Harvard-MIT Division of Health Sciences and Technology
HST.176: Cellular and Molecular Immunology
Course Director: Dr. Shiv Pillai
AICD
Naive
Activated
Activated
Effector
Memory
Effector
AICD
Naive
Activated
• INNOCUOUS
ANTIGEN
Memory
• PATHOGEN
• No Danger- very low • Danger induces
expression of
costimulatory ligands
costimulatory ligands • Signal One + Signal
• Signal One Only
Two
– Non-responsiveness
– Tolerance (anergy)
– Lymphocyte
Activation
INNOCUOUS ANTIGENS
• Commensal microbes
• Food antigens
• Host cell proteins that have not induced
thymic deletion
• Fetal antigens?
Choices during T cell activation
• INITIALLY:
– Anergy OR
– Activation into Effector state
• RESTIMULATION OF EFFECTORS
– Activation Induced Cell death OR
– Memory
– Differentiation e.g.Th1 versus Th2
– Other regulatory subsets
Issues in T cell activation - I
• Crosslinking versus Coreceptors
• Is the Affinity of the TCR for MHC Too
Low?
• Lipid rafts and the need for Immunological
Synapse generation
• Interfering with Signal Oneimmunosuppression and how it works
Issues in T cell Activation -II
• Anergy versus Activation
• How is the quality of an immune response
modulated - Th1 versus Th2 for instance?
α
γ
ε
β
ε
δ
ζζ
ITAM
α2
β2
α1
β1
MHC class II
peptide
α
γ
ε
β
ε
T cell receptor
δ
ζζ
polysaccharide
monosaccharide
antigen receptor
NO SIGNAL
SIGNAL TRANSDUCTION
TCR
α
CD4
γ
ε
β
ε
δ
ζζ
Fyn
Lck
Zap-70
TCR-MHC interactions
• 1. Few MHC-peptide complexes on an APC
specific for a given TCR
• 2. Affinity of TCR for specific MHCpeptide combo is pretty low - 10-4 to 10-6 M
• 3. How then does a T cell receive specific
signals?
It Takes Two to Tango…..?
• SLC (secondary lymphoid chemokine)
draws naïve T cells and activated dendritic
cells through the HEV into lymph nodes
• Signaling through CCR7 activates integrins
for adhesion but also induces T cell
polarization via cytoskeletal rearrangements
• “Leading edge” of T cell slides alongside
any dendritic cell it sees and a dance begins
Just a brief romance……..?
• Adhesion molecules on T cell bind to their
counterparts on the DC; LFA-1 to ICAM1,VLA-4 to VCAM-1 (LFA-1 and VLA-4
are integrins), CD2 to CD48 and so on
• CCR7 mediated adhesion is brief….minutes
The TCR knows…..
• The right TCR-MHC/peptide interaction
sustains a long term relationship…. For a
few hours
• True synapse formation is initiated
Lipid rafts and Immunological
Synapses
• Signaling initiated from specialized
membrane microdomains- lipid rafts aka
DIG domains or GEMs (these contain
acylated proteins, GPI anchored proteins,
PIP2 etc).
• Signaling through TCR induces cytoskeletal
rearrangements and fusion of lipid rafts
forming immunological synapses.
~40 nm
15 nm
CD28
T
B-7
CD4
TCR
MHC class II
CD2AP
CD48/CD58
APC
CD2
LFA-1
ICAM-1
Outer ring
"Quiescent" APC
Low levels of B7-2/CD86
MHC class-II
CD4
Co-receptor
γ
ε
ε
δ
ζζ
ITAM
CD28
Signal One
T cell
Minimal IL-2 Transcription
No activation
Induction of anergy
"Activated" APC
High levels of B7-1/CD80and B7-2/CD86
MHC class-II
CD4
Co-receptor
γ
ε
ε
δ
ζζ
ITAM
CD28
Signal One
T cell
Signal Two
IL-2 transcription
Also induction of IL-2R, CD40L, FasL, and CTLA-4
Some of the events turned on by
Signals One and Two
• Induction of IL-2R and IL-2 expression
• Induction of cyclins D2 and D3 and CDK4
and degradation of p27 CDK inhibitor cells divide
• Induction of CD40L
• Induction of FasL and of higher levels of
CTLA-4
B7 costimulation III: terminating responses
High levels of B7.1 and B7.2
MHC class-II
CD4
coreceptor
δ
ε
ε
γ
ζζ
ITAM
CD28
CTLA-4
High levels of CTLA-4
induced by TCR, CD28,
and IL-2R signals
Signal Two
T cell
Signal One
CD4
CD28
TCR
Lck
Fyn
ZAP-70
LAT
PI3 Kinase
Vav
PLCγ1
Cleaves PIP2
DAG
Grb2
SOS
Ras/Raf/ERK
IP3
Ca++
Rac/SEK/JNK
Nil-2-a
Cytoplasmic NFAT
Calcineurin
(phosphatase)
Nuclear NFAT
ERK P
JNK
IL-2 transcription
Fos/Jun (AP-1)
(Repressor
of IL-2
transcription)
ACTIVATED
P
P
P
P
P
P
P
P
P
P
IL-2
A
NF-AT AP-1 Oct-1
NFκB/ AP-1 NFAT AP-1
NFAT AP-1 Oct-1
NFAT
NFκB/ AP-1
c-Rel
p65
Nil-2-a
Egr-1/
HMGI/Y
SP-1
Egr-1/
SP-1
TATA
ANERGIZED
X
A
NF-AT AP-1 Oct-1
NFκB/ AP-1
p65
Egr-1/
SP-1
NFκB/ AP-1 NFAT AP-1
c-Rel
HMGI/Y
-285 -275 -250 -220
-205 -180
NFAT AP-1 Oct-1
Nil-2-a
NFAT
TATA
Egr-1/
SP-1
-160 -150 -135 -125-105 - 9 0 - 8 5 - 7 5 - 6 0 - 4 5
-30
No Activation
Anergy
TCR
Naive
CD4
Activation induced
cell death
Activated
Effector
TCR/CD28
Restimulation
Chronic
stimulation
and phenotypic differentiation
IL-12
IL-18
No restimulation
IL-4
Memory
Th1
Th2
Receptor clustering
Activation of Src-family kinases
ITAM tyrosine phosphorylation
Recruitment of Syk family tyrosine kinases to
ITAMs and activation of these kinases
Recruitment of adaptors to the membrane
Tyrosine phosphorylation of adaptor proteins and
activation of downstream pathways
SOS 1, 2
(Guanine nucleotide exchange factors)
Inactive GDP
Ras
Active
Ras
GTP
Effectors
p120GAP
Neurofibromin
(RasGAPs)
c-Raf
ERK activation
1. Crosslink receptor
YP
YP
YP
2. Activate tyrosine kinase
GDP
GTP
3. Phosphorylate tyrosines on ITAMs and
other adaptors
MAPKKK
(Raf)
4. Recruit signal effectors such as GEFs
5. Convert G protein from inactive GDP bound
form to active GTP bound form
MAPKK
(MEK)
6. Recruit and activate MAP Kinase Kinase Kinase
(sometimes referred to as a MEK kinase)
MAPK
(ERK)
7. MAPKKK is a serine/threonine kinase which
activates a MAP Kinase Kinase
8. MAPKK is a dual specificity kinase which
phosphorylates MAP Kinases on threonine
and tyrosine residues. MAPK translocated
to the nucleus
TP YP
9. MAPK phosphorylates targets
in nucleus which may regulate
transcription or message
stability
Nucleus
CTLA-4
CD4
CD28
TCR
Lck
Fyn
ZAP-70
X
LAT
PI3 Kinase
Vav
PLCγ1
Cleaves PIP2
DAG
FK506
Ras/Raf/ERK
IP3
Ca++
Cyclosporin
Grb2
SOS
X
Rac/SEK/JNK
Nil-2-a
Cytoplasmic NFAT
Calcineurin
(phosphatase)
Nuclear NFAT
ERK P
P
JNK
Fos/Jun (AP-1)
IL-2 transcription
(Repressor
of IL-2
transcription)
IL-12
Thp
Inflammation
IL-1
TNF
IL-6
Fever
Acute phase response
Th1
IFN-γ
IL-2
Lymphotoxin
Th1/Th2
1. TCR Signal strength - nature of
antigen and amount
2. Route of immunization and cytokine
milieu
3. Signals induce transcriptional patterns
that influence differentiation
The lists of transcription factors on the next image
are included to paint the picture more completely
- you do NOT need to memorize them
IL-4R
CD28 TCR
IL-12R
IL-18R
SLAM
JNK1
JNK2, p38
JNK1
STAT6
c-Maf
GATA-3
NIP-45
NFATc
JunB
NF-IL6
etc
Th2 phenotype
1. Transcriptional Induction
of IL-4, IL-5, IL-6, IL-9, IL-10, IL-13
NFATp
NFAT4
Bcl-6
JNK2, p38
T-bet
STAT4
? ATF-2
Th1 phenotype
1. Transcriptional induction of
IFN-γ, IL-2, LT
2. Shut off of IL-12Rβ2
2. Synthesis of IL-12Rβ2
3. Expression of CCR3
3. Expression of CCR5
Cytokine
receptor
Cytokine
Y
Y
Y
Y
Y
Y
Y
Y
Jak
SH2 domain
STAT
Y
STAT dimer
Transcription of
target gene
Y
STAT site
IL-7Rα
CC
CC
CC
CC
γc
WSXWS
Src
family
kinases
Jak3
PI3Kinase
Accessibility for
V(D)J recombination
Survival/proliferation
Induction of Bcl-2 in pro-T cells
γc and X-linked SCID
γc or the common gamma chain is a
component of the IL-2, IL-4, IL-7,
IL-9, and IL-15 receptors
The γc gene is encoded on the Xchromosome
IL-2Rα
IL-2Rβ
γc
CC
CC
"sushi"
WSXWS
Box 1
Box 2
Syk
Jak1
c-myc
mTOR/
FRAP
Jak3
Lck
Shc
Ras/
Raf/ERK
PI3K
Y
STAT dimer
(STAT 5 and STAT 3 are activated)
Bcl-2
Activated B and Th2 cells
Th1 cells
S
S
S
S
S
S
High affinity receptor
IL-12Rβ1
(binding subunit
in mouse; low affinity
receptor in man)
IL-12Rβ2
(signaling
subunit)
IL-12Rβ1
Jak2
Tyk2
Y
STAT 4 dimer
Igα
YY
Src-family kinase
YY
Igβ
ITAM
Igα
YY
YY
YY
Activated Syk
YY
Igβ
ITAM
Inactive Syk
Btk is recruited to the membrane by PI3K
PH
TH
PI3K generates PIP3
PR
YP(223)
SH3
SH3
SH2
Autophosphorylation
site
PR
SH2
TH
KINASE
PH
YP(551)
KINASE
Y551
Activation segment
tyrosine phosphorylated
by Src family kinases
CR2/CD21 is a coreceptor and positive regulator of BCR signaling
Receptor for EBV
CR2/CD21
YYYY
Immune complex
BCR
CD19
β α
CD81
Lyn
Fyn
Syk
Vav
PI3K
CD22 is a negative regulator of BCR signaling
Sia α2-6Gal β1-4GlcNAc
CD22
Igβ Igα
Lyn
SHP-1
BCR
Y
Ιgβ
Y
immune complex
Y YY
FcγRIIB1
Ιgα
ITIM
PI3K
SHIP
ITAM
Btk, Akt, PLC γ1, etc
Ca++
activated
TCR
T
CD4
gp 39/CD40L
YBCR
CD40
MHC
class II
(loaded)
Y
B
CD40
TRAF-N
Ring
TRAF2
TRAF3
TRAF5
TRAF6
Zn finger
TRAF-C
JNK/SAPK activation
Activation of IKK complex
IκB degradation
NF-κB nuclear translocation
CD8+cell
IL-2
CD28
CD8
CD4+ cell
MHC class II
B7
CD4
MHC classI
APC
CD8+cell
CD4+ cell
CD28
CD40L
CD8
CD4
MHC class II
MHC class I
B7
B7
CD40
"Unlicensed APC
"Licensed APC"