Allogenic MHC activates T cells'

1
Allogenic MHC activates T cells.
Syngeneic identical in all genetic loci.
Congenic genetically identical except at a
single loci (e.g. MHC).
2
NK cells cause hybrid resistance in bone marrow
transplant.
Bone marrow
H-2a/b
H-2a
NK (Ly49 for H-2a)
rejection
NK (Ly49 for H-2b)
H-2b
H-2a/b
NK (Ly49 for H-2a)
rejection
NK (Ly49 for H-2b)
H-2a/b
H-2a/b
NK (Ly49 for H-2a)
acceptance
NK (Ly49 for H-2b)
NK cells make little contribution to rejection of
solid tissues.
3
Complete matching of MHC is rare.
Twins
Sibling (1/4)
4
Minor Histocompatibility Antigens
Non-MHC alloantigens capable of eliciting a T
cell mediated response
One set of minor histocompatibility antigen is
encoded on the Y chromosome (H-Y). Autosome
minor histocompatibility antigens also exist.
5
Acute graft rejection
Injury induces inflammation
graft
Donor APC (direct activation)
Perforin Granzyme FasL-Fas
CTL
Recipient APC (indirect activation)
M?
TH1
IFN-?
Lytic enzymes NO
IL2
CD4 T
CD8 T
Conditioning of APC
CD40L-CD40
Lymph node
6
Graft versus host disease (GVHD)
Bone marrow transplant is used to treat leukemia,
lymphoma, and immunodeficiency diseases.
recipient
Lymphocytes depleted by radiation or radiomimetic
drugs.
Donor bone marrow (contaminated by T cells)
HSC
T cell
Recipient APC
BM
Lymph node
Tissue damage, GVHD
effector T cells
Kill leukemia cells (graft versus leukemia
effect)
Depletion of T cells in donor bone
marrow. Immunosuppression drug.
thymocyte
thymus
tolerized T cells
7
Immunosuppression drug
TCR/CD3
ZAP-70
Phosphorylation (kinases/phosphatases)
GEF
PLC-??
Ras
PIP2
IP3
DAG
MAP kinase cascade
PKC
Ca2
Cyclosporin A (CsA)
FK506
AP-1
NF-?B
transcription
NFAT
Expression of genes involved in proliferation and
T cell effector functions
8
CsA and FK504 (tacrolimus) inhibit IL2 production.
TCR
cyclophilin
Immunophilin peptidyl-prolyl cis-trans isomerases
Cytoplasm
Ca2
FKBP
calmodulin
Ca2/calmodulin
CsA / cyclophilin
FK506 / FKBP
Calcineurin (Ser/Thr phosphatase)
NF-AT
Nuclear factor of activated T cells
NF-AT
active
inactive
IL2 gene
NF-AT
nucleus
9
Rapamycin (sirolimus) blocks T cell proliferation.
APC
CD4 T cell
CsA / cyclophilin FK506 / FKBP
Activated CD4 T cell
Rapamycin / immunophilin complex inhibits a
protein kinase (mTOR mammalian target of
rapamycin). mTOR regulates translation, and is
essential for G1 to S phase transition.
Rapamycin / immunophilin
Proliferation
G1
S
Balance between graft tolerance and infection.
Toxic to many tissues (kidney)
10
Corticosteroids
11
Cytotoxic drugs inhibit DNA synthesis.
Lymphocytes are very sensitive to DNA synthesis
inhibitors.
Azathioprine, mycophenolate, and methotrexate
interfere with the synthesis of nucleotides.
Cyclophosphamide is metabolized to phosphoramide
mustard, which alkylates DNA.
Highly toxic. Used at lower dose in combination
with other drugs.
Radiation
Lymphocytes are very sensitive to X-rays.
Anti-T cell antibodies
Anti-CD3 antibodies, anti-CD2 antibodies,
anti-ICAMI antibodies.
12
Azathioprine and corticosteroids
Cyclosporin A and corticosteroids
13
Tolerance induction
Mixed chimerism
recipient
T cells depleted by anti-T cell antibodies,
radiation
Donor BM
HSCd
HSCr
HSCd
thymus
APCd
APCr
Td
Tr
Donor organ
Tolerized to both donor and recipient antigens
14
Peripheral tolerance induction
15
Chronic graft rejection
Arteriosclerosis of graft blood vessels,
accompanied by fibrosis and atrophy.
Chronic rejection is frequently associated with
anti-donor antibodies.
Refractory to increases in immunosuppressive
therapy.
16
Major limitation availability of donor organ
Xenograft
Pig as organ donor
Non-human cells contain a disaccharide antigen
(galctosyl-1,3-?-galactose) that is not present
on human cells.
This sugar is present in microbes. Human has
preformed antibodies against it.
RCA does not work well across species.
Transgenic pig expressing human RCA.
17
Immunodeficiency
Primary immunodeficiency genetic mutation
Secondary immunodeficiency other disease
18
Evaluation of immune function
Cell number in blood
Hematology
Flow cytometry
19
Measurement of immunoglobulin levels
Enzyme-Linked Immunosorbent Assay (ELISA)
20
Serum Ig levels can be measured by sandwich ELISA.
P
S
Detection antibody alkaline-phosphatase
(AP)-anti-human Ig
Human antibody
Coating antibody anti-human Ig
OD
Standard curve
Log10 (Ab concentration)
21
Complement Activities
CH50 the quantity of complement required for 50
lysis of 5 x 108 antibody-coated sheep red blood
cells in 1 hour at 37oC.
Complement titer is expressed as the number of
CH50 units contained in 1 ml of undiluted serum.
125-300 CH50 / ml
Serum volume
1 50 lysis
y/(1-y)
Y percentage lysis
22
Lymphocyte activity
Lymphocyte proliferation (3H-thymidine)
Polyclonal mitogens
Phytohemagglutinin (PHA)
T cells
lectins
Concanavalin (ConA)
T cells
B cells
Pokeweed mitogen (PWM)
LPS
B cells (mouse)
23
Severe combined immunodeficiency (SCID)
Deficiencies in T, B, and NK cells
T-B-NK
T-B-NK-
T-BNK-
T-BNK
Low or no Ab
lymphopenia
Small thymus
No lymph nodes or tonsils
Persistent infections in the first few months of
life. Fatal in the first two years of life.
24
Lymphocytes fail to respond to mitogens,
antigens or allogenic cells.
25
Mutations in nine genes can cause SCID.
170 patients
26
Common Gamma Chain Deficiency (46) (X-linked
severe combined immunodeficiency, SCID-X1)
Mutations in common gamma chain (gc) shared by
receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and
IL21.
T-BNK-
IL7R T cells
IL15R NK cells
IL-7 Receptor Alpha Chain Deficiency (10)
(IL-7R?-Deificient SCID)
T-BNK
27
Janus Kinase 3 Deficiency (Jak-Deficient SCID, 6)
T-BNK-
Jak3 transduces the signal From ?c.
28
Adenosine Deaminase (ADA) Deficiency (17)
T-B-NK-
CDP, TDP, ADP, GDP
ATP
dATP
Ribonucleotide reductase
Accumulates in ADA deficiency
dATP
ADP
dADP
AMP
dCDP, dTDP, dADP, dGDP
dAMP
5 nucleotidase
adenosine
deoxyadenosine
adenosine deaminase
inosine
deoxyinosine
dCTP, dTTP, dATP, dGTP
Uric acid
Lymphocytes are deficient in 5 nucleotidase.
DNA synthesis
29
Deficiency in purine nucleotide phosphorylase
(PNP)
PNP
inosine
Guanosine
GMP, GDP, GTP, dGMP, dGDP, dGTP accumulate.
30
Deficiency in V(D)J recombination
T-B-NK
RAG deficiency (3)
Artemis deficiency (1)
heptamer
5
3
TCCACAGTG
CACTGTGTA
J
D
RSS
RSS
3
AGGTGTCAC
GTGACACAT
5
RAG-1,2
Hairpin
Hairpin
5
CACAGTG
CACTGTG
3
TA
J
TC
RSS
RSS
D
GTGTCAC
GTGACAC
AT
3
AG
5
Artemis (nuclease cuts around hairpin)
P (palindrome) nucleotide
3
TA
J
5
TC
GA
AT
5
D
3
TdT (terminal deoxynucleotide transferase) Add
random nucleotides to 3 OH
5
3
J
TA
TC
GATC
D
AT
GGC
3
5
31
scid mouse
Deficient in B and T cells
Defect in DNA-dependent protein kinase (DNA-PKcs)
DNA-PKcs
Phosphorylation of Artemis
Cleavage of hairpin