removed from circulation before mother can initiate an immune response
Subsequent Rh pos fetuses remain unaffected

Front 32

Other blood groups

Back 32

Approximately 25 other minor blood groups
Can be responsible for minor transfusion reactions
Include the following
Lewis
MN
Kell
Duffy

Front 33

Laboratory methods used in transfusion services

Back 33

Do not cause agglutination of RBC antigen-antibody complexes
Difficult to visualize in the lab
Problem overcome by Coombs in 1945 with preparation of antihuman globulins (AHG) in rabbits

Front 34

antihuman globulins (AHG)

Back 34

Production of AHG is based on 2 facts:
2.Igs of one species (e.g., human) are immunogenic when injected into another species (e.g., rabbit)
-Production of Abs against the Igs
2. Anti-Igs bind with the antigenic determinants present of the Fc portion of the Ab
-Leaves the Fab portions free to react with Ag

Front 35

Development of the AHG test

Back 35

Development of the AHG test marked the
beginning of modern (immunohematology)
blood banking
Serological testing permits the detection of incomplete IgG antibodies
Safer transfusions with decreased risk of transfusion reaction

Front 36

Direct antiglobulin test (DAT)

Back 36

Purpose: To detect RBCs coated with IgG or complement

Front 37

Procedure - DAT

Back 37

1. Anti-human Ab is added to washed patient RBCs (Ag)
2. Suspension is centrifuged to enhance agglutination
3. Observe for agglutination

Front 38

Uses of DAT

Back 38

Detection of auto-Abs in autoimmune hemolytic anemia
Diagnosis of HDN
Detection of drug-induced hemolytic anemia
Investigation of transfusion reactions

Front 39

Principle-Indirect antiglobulin test (IAT)

Back 39

Detection of possible patient Abs to potential donor RBCs

Front 40

Uses of IAT

Back 40

Compatibility testing (cross match) prior to transfusion
Detect presence of unexpected Abs in patient serum
Identification of Abs
Use panel of RBCs with known Ag specificity

Front 41

Procedure- IAT

Back 41

1. Combine patient serum with reagent or donor RBCs
2. Incubate at 370C
3. Wash to remove unbound Ab
4. Add anti-human globulin
5. Centrifuge
6. Observe for agglutination

Front 42

Transfusion reactions

Back 42

Occurrence of transfusion reactions are rare
<5% of all transfusions
Can range from mild to severe

Front 43

Transfusion reactions

Back 43

febrile
allergic
hemolytic

Front 44

Febrile reactions

Back 44

Temperature increase > 2ºC occurs within 1 hour of transfusion
Very common, rarely causes major clinical reactions
Caused by reaction of:
Cytokines
WBC Ags
Platelet Ags

Front 45

Allergic reactions

Back 45

Characterized by hives, itching
Rarely causes clinical problems
Cause is unknown

Front 46

Hemolytic reactions

Back 46

Characterized by muscle pain, headache, vomiting, fever, drop in blood pressure
Occurs within minutes of transfusion
Most serious consequence of blood transfusion (may be life-threatening)
Either intravascular or extravascular

Front 47

Intravascular hemolysis

Back 47

Occurs within blood vessels
Due to A,B,O compatibility
Human error, usually clerical
Lab technical errors are rare
RBC destruction by MAC of complement system

Front 48

Extravascular hemolysis

Back 48

RBCs are destroyed by fixed macrophages throughout the body
Due to minor blood group incompatiblity
May be delayed up to 1 week before reaction occurs

Front 49

transplantation

Back 49

Organ damage is irreversible or other treatments are not applicable
Disease must not recur
-Not recommended for Goodpasture’s syndrome (anti-glomerular basement membrane Abs)
Chances for rejection must be minimized
-ABO compatibility between donor & recipient
-No recipient anti-donor HLA Abs
-Donor and recipient have as close an HLA match as possible

Front 50

Principle targets of rejection

Back 50

The antigens of allografts that serve as
the principle targets of rejection are the
proteins encoded in the major histo-
compatibility complex (MHC).

Front 51

MHC genes are highly pleomorphic

Back 51

Each person expresses 6 class I alleles
-One allele of HLA-A, -B and –C from each parent
-~ 120 alleles of HLA-A genes
-~ 250 alleles of HLA-B genes
HLA-C and HLA-DP
-Limited polymorphism
-Minor significance during transplant

Front 52

strongest known immune responses

Back 52

Recognition of MHC Ags on another individual’s cells (allograft) is one of the strongest known immune responses

Cross reaction in which a T cell specific for a self MHC molecule-foreign peptide also recognizes an allogeneic MHC molecule whose structure resembles the self MHC molecule-foreign peptide complex

Front 53

Direct recognition

Back 53

Occurs when T cells in the recipient recognize donor allogeneic MHC molecules on graft dedritic cells
T cells become activated, stimulate the development of CTLs which attack the cells of the graft

Front 54

Indirect recognition

Back 54

If graft cells are ingested by dendritic cells in recipient
Donor Ags are processed and presented by self MHCs on recipient APCs

Front 55

Pretransplant testing for donor selection

Back 55

ABO grouping
HLA typing
Ab screen
Testing prior to transplantation is dependent upon type of organ being transplanted and donor source (cadaver vs. living donor)
-Extended pre-transplant testing is done if time allows
-Includes testing for presence of latent viruses, HIV, hepatitis, etc.

Front 56

ABO blood group typing

Back 56

ABO blood group typing
Incompatibility leads to complement-mediated lysis of cells/tissue
Rejection within minutes

Front 57

HLA typing- Class 1 and 2 Ag

Back 57

Class I antigens:
-Detected by serological testing or PCR
-Determines HLA-A, -B and –C types
Class II antigens:
-Detected by PCR or mixed lymphocyte culture reactions
-Determines HLA-D types
-Most important determination

Front 58

HLA typing microcytotoxicity test

Back 58

Monoclonal Abs of known HLA specificity are reacted with patient B and T cells
Ag-Ab complex will form on cell surface if Ag and reagent Ab are of same specificity
Complement is added
Lysis occurs if Ag-Ab complex is present
Visible by uptake of stain

Front 59

Lymphocyte matching:mixed leukocyte reaction (MLR)

Back 59

Used to determine degree of compatibility between recipient (responder) T, B and NK cells and donor (stimulator) cells
Incompatibility of HLA types between the 2 cell populations results in recipient immune response against donor cells

Front 60

Immunologic response to transplant shows

Back 60

Specificity
Memory
Recognition of self from non-self
The more foreign the graft (xenograft), the
greater the immune response.

Front 61

Symptoms of graft rejection

Back 61

Depends on type of graft rejection
Can involve
Thrombosis
Necrosis
Fever
Leukocytosis
In cases of renal transplant: decreased urine output
May eventually lead to graft failure

Front 62

Graft rejection

Back 62

Graft rejection is classified based on clinical and pathological features
Hyperacute
Acute
First stage
Second stage
Chronic

Front 63

Hyperacute rejection

Back 63

Occurs minutes/hours after transplant
Due to pre-existing host Abs specific for donor graft Ags
-Previous transplant, transfusion or pregnancy
Host Ab binds on vascular epithelium of graft
Activation of complement and coagulation cascades
Almost immediate rejection

Front 64

Acute first set rejection

Back 64

First 7-10 post-transplant, no problems seen
After ~ 2 weeks, rejection starts
-Mediated mostly by T cells
Seen with:
-Incompatible (mismatched) organs
-Insufficient immunosuppressive therapy

Front 65

Mechanisms

Back 65

Cytotoxic T cells directly destroy graft tissue
-Cell damage and inflammation
T cells and Abs react against graft blood vessel endothelial cells
-Vascular damage

Front 66

Second set rejection

Back 66

Occurs with second transplanted organ
-Either from same donor or donor with Ags similar to the 1st donor
Process of rejection is accelerated
-Within a week of transplant
Due to anamnestic (memory) response

Front 67

Chronic rejection

Back 67

Occurs over months to years
Progressive loss of graft function
Graft arteriosclerosis
-Gradual fibrous narrowing of graft vessels
T cells secrete cytokines
-Stimulate proliferation of fibroblasts and vascular smooth muscle cells in graft

Front 68

Stem cell transplant (SCT)

Back 68

Bone marrow transplant – a risky procedure
Even with well matched donors, mortality can reach 20%
Used to restore myeloid and lymphoid cells

Front 69

Circumstances requiring SCT

Back 69

Hematological malignancy
-Especially if there is a high risk of relapse
Reduced or abnormal myeloid cell production
-Aplastic anemia
Primary immunodeficiencies
-Severe combined immunodeficiency (SCID)
Genetic diseases

Front 70

Sources of stem cells

Back 70

Bone marrow
--Aspiration of large amounts of donor marrow acquired under general anesthesia
Peripheral blood
--Donor is treated with colony stimulating growth factors
--Release of stem cells into circulation
Cord blood

Front 71

Cord blood

Back 71

Large numbers of stem cells can be frozen
Advantage
-Immature lymphocytes are less likely to cause graft versus host disease
Disadvantage
-Only enough cells for children or small adults

Front 72

Recipient conditioning:myeloablation therapy

Back 72

High dose chemotherapy and/or radiotherapy
-Destroys recipients stem cells
Provide space in marrow for donor cells to engraft
Prevents rejection of grafted cells by recipient’s T cells
After treatment, patient cannot survive without SCT

Front 73

Hematopoietic system

Back 73

Hematopoietic system starts to be reconstituted within a few weeks after transplant
-Innate immunity cells (granulocytes and NK cells) recover first
When fully recovered, patient is a chimera
-Hematopoietic cells: donor genotype
-All other cells: recipient genotype

Front 74

Graft versus host disease (GVHD)

Back 74

Occurs when donor T cells respond to allogeneic recipient Ags

Front 75

Sites targeted for GVHD

Back 75

Pre-transplant conditioning also destroys tissues with rapidly dividing cells
Skin
Intestinal epithelium
Hepatocytes in liver

Front 76

Symptoms of GVHD

Back 76

Skin rash
-Starts 10-28 days after transplant
-Begins on palms and soles and spreads to head and trunk
Intestinal reaction
-Cramps and diarrhea
Inflammation of bile ducts
-Hyperbilirubinemia and ↑ liver enzymes
Methotrexate and cyclosporin A used to reduce incidence and severity

Front 77

immunosuppressive drugs

Back 77

Allogeneic transplantation is possible by the use of immunosuppressive drugs
Three types of drugs
1. Corticosteroids
2. Cytotoxic drugs
3. Protein synthesis inhibitors
All inhibit normal immune function against foreign microbes
↑ risk of infection
All are toxic to other tissues
Drugs are generally used in combination to lessen side-effects

Front 78

Corticosteroids

Back 78

Act at intracellular receptors and modulate the transcription of many genes
Inhibit protein synthesis
Impairs IL-1 and IL-2 production
Result: impairment of activation of:
-Macrophages
-CD4+ and CD8+ T cells
-B cells
Prednisone, prednisolone, methylprednisolone

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Side-effects: steroids

Back 79

Fluid retention
Weight gain
Diabetes
Thinning of skin
Hair loss

Front 80

Cytotoxic drugs

Back 80

Kill proliferating cells
-Originally used to treat cancer
Affect tissues active in cell division
-Bone marrow → anemia, leukopenia, thrombocytopenia
-Intestinal epithelium → intestinal damage
-Hair follicles → hair loss

Front 81

Types of cytotoxic drugs

Back 81

Azathioprine and mercaptopurine
-Interfere with RNA and DNA synthesis
Cyclophosphamide and chlorambucil
-Interfere with DNA metabolism
Methotrexate
-Inhibits dihydrofolate reductase, essential for thymidine synthesis

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Protein synthesis inhibitors

Back 82

Interfere with signal transduction pathways needed for clonal proliferation of lymphocytes
-Inhibit IL-2 synthesis
Cyclosporin A and tacrolimus
Side-effects
-Renal toxicity
-↑ risk of cancer with long term use

Front 83

Antibody therapy

Back 83

Antibodies specific for T cells are used to control acute rejection
Made in sheep or goats immunized with human thymocytes (anti-thymocyte globulin: ATG) or lymphocytes (anti-lymphocyte globulin: ALG)
Cause destruction of cells to which they bind

Front 84

Xenotransplantation

Back 84

Pigs are the most suitable animal donors for humans:
Of similar size
Pigs are already farmed and consumed by humans in large numbers
Problems:
Most humans have circulating Abs that bind to pig endothelial cells → hyperacute rejection
Possibility of pig viruses crossing the species barrier