The document provides an overview of antigen receptor signaling and the immunological synapse between T cells and antigen presenting cells (APCs). It discusses how T cell receptor (TCR) signaling is initiated upon antigen binding and transduced through invariant CD3 and ζ proteins containing immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs are phosphorylated by Src family kinases, recruiting Syk/ZAP-70 kinases and initiating downstream signaling cascades involving transcription factors like NFAT, NF-κB, and AP-1. The interaction between T cells and APCs results in the formation of the immunological synapse, containing central, peripheral, and distal supramolecular activation complexes where receptor clustering

1. 1.
An&gen
Receptor
Signaling
with
focus
on
TCR
signaling
&
the
Immunological
Synapse
BIOM
514,
Cell
Signaling
Apr.
1
&
6,
2016
Aaron
Neumann,
Ph.D.
(Pathology)
CRF327,
akneumann@salud.unm.edu
Abbas, Lichtman & Pillai, 8th ed., chapters 7, 9 and parts of 4 & 12

2. OVERVIEW
OF
LECTURE
MATERIAL
• Lecture
1:
An&gen
Receptor
Signaling
and
the
T
cell
Immune
Synapse
• Lecture
2:
Cytokine
Receptor,
Notch
and
Innate
Immunoreceptor
Signaling.
Regula&on
of
signal
dynamics
• Problem
Set:
Spillane
&
Tolar

3. Basic
Concepts
in
Signal
Transduc&on

4. Common events that govern signaling

5. • Signal
ini&a&on
• Ligand
binding
to
membrane
receptors
leads
to
receptor
conforma&onal
changes
and/or
aggrega&on,
resul&ng
in
phosphoryla&on
of
receptors
(or
associated
intracellular
signaling
proteins)
• Signal
propaga&on
• Recruitment
of
adaptor
proteins
to
phosphorylated
receptors
ini&ates
signaling
cascades
that
take
the
signal
to
the
nucleus
• Structural
mo&fs
allow
for
specificity
of
protein
interac&ons
• Signal
termina&on
• Phosphatases
can
block
signaling
• Proteins
are
degraded
(ubiqui&na&on)
General principles of membrane receptor signaling

6. Types of receptors that regulate
immune cell signaling

7. Protein Phosphorylation
KINASE
PHOSPHATASE
Serine
Threonine
Tyrosine
Kinases: Transfer the terminal phosphate of ATP to the hydroxyl group of a
tyrosine residue in the substrate protein
Phosphatases: Remove phosphate moieties from tyrosine residues (dephosphorylation)
Phosphoryla&on
regulates
protein
ac&vity
and
provides
a
binding
site
for
other
proteins.

8. Motifs Bind to Other Motifs in Proteins
3 examples
Src homology 3-domain (SH3)
Src homology 2-domain (SH2)
Regulatory domain Binds to:
Phospho-
tyrosine
P X X P Proline-rich
stretch
Pleckstrin homology domain (PH) PIP3
Phospho-
inositols
P X X X
These motifs become very important in building local assemblies of signaling
proteins on adaptor proteins.
How
do
mul)ple
protein/protein
interac)on
domains
improve
cell
signaling?

9. The importance of protein domains/motifs for signaling.
Fig 6.3 Janeway © 2001 Garland
400 - 500 nm
1.0-1.5um
Illustration of the Problem
Confocal Observation Volume for
Fluorescence Correlation Measurements
of Cytosolic Signaling Protein Dynamics
To scale, a typical membrane associated protein
signaling domain would be about this size…pretty tiny.
The concentration of cytosolic signaling intermediaries
is typically such that there are only 1-3 molecules
in this volume!
Volume:
~1 fL
The probability of receptor-intermediary interaction is
quite low (takes ms to diffuse to receptor in this volume,
but binding/unbinding is on µs timescale.)
Adaptor proteins form signaling complexes that
promote and stabilize protein-protein and protein
lipid interactions.

10. Overview
of
ITAM
signaling
systems
• Ini&a&on:
Membrane
proximal
signaling
events
• Output:
Transcrip&on
factor
ac&va&on
• Finally,
we
will
focus
in
detail
on
the
process
of
T
cell
ac&va&on
via
the
TCR
to
inves&gate
how
one
important
ITAM
signaling
system
works.

11. Antigen receptors

12. ITAM
Immunoreceptor
Tyrosine-­‐based
AcAvaAng
MoAf
YXXL/I(X)6-8YXXL/I
ITAM Sequence
Tyrosine
Leucine/
Isoleucine
*ITAMs do not have intrinsic kinase activity
*The phosphorylated ITAM motif recruits kinase

13. There are many more ITAM receptors…
Note that many of these non-TCR/BCR ITAM signaling systems use
FcRgamma chain or DAP12 as the signaling partner that actually provides the
ITAM motif.
A receptor/signaling chain complex must form.

14. What are the early, receptor proximal events in ITAM signaling generally?
ITAM/receptor complex Src family kinase
ITAM pTyr
ITAM Recruits active Syk/ZAP-70
Phosphorylate downstream adaptors and signaling proteins

15. ITAMs are phosphorylated by Src Family kinases
Including: Src, Yes, Lyn, Fyn, Blk, Lck
pTyr in C-term interacts
with SH2 domain, locking
protein in an inactive
conformation
(CskàpY505 on lck)
Dephosphorylation of this
Tyr (CD45, SHP-1) leads to
conformational change that
allows for unfolding &
kinase activity
Myristoylation:
promotes membrane localization
Regulatory tyrosine

16. • Phosphorylation of the ITAM domains
creates binding sites for
other protein domains called SH2 (Src
Homology 2 domain)
• This enables recruitment of signaling
proteins to activated receptors
– Signaling proteins are brought
near the kinases and can in turn be
activated
– Or binding to a phosphotyrosine
may activate the proteins by altering
protein conformation (allosteric
activation)
– Syk, ZAP-70
• Proteins can be phosphorylated on
three classes of amino acids:
– tyrosines (receptors), serine/
threonine (downstream), or histidine

17. These kinases are ESSENTIAL for
immune responses. ZAP-70 defects
lead to Severe Combined
Immunodeficiency (autosomal
recessive).
Syk KO mice die after birth of severe
haemorrhaging. B-lineage cells cannot
form (lack of signaling from pre-BCR
complex, no clonal expansion or
maturation of pre-B cells).
Syk & ZAP-70
are key kinases in
ITAM cascades
TWO
Src homology 2 domains
www.nimr.mrc.ac.uk/.../thirty2/kinases/
Mouse embryos that lack the
cytoplasmic tyrosine kinase Syk develop
lethal hemorrhages at midgestation
www.mpi-muenster.mpg.de/nvz/kiefer.shtml

18. Mocsai, et al. Nature Reviews Immunology 10, 387-402
SHP-1
Cbl
ITAM/Syk Signaling Engages Many Downstream Effectors

19. Mocsai, et al. Nature Reviews Immunology 10, 387-402
Syk’s many pTyr sites control its activity and interactions,
both on and off of the ITAM-containing receptor

20. Where is this all going?
Nuclear Translocation of Transcription Factors
Figure 9-14 Abbas © 2005 Elsevier

21. Figure 9-14 Abbas © 2005 Elsevier
NFkB and NFAT are
activated in the cytosol
and translocate into the
nucleus to begin
transcriptional
activation.
AP-1 is composed of fos
and jun. Jun is
activated in the cytosol
and translocates. Fos
must be transcribed by
Elk, which is
translocated.

22. Receptor ligation
ITAM
Syk/ZAP70
Cell Type
Specific
Program
NFAT
NFkB
AP-1
activation
We will examine TCR signaling as a model to understand what lies in the
middle and how this sensitive immunoreceptor system gets triggered.
input
output

23. TCR signaling
• The biochemical signals that are triggered
in T cells by antigen recognition are
transduced not by the TCR itself but by
invariant proteins called CD3 and ζ which
are non-covalently linked to the antigen
receptor to form the TCR complex.
– Highly variable antigen receptor + invariant
signaling proteins
• What advantages does this give?

24. Peptide binding pocket
Variable region /
Complementrarity-determining
region (CDR)
Disulfide bond
Positively charged amino acids:
Lysine in α chain
Lysine + arginine in β chain
• Interact with neg residues in
CD3 and ζ
TCR Structure

25. Antigen affinity (Kd): 10-5 to 10-7 M (i.e., OT-I TCR 5.9 uM; Immunity. 1999;10:227–37.)
Low affinity = need for adhesion molecules
TCR Structure

26. The TCR complex

27. • Facilitate signaling
• Provide secondary signals
• Adhesion molecules
Accessory molecules
See also Abbas Figure 7-9B

28. Coreceptors: bind MHC molecules and enhance TCR signaling
• CD4+ respond to Class II MHC: cytokine-production helper cells, extracellular microbes
• CD8+ respond to Class I MHC: CTLs, eradicate intracellularly infected cells
• Signal transduction via Lck:
Interaction of CD4/CD8 with MHC brings CD4/CD8-associated Lck close to TCR
the complex, which then phosphorylates the ITAMs of CD3 and ζ

29. • Close proximity of CD4/CD8-
associatedLck activated ITAMs
• CD3 associated Fyn may
activate ITAM : TCR binding to MHC
may transduce conformational change
to activate Fyn
• Zap-70 is related to Syk in the BCR
system
More details…

30. Signaling pathways triggered by TCR activation

31. CosAmulatory
receptors:
recognize
molecules
on
APC
and
iniAate
second
signals
• Receptor tails have structural
motifs that serve as docking sites
for adaptor molecules (such as
PI-3 kinase or Grb2) once
tyrosines are phosphorylated
• Functional consequences are
different for each receptor
**Can be activating or inhibitory

32. Example of Costimulatory receptor function:
After binding ligands for TLRs, Dendritic cells will express B7

33. • TCR provides specificity
• Coreceptors enhance signaling by bringing Lck in close
proximity of ITAMs
• Costimulatory receptors initiate activating or inhibitory
signals and play a key role in the outcome of APC
interaction
Activation of T cells involves the integration of
signals from multiple receptors…

34. OrganizaAon
of
signaling
at
the
T-­‐APC
contact
On
the
scales
that
are
accessible
within
the
limits
of
conven&onal
fluorescence
imaging,
we
realize
that
the
T-­‐APC
contact
forms
an
organized
(but
very
dynamic!)
structure
termed
the
“immunological
synapse”.
Supramolecular
Ac&va&on
Complex
(SMAC)
Distal
(dSMAC)
Peripheral
(pSMAC)
Central
(cSMAC)
Many
important
intermolecular
and
intercellular
events
happen
in
the
SMAC.
We
will
start
at
the
molecular
level
and
build
our
way
up,
focusing
on
the
literature
on
T
cell
immunological
synapses
from
the
past
decade.
Huppa,
Davis.
Nature
Reviews
Immunology
3,
973-­‐983
(December
2003)

35. Molecular
&
Cellular
Interac&ons
Relevant
to
TCR
Triggering
• TCR-­‐CD3
complex
structure
• Models
of
TCR
ac&va&on
by
cognate
pMHC
• Biophysical
considera&ons
regarding
forma&on
of
ac&va&ng
T-­‐APC
interfaces
• Why
it
all
maeers

36. The TCR complex
Immunity
Volume
24,
Issue
2
2006
133
-­‐
139
Michael
S.
Kuhns
,
Mark
M.
Davis
,
K.
Christopher
Garcia
If you had to experimentally demonstrate that
this is the correct TCR complex structure, how
would you do it?
Call, et al, Cell. 2002 Dec 27;111(7):967-79.

37. Kuhns
&
Davis,
Cell.
2008
Nov
14;
135(4):
594–596;
Xu,
et
al.
Cell.
2008
Nov
14;135(4):702-­‐13.
Basic
residues
(+)
in
the
zeta
and
epsilon
tails
promote
membrane
associa&on
in
inac&ve
TCR-­‐CD3
complexes
by
binding
to
acidic
lipids
(e.g.,
phospha&dylserine).
Membrane
associa&on
buries
the
ITAM
tyrosines
in
the
membrane
and
denies
kinase
access,
preven&ng
ac&va&on
in
the
absence
of
pMHC.

38. P.
Anton
van
der
Merwe
&
Omer
Dushek
Nature
Reviews
Immunology
11,
47-­‐55
(January
2011)
Ini&a&on
of
TCR
signaling
is
likely
to
require
mechanical
forces
or
reorganiza&on
of
the
lipid
bilayer
to
relieve
the
associa&on
of
CD3
complex
ITAM
domains
with
the
membrane.
Mechanical
ac&va&on
Note
that
in
this
example,
the
force
is
applied
perpendicular
to
the
membrane
(piston-­‐like
mo&on).
More
recently
tangen&al
force
models
have
been
considered
(covered
later).
Lipid
reorganiza&on
Ca2+
influx
may
also
be
involved
in
amplifying
ITAM
signaling
by
locally
compe&ng
the
electrosta&c
interac&ons
between
CD3
tails
and
membrane.

39. Problem:
How
much
cognate
pMHC
does
it
take
to
sAmulate
a
T
cell,
parAcularly
if
the
pMHC
is
rare?
The
story
starts
in
Switzerland
in
the
mid
90s…

40. The
Serial
Triggering
Model
Observed
by
flow
cytometry:
• Number
of
pMHC
on
an
APC
• Number
of
internalized
TCR
(assumed
internalized
=
ac&vated)
àCalculated
#
TCR
ac&vated
per
pMHC
present
Determined:
Each
pMHC
must
ac&vate
and
cause
the
internaliza&on
of
up
to
200
TCR.
àThis
led
to
the
Serial
Triggering
model
where
one
pMHC
could
serially
ligate
and
ac&vate
many
different
TCRs.
Implies:
agonis&c
pMHC
needs
short
bound
life&me
to
engage
many
TCRs
during
the
course
of
a
T-­‐APC
encounter.
T
cells
scan
over
DCs
for
cognate
pMHC.
They
must
find
it
in
<10min
(50%
of
contacts
last
<
~2.5m)
if
they
are
going
to
stop
and
be
ac&vated.
Is
serial
triggering
realis8c
during
scanning
T-­‐APC
interac8ons?
Must
know:
What
is
the
life8me
of
an
agonis8c
TCR-­‐MHC
interac8on?
Celli,
et
al.
JEM
vol.
202
no.
9
1271-­‐1278
(Valituu
et
al.
Nature.
1995
May
11;375(6527):148-­‐51.)
Dura&on
of
T-­‐APC
encounters
With
cognate
pMHC
Without
cognate
pMHC

41. Stone,
et
al.
Immunology.
2009
Feb;
126(2):
165–176.
TCR-­‐pMHC
Bound
life&me
t1/2
range
from
less
than
1
s
to
~30
s
Solu&on
binding
data
for
various
TCR/pMHC
complexes
The
TCR-­‐pMHC
with
fastest
kine&cs
could
account
for
serial
triggering
leading
the
T
cell
to
stop,
but
many
agonis&c
pMHC
seem
too
slow.
For
instance,
a
pMHC
with
20
s
bound
life&mes
could
visit
<30
TCR
during
a
T-­‐APC
encounter,
not
hundreds.

42. Xie,
et
al.
Nature
Immunology
13,
674–680
(2012)
Is
serial
triggering
really
necessary?
Photocrosslinkable
pMHC
were
aeached
covalently
to
TCR
during
a
T-­‐APC
interac&on.
àThey
can’t
dissociate,
so
if
serial
triggering
is
required,
these
“pMHC
(XL)”
should
be
less
s&mulatory
than
their
standard,
non-­‐crosslinked
counterparts,
“pMHC
(Std)”.
Result:
pMHC
(XL)
is
more
s8mulatory
than
pMHC
(Std).
• More
prolonged
Ca2+
flux
• Greater
IL-­‐2
secre&on
While
serial
triggering
may
happen
to
some
extent,
it
does
not
appear
to
be
so
essen&al
as
originally
thought.

43. Are
there
any
thermodynamic
or
kineAc
parameters
derived
from
soluAon
measurements
of
TCR-­‐pMHC
binding
that
correlate
with
sAmulatory
potency?
• When
it
became
generally
feasible
to
measure
solu&on
binding
kine&cs
of
TCR-­‐
pMHC
with
commercially
available
SPR
instruments,
there
was
much
interest
correla&ng
affinity,
kon
and
koff
of
pMHC
for
TCR
with
biological
ac&vity
• The
affinity
(in
solu&on
binding
measurements)
of
TCR
for
agonist
pMHC
is
rela&vely
weak,
typically
in
the
Kd=1-­‐100uM
range.
Mod.
From:
Stone,
et
al.
Immunology.
2009
Feb;
126(2):
165–176.
Bound
state
life&me
There
were
correla&ons
between
binding
affinity
and
kine&cs
in
some
limited
systems
but
overall
there
was
no
universally
strong
correla&on
between
solu&on
binding
parameters
and
ac&vity.
Agonist,
weak
agonist
and
antagonist
pMHC
can
have
very
similar
solu&on
binding
affinity
and
rate
constants

44. More
recently,
single
molecule
imaging
invesAgaAon
has
pushed
the
limit
of
resoluAon
to
allow
tesAng
triggering
capability
of
ever
smaller
numbers
pMHC
Manz,
et
al.
(groves
lab)
PNAS
May
31,
2011
vol.
108
no.
22
9089-­‐9094
Circles
and
triangles:
T
cells
with
two
different
TCRs
Titrated
bio&nylated
MCC-­‐pMHC
and
measured
number
pMHC
at
synapse
(x
axis)
Recorded
Ca2+
flux
in
T
cells
Irvine,
et
al
(Davis
Lab)
Nature
419,
845-­‐849(24
October
2002)
Ca2+
fluxes
can
be
triggered
by
<10
cognate
pMHC
in
an
immune
synapse
Using
a
microfabricated
ar&ficial
bilayer
that
constrained
the
number
of
pMHC
that
TCRs
could
see
to
only
a
few,
Ca2+
flux
in
the
T
cell
required
very
small
numbers
of
pMHC

45. How
does
the
TCR
know
to
acAvate
signaling
in
response
to
very
low
levels
of
pMHC?
Pseudodimer
model:
dimers
of
congnate
and
non-­‐congate
pep&de
pMHC
promote
signaling
Molecular
MechanotransducAon:
pMHC
applies
a
torque
to
TCRαβ,
which
is
transmieed
to
CD3εγ
and
CD3εδ
Dwell
Time:
What
determines
if
a
pMHC-­‐TCR
interac&on
is
s&mulatory
is
how
long
the
pMHC
“dwells”
near
a
single
TCR,
repe&&vely
binding
it.
We’ll
look
at
three
mechanis&c
models
of
TCR
s&mula&on
arising
from
the
recent
literature.
Note
that
these
models
are
not
mutually
exclusive
alterna&ves
but
may
be
looking
at
different
aspects
of
the
same
process
as
different
research
groups
look
at
TCR
triggering
from
diverse
backgrounds
and
perspec&ves.

46. How
does
the
TCR
know
to
ac8vate
signaling
in
response
to
very
low
levels
of
pMHC?
Pseudodimer
model:
dimers
of
cognate
and
non-­‐cognate
pep&de
pMHC
promote
signaling
P.
Anton
van
der
Merwe
&
Omer
Dushek
Nature
Reviews
Immunology
11,
47-­‐55
(2011)
CD4
contacts
an&genic
pMHC
but
also
brings
lck
into
proximity
to
TCR
interac&ng
with
dimerized
“self”
pMHC,
ac&va&ng
both
TCRs’
signaling
Cohran
et
al.
Immunity.
2000
Mar;12(3):241-­‐50.
Soluble
single
chain
MHC
have
been
engineered
and
loaded
with
defined
pep&des
that
are
chemically
x-­‐linked
to
the
MHC
monomeric
>=dimeric
Monomeric
pMHC
bind
TCR
but
are
not
s&mulatory,
but
>=dimer
pMHC
is
s&mulatory.
à
Suggests
that
single
pMHC
are
not
s&mulatory
and
you
have
to
at
least
present
a
dimer
of
pMHC
(but
this
is
soluble
pMHC,
not
in
a
T-­‐APC
contact)

47. How
does
the
TCR
know
to
ac8vate
signaling
in
response
to
very
low
levels
of
pMHC?
Pseudodimer
model:
dimers
of
cognate
and
non-­‐cognate
pep&de
pMHC
promote
signaling
PepAdes
to
be
presented
to
5C.C7
(MCC
reacAve)
T
cells
Krogsgaard
et
al.
Nature
434,
238-­‐243(10
March
2005)
Standard
agonis&c
pep&de
Single
AA
subs&tu&ons
(altered
K5)
ER
chaperone
that
binds
this
MHC
prior
to
an&gen
pep&de
loading
Self
pep&des
that
bind
this
MHC
(Ca2+
flux)
dimeric
pMHC
• K5
monomer
is
not
s&mulatory,
but
K5-­‐K5
is.
• Some
of
the
altered
K5
and
self
pMHC
(those
that
are
recruited
to
T-­‐APC
contacts)
can
support
signaling
from
heterodimers
with
K5.
• These
self
pMHC
are
not
agonis&c
when
homodimerized.
• Some
altered
K5
and
self
pMHC
can’t
support
signaling
with
K5
(i.e.,
b2m).

48. How
does
the
TCR
know
to
ac8vate
signaling
in
response
to
very
low
levels
of
pMHC?
Molecular
MechanotransducAon:
pMHC
applies
a
torque
to
TCRab,
which
is
transmieed
to
CD3εγ
and
CD3εδ
N-­‐glycans
top
side
FG
loop
of
b
chain
TCRab
rises
above
shorter,
more
rigid
CD3
dimers
FG
loop
of
TCRβ
C
domain
is
important
for
TCR
ac&va&on,
is
well
structured
and
approaches
CD3εγ
Op&cal
traps
are
used
to
apply
force
through
bead-­‐bound
ligands
to
the
TCR.
Forces
applied
are
in
the
10s
of
pN
range,
typical
of
the
forces
applied
at
cell-­‐cell
and
cell-­‐
substrate
interfaces
Kim
et
al.
Front.
Immunol.,
18
April
2012
|
doi:
10.3389/fimmu.2012.00076
Wang
et
al.
Immunol
Rev.
2012
Nov;
250(1):
102–119.

49. How
does
the
TCR
know
to
ac&vate
signaling
in
response
to
very
low
levels
of
pMHC?
Molecular
MechanotransducAon:
pMHC
applies
a
torque
to
TCRab,
which
is
transmieed
to
CD3εγ
and
CD3εδ
Tangen&al
force
applied
through
a
trapped
ligand-­‐bead,
measured
Ca2+
flux
17A2=Non-­‐agonis8c
an8-­‐CD3
With
force
Without
force
Ligand
on
bead:
non-­‐agonis&c
mAb
pMHC
Kim
et
al.
J
Biol
Chem.
2009
Nov
6;284(45):31028-­‐37.
For
mAb
and
pMHC,
you
only
see
Ca2+
signal
if
you
pull
tangen&ally
on
the
bead-­‐ligand
Model:
• TCR-­‐pMHC
bonds
form
• T
cell
translates
rela&ve
to
APC
• Tangen&al
force
on
TCR
complex
via
pMHC
(yellow)
• TCR
Cβ
(blue)
pivots
on
its
TM
domain
• FG
loop
(magenta)
pushes
down
on
CD3
dimers
• Presumably
causes
changes
in
CD3
tails
that
promote
their
ac&va&on
Ca2+
flux

50. How
does
the
TCR
know
to
ac8vate
signaling
in
response
to
very
low
levels
of
pMHC?
Dwell
Time:
What
determines
if
a
pMHC-­‐TCR
interac&on
is
s&mulatory
is
how
long
the
pMHC
“dwells”
near
a
single
TCR,
repe&&vely
binding
it.
• This
concept
is
different
from
serial
triggering
(one
pMHC
binds
and
ac&vates
many
different
TCR
in
series).
• Dwell
&me
considers
the
&me
that
a
pMHC
spends
bound
to
a
single
TCR,
dissociates,
but
then
rebinds
that
TCR
before
diffusing
away.
• In
this
model,
a
strongly
agonis&c
pMHC
would
be
able
to
dwell
for
a
long
&me,
thus
integra&ng
a
lot
of
signaling
through
the
bound
TCR.
Dwell
&me
depends
on:
• TCR/pMHC
binding
kine&cs
(determines
half-­‐life
of
bound
complex)
AND
• Diffusion
of
TCR
and
MHC
in
the
membrane
(determines
how
likely
it
is
that
TCR
and
MHC
will
move
too
far
apart
to
bind
before
rebinding
can
occur)
Govern
&
Chakraborty.
Immunity.
2010
Feb
26;32(2):141-­‐2.

51. How
does
the
TCR
know
to
ac8vate
signaling
in
response
to
very
low
levels
of
pMHC?
Dwell
Time:
What
determines
if
a
pMHC-­‐TCR
interac&on
is
s&mulatory
is
how
long
the
pMHC
“dwells”
near
a
single
TCR,
repe&&vely
binding
it.
• In
solu&on,
you
would
never
see
TCR
dwelling
on
pMHC
because
solu&on
diffusion
is
much
too
fast.
Typically
approx
tens
to
100
um2/s
for
proteins.
• In
a
membrane,
transmembrane
protein
diffusion
is
much
slower
(and
constrained
to
the
2D
bilayer).
Typical
protein
diffusion
in
membranes
is
~0.1
um^2/s.
Known:
kon
and
diffusion
coefficients
Based
on
a
model
of
binding+diffusion
in
a
membrane,
calculated
a
predicted
average
number
of
rebindings.
Note
that
the
predicted
number
of
rebindings
is
rela&vely
small
Govern
et
al
used
two
different
TCRs
and
a
variety
of
pMHC
with
known
affini&es
and
binding
rate
constants
for
those
TCRs
as
well
as
known
ability
to
s&mulate
T
cell
prolifera&on
and
cytokine
produc&on.
Govern
et
al.
Proc
Natl
Acad
Sci
U
S
A.
2010
May
11;107(19):8724-­‐9.

52. How
does
the
TCR
know
to
ac8vate
signaling
in
response
to
very
low
levels
of
pMHC?
Dwell
Time:
What
determines
if
a
pMHC-­‐TCR
interac&on
is
s&mulatory
is
how
long
the
pMHC
“dwells”
near
a
single
TCR,
repe&&vely
binding
it.
Govern
et
al.
Proc
Natl
Acad
Sci
U
S
A.
2010
May
11;107(19):8724-­‐9.
They
knew
the
kon
and
diffusion
coefficients
of
the
TCR
and
pMHC
used.
From
this
they
could
use
a
model
of
binding/diffusion
in
a
2D
membrane
to
calculate
the
dwell
&me
of
each
pMHC/TCR
(
“ta”).
ta
for
TCR/pMHCs
was
the
best
predictor
of
T
cell
prolifera)ve
response.
EC50
(uM)
For
prolifera&on
pMHC
agonis&c
strength:
Strongest
Moderate
weakest

53. Dwell
&me
theory
of
TCR-­‐pMHC
interac&ons
• Depends
on
– Forward
kine&c
rate
constant
for
binding
– Diffusion
of
TCR
and
pMHC

54. Dwell
Time
Modeling:
Goals
• How
does
the
membrane
diffusion
environment
influence
TCR-­‐pMHC
interac&on
dynamics?
– Does
changing
D
(diffusion
coefficient)
from
solu&on
values
(100
um^2/s)
to
membrane
values
(0.1
um^2/
s)
impact
molecular
dwell
&mes
• How
does
changing
the
forward
rate
constant
for
binding
influence
TCR-­‐pMHC
interac&on
dynamics?
– Does
changing
pON
(probability
of
binding
in
one
model
step)
from
lowàmoderateàhigh
values
impact
molecular
dwell
&mes

55. How
the
model
sets
up
and
runs
Ini&alize
and
execute
model
Control
how
binding
and
diffusion
works
Quan&ta&ve
outputs
about
binding
Size
and
&me
informa&on
about
model
you’ve
run
Free
TCR
(red
circle)
Bound
TCR
(yellow
circle)
Free
pMHC
(green
square)
Agent
based
model
of
TCR
and
pMHC
binding
in
apposed
membranes

56. Run
the
Netlogo
model
of
TCR-­‐pMHC
interac&on
with
• D=0.1,
100
um^2/s
• Low,
moderate
and
high
probability
of
binding
RESULTS

57. Triplicate
runs
pOFF=0.01
in
all
250s
dura&on,
10ms
&me
resolu&on
10nm
binding
radius
100
TCRs
and
pMHCs
in
~15
um^2
membrane
D
Diffusion
Coefficient
(um^2/s)
Membrane
Solu&on

58. Now
that
we’ve
considered
the
state
of
knowledge
regarding
molecular
TCR-­‐pMHC
interacAons,
let’s
think
about
the
cell-­‐cell
interface
environment
in
which
these
interacAons
occur…
What
are
some
important
features
of
this
environment
that
bear
on
signaling
processes?
Achieving
close
cellular
apposiAon
Micro/nanoscale
organizaAon
of
signaling
components
Mechanics
of
the
Immune
Synapse

59. Achieving
close
cellular
apposiAon.
What
are
the
challenges?
Huppa,
Davis.
Nature
Reviews
Immunology
3,
973-­‐983
(December
2003)
Molecular
InteracAons
at
the
T-­‐APC
immunological
synapse
This
figure
helps
to
summarize
cell
biological
informa&on,
and
to
be
fair,
that
was
all
it
was
intended
to
do
in
the
review
cited
below.
But
from
the
standpoint
of
physical
interac&on
between
two
cells,
it
has
some
major
omissions
and
inaccurate
representa&ons.
What
aspects
of
the
immune
cell
interface
are
not
represented
or
not
accurately
represented
here?

60. Achieving
close
cellular
apposiAon.
What
are
the
challenges?
Casasnovas
et
al.
Proc
Natl
Acad
Sci
U
S
A.
1998;95(8):4134-­‐9.
Yin
et
al.
Proc
Natl
Acad
Sci
U
S
A.
2012
Apr
3;109(14):5405-­‐10.
LFA-­‐1
ICAM-­‐1
dimer
There
is
no
glycocalyx
illustrated
on
either
cell,
but
this
is
an
important
repulsive
barrier
that
resists
apposing
two
cells
closer
than
~100nm.
40
nm
T-­‐APC
adhesion
via
LFA-­‐1/ICAM-­‐1
is
important
for
stabilizing
the
cell-­‐cell
interface,
but
this
would
space
the
cell
membranes
~40nm
apart
The
TCR-­‐pMHC
complex
is
only
~15nm,
so
engagement
of
TCR
will
require
membranes
to
be
pushed
into
~15nm
separa&on.

61. Achieving
close
cellular
apposiAon.
What
is
the
solu8on?
T
cell
invadopod
like
protrusions
(ILP)
Sage
et
al.
J
Immunol.
2012
Apr
15;188(8):3686-­‐99.
Top:
T
cells
siung
on
an
Ag-­‐pulsed
APC
probe
the
APC
with
ILPs
even
before
Ca2+
flux.
Red
arrows
show
sites
where
membrane
targeted
fluorescent
protein
in
the
T
cell
is
pushed
into
the
APC
membrane.
Le€:
TEM
image
shows
the
interdigita&on
of
the
synapse
between
APC
and
T
cell,
including
sites
of
apparent
T
cell
ILP
ac&vity
(red
arrows).
ILPs
are
ac&n
dependent
and
are
thought
to
push
the
T
cell
and
APC
membranes
close
enough
to
engage
TCR-­‐pMHC
binding.

62. Achieving
close
cellular
apposiAon.
What
is
the
solu8on?
Kine&c
segrega&on.
Sequen8al
engagement
of
different
sized
receptors
accompanied
by
reorganiza8on
of
their
membrane
distribu8ons.
ICAM-­‐1
LFA-­‐1
T
APC
TCR
pMHC
CD45
James,
vale.
Nature.
2012
Jul
5;487(7405):64-­‐9.
ICAM-­‐1
LFA-­‐1
T
APC
TCR
pMHC
CD45
During
ini&al
T-­‐APC
adhesion,
large
ICAM-­‐1/LFA-­‐1
interac&ons
predominate.
TCR
can’t
be
engaged
at
this
distance.
Large
nega&ve
regulatory
phosphatases
are
allowed
in
the
contact.
ILPs
form
small
close
contacts
where
TCR-­‐pMHC
can
engage.
Adhesive
interac&ons
between
CD2
and
CD58
can
stabilize
these
close
contacts
(CD2/
CD58
is
about
the
same
intermembrane
length
as
TCR/
MHC).
As
close
contacts
grow,
the
energy
cost
of
bending
membranes
is
balanced
by
mul&ple
binding
interac&ons
to
stabilize
and
enlarge
areas
of
TCR
engagement.
Large
ectodomain
proteins
are
excluded
(e.g.,
CD45)

63. Micro/nanoscale
organizaAon
of
signaling.
Recruitment
of
LAT
to
ac&vated
TCR
complexes
is
a
key
early
event
in
TCR
signaling.
How
do
we
prevent
LAT
from
prematurely
assembling
at
TCR
complexes?
Methods: fluorescence super resolution imaging and TEM with gold beads on membrane sheets and
whole cells.
Both methods are in good agreement, so I’m showing just the TEM immunogold.
• TCR complex has been shown to form nanoclusters in resting T cells.
• Lat forms similarly sized clusters.
• The size of both clusters is in the 40-300nm range.
• Estimated 5-20 TCR per cluster
Small
CD3ζ
Large
Lat
Non-­‐ac&vated
T
cell
membrane
ac&vated
T
cell
membrane
Lillemeier,
et
al.
Nat
Immunol.
2010
Jan;11(1):90-­‐6.

64. Micro/nanoscale
organizaAon
of
signaling.
Top:
Ripley’s
func&ons
and
their
deriva&ves
are
a
way
to
show
that
objects
(i.e.,
small
and
large
immunogold
beads)
are
clustered
on
certain
length
scales.
• The
peak
of
the
Ripley’s
L(r)-­‐r
curve
is
the
length
scale
of
greatest
clustering.
• Ac&va&on
on
cognate
pMHC
surfaces
increases
the
amount
and
length
scale
of
clustering
for
CD3
and
Lat.
Boeom:
Bivariate
Ripley’s
sta&s&cs
show
the
probability
that
CD3
and
Lat
are
clustered
together
at
different
length
scales.
• The
shaded
areas
represent
the
regions
of
the
plot
where
the
observed
distribu&on
of
CD3
and
Lat
are
not
dis&nguishable
from
random
with
99%
confidence.
• If
the
lines
go
outside
of
these
regions,
then
the
paeern
colocaliza&on
of
CD3
and
Lat
shows
evidence
of
clustering
(above
the
shaded
area)
or
segrega&on
(below
the
shaded
area).
• On
a
non-­‐ac&va&ng
surface,
Lat
clusters
are
segregated
from
CD3.
• On
an
ac&va&ng
surface,
they
become
co-­‐aggregated

65. Micro/nanoscale
organizaAon
of
signaling.
Complimentary
evidence
of
Lat
and
TCR
complex
co-­‐
aggrega&on
a€er
ac&va&on
came
from
fluorescence
cross-­‐correla&on
spectroscopy
(FCCS)
measurements.
Orange
lines
show
the
likelihood
of
observing
co-­‐
diffusion
of
Lat
and
CD3ζ
in
living
cell
membranes.
• CD3/Lat
co-­‐diffusion
is
not
seen
in
T
cells
on
a
non-­‐
ac&va&ng
ar&ficial
membrane.
• Placing
agonis&c
ligand
on
the
bilayer
markedly
increases
the
co-­‐diffusion
of
TCR
complex
and
Lat.
Therefore,
several
lines
of
evidence
show
that:
1. TCR
and
the
key
signaling
adaptor
Lat
are
preclustered
in
T
cell
membranes
on
tens
to
hundreds
of
nanometers
scales
.
2. Prior
to
s&mula&on,
Lat
and
TCR
complex
are
segregated
from
one
another
in
separate
nanostructures.
3. When
a
cognate
pMHC
is
seen,
Lat
becomes
co-­‐aggregated
with
TCR
nanoclusters.

66. Mechanics
of
the
immune
synapse.
So,
TCR
organizes
into
clusters
when
ac&vated.
What
happens
to
TCR
clusters
in
the
immunological
synapse?
cSMAC
pSMAC
dSMAC
TCR
TCR
The
cSMAC
is
rich
in
TCR,
while
CD45
and
other
nega&ve
regulatory
phosphatases
tend
to
be
in
the
dSMAC.
This
ini&ally
led
to
the
idea
that
TCR
signaling
was
occurring
in
the
cSMAC.
CD45
Subsequent
imaging
work
at
high
spa&al
and
temporal
resolu&on
clarified
that
signaling
from
the
TCR
actually
happens
as
ac&vated
TCR
forms
into
microclusters,
largely
in
the
pSMAC,
during
the
ini&al
spreading
phase
of
synapse
forma&on.
In
this
phase
TCR
is
sampling
for
an&gen
and
forming
microclusters.
These
TCR
microclusters
then
move
laterally
into
the
cSMAC.
This
happens
when
the
synapse
stops
spreading
and
develops
a
strongly
contrac&le
pSMAC.
In
this
phase,
TCR
is
signaling,
causing
the
cell
to
stop
and
integrate
signal
and
eventually
being
downregulated.
We
will
consider:
How
do
TCR
microclusters
move?
What
actually
happens
in
the
pSMAC?

67. How
do
TCR
microclusters
move?
An
actomyosin
flow
develops
in
the
pSMAC.
This
is
similar
to
the
“tractor”
that
forms
at
the
leading
edge
of
migra&ng
cells
and
moves
them
forward.
F-­‐ac&n
forms
into
bundles
at
the
edge
of
the
synapse
and
myosin
IIA
mediated
contrac&on
causes
the
F-­‐ac&n
to
contract
inward
toward
the
cSMAC.
Recruitment
of
GEFs
to
the
synapse
is
important
for
upregula&ng
Rac
and
Cdc42
GTPase
dependent
ac&n
nuclea&on
(e.g.,
via
Arp2/3
complex).
Rho
GTPase
regulates
myosin
IIA
mediated
contrac&on
in
the
pSMAC.
Babich
et
al.
2012
//
JCB
vol.
197
no.
6
775-­‐787
Kumari
et
al.
Biochim
Biophys
Acta
2014
Feb;1838(2):546-­‐56.
Mechanics
of
the
immune
synapse.

68. How
do
TCR
microclusters
move?
Ar&ficial
membranes
have
been
made
with
underlying
metal
barriers.
When
transmembrane
proteins
(i.e.,
TCR
complex)
encounter
the
barrier,
they
must
go
around,
not
through.
Examples
of
TCR
microcluster
trajectories
as
they
move
toward
the
cSMAC
and
encounter
barriers
show
that
the
TCR
gets
hung
up
on
the
barriers
and
must
slide
along
the
barriers
to
con&nue
centripetal
mo&on.
In
contrast,
actomyosin
mo&on
is
beneath
the
membrane
and
thus
not
effected
by
the
barriers.
Ac&n
flows
past
the
barriers.
This
suggests
“fric&onal
coupling”
of
the
actomyosin
flow
to
the
TCR
complex
as
a
mechanism
of
TCR
cluster
mobility.
The
exact
mechanism
of
coupling
is
currently
unknown.
Mechanics
of
the
immune
synapse.
Demond
et
al.
Biophys
J.
2008
Apr
15;94(8):3286-­‐92.

69. Do
moving
TCR
clusters
transduce
force
as
part
of
their
signaling?
Mechanics
of
the
immune
synapse.
TCR-­‐pMHCs
being
dragged
from
pSMAC
to
cSMAC
by
actomyosin
flow
will
experience
force
in
the
piconewton
range.
T
cells
respond
with
greater
TCR
signaling
when
presented
with
an&-­‐CD3
on
s&ff
surfaces
rela&ve
to
so€er
surfaces,
which
may
relate
to
the
amount
of
force
experienced
by
the
TCR.
Huppa,
etal.
Nature.
2010
Feb
18;463(7283):963-­‐7.
Time
(sec)
of
TCR-­‐agonist
pMHC
interacAon
Huppa
et
al
used
a
FRET
probe
between
a
pMHC
and
an&-­‐
TCR
monovalent
an&body.
FRET
signal
was
only
observed
when
the
TCR-­‐pMHC
complex
was
bound,
allowing
measurements
of
TCR-­‐pMHC
binding
&mes.
Found
that
synap&c
TCR-­‐pMHC
binding
&mes
are
3-­‐12
&mes
shorter
than
the
solu&on
value.
F-­‐ac&n
inhibitors
(prevents
actomyosin
transport
of
TCR
clusters)
increased
TCR-­‐pMHC
binding
&mes.
Suggested
that
the
force
applied
to
TCRs
in
the
synapse
as
they
are
dragged
by
actomyosin
flow
can
shorten
their
half
&me
of
binding
to
pMHC.

70. What
is
the
mechanical
nature
of
the
TCR-­‐agonist
pMHC
bond?
Mechanics
of
the
immune
synapse.
Depoil
and
dus&n.
Trends
Immunol.
2014
Nov
17;35(12):597-­‐603.
Liu
et
al.
Cell.
2014
Apr
10;157(2):357-­‐68.
OT1
Types
of
bonds
• Slip
bonds
are
linearly
more
likely
to
break
(shorter
life&me)
with
increasing
force.
• Catch-­‐slip
bonds
become
stronger
(longer
life&me)
with
increasing
force,
but
only
up
to
their
rupture
force
(then
they
become
slip
bonds).
Biomolecular
Force
Probe
(BFP)
• A
rigid
bead
is
coated
with
a
low
density
of
pMHC
such
that
only
a
single
pMHC
will
contact
a
T
cell
when
the
bead
touches
it.
• The
bead
is
aeached
to
an
RBC.
• The
RBC/bead
is
repeatedly
brought
up
to
the
T
cell
membrane,
then
retracted.
• The
force
applied
to
the
TCR
can
be
calculated
from
the
RBCs
membrane
deforma&on
during
retrac&on.

71. What
is
the
mechanical
nature
of
the
TCR-­‐agonist
pMHC
bond?
Mechanics
of
the
immune
synapse.
Liu
et
al.
Cell.
2014
Apr
10;157(2):357-­‐68.
Antagonist
pMHC
Agonis&c
pMHC
Agonist
pMHC
strength
Le€:
Agonis&c
pMHC
consistently
display
catch-­‐slip
bonding
with
TCR.
The
rupture
force
is
correlated
with
the
agonis&c
“strength”
of
the
pep&de.
Right:
Antagonis&c
pep&des
form
slip
bonds
with
TCR.
T
cells
integrate
the
total
&me
single
TCRs
are
subjected
to
catch
bonded
pulling
force
(10
pN,
OVA
pep&de).
Strong
T
cell
Ca2+
fluxes
require
integra&on
of
10
s
of
catch
bonded
&me
per
minute
of
total
BFP
s&mula&on.

72. Why
does
it
all
ma]er?
Mechanis&c
Underpinning
for
T
cell
Sensi&vity
These
studies
help
us
to
understand
the
physical
parameters
that
define
whether
a
pMHC
will
be
s&mulatory
or
not.
They
also
help
us
to
understand
the
exquisite
sensi&vity
of
T
cells
for
low
densi&es
of
agonist
pMHC.
Sensi&vity
is
important
because
professional
APCs
like
DCs
might
not
express
large
densi&es
of
a
par&cular
cognate
pMHC
for
a
T
cell.
Also,
a
scanning
T
cell/APC
interac&on
in
the
lymph
node
is
rather
short
lived
(~minutes),
so
the
T
cell
needs
to
be
able
to
signal
and
stop
migra&ng
if
even
a
small
amount
of
cognate
pMHC
is
found.
Possible
Mechanism
of
Quan&ta&ve
T
cell
Help
for
B
cell
Matura&on.
Review
re:
role
of
IS
in
T
cell
help
for
B
cells-­‐-­‐Dus&n.
Mol
Cell.
2014
Apr
24;54(2):255-­‐62.
B
T
Ag
Y
Y
AnAgen
gathering
Efficiency
α
BCR
affinity
AnAgen
presentaAon
#
pMHC
α
BCR
affinity
TCR
signals,
acAvaAon
#
CD40L
α
#
pMHC
seen
T
B
T
cell
help
#
CD40L
α
B
cell
prolif
A
2014
ar&cle
suggested
that
exocytosis
of
TCR
in
the
cSMAC
may
contribute
to
long-­‐term
regula&on
of
B
cell
responses
to
T
cell
help
las&ng
beyond
the
&me
frame
of
direct
T-­‐B
interac&ons.

73. Possible
Mechanism
of
Quan&ta&ve
T
cell
Help
for
B
cell
Matura&on.
Choudhuri
et
al.
Nature.
2014
Mar
6;507(7490):118-­‐23.
TCR
F-­‐acAn
3um
TCR
int.
overlay
TEM
500nm
Vesicles
T
cell
PM
Fluorescence-­‐TEM
correla&ve
study:
TCR
intensity
is
found
where
cSMAC
microvesicles
are
seen
Tg
TCR
T
cell
on
ar&ficial
membrane
with
cognate
pMHC
and
ICAM-­‐1.
Microvesicles
are
present
outside
the
T
cell
near
the
cSMAC
loca&on.
Microvesicles
are
produced
by
ESCRT-­‐dependent
exocytosis
(similar
to
HIV
budding).
B
cells
expressing
cognate
pMHC
internalize
Tg
TCR
at
immunological
synapses
and
ac&vated
PLCg
is
found
in
proximity
B
cell
captured
TCR.
Purified
microvesicles
display
total
TCR
in
propor&on
to
the
density
of
cognate
pMHC
used
to
ac&vate
the
T
cell.
Germinal
Center
Light
Zone
Dark
Zone
T
B
Hypothe)cal
Model

74. 2.
Cytokine
Receptor,
Notch
and
Innate
Immunoreceptor
Signaling.
Regula&on
of
signal
dynamics
BIOM
514,
Cell
Signaling
Apr.
3,
2015
Aaron
Neumann,
Ph.D.
(Pathology)
CRF327,
akneumann@salud.unm.edu
Abbas (7th ed.) Chapter 7,9, parts of 11

75. OVERVIEW
• Cytokine
Receptors—Jak/STAT
signaling
• Notch
Signaling
• NFkB—a
central
immune
response
transcrip&on
factor
• Innate
Immunoreceptor
Signaling:
Fc
Receptors,
Toll
like
Receptors,
C
type
lec&n
receptors
• ITIM
antagonism
of
ITAM
signaling
• Dynamical
considera&ons
in
signaling

76. Cytokine receptors

77. JAK/STAT Pathway
Type I and II cytokine receptors
• Cytoplasmic tail contains tyrosine
• Jak is attached to receptor tail
• Clustering of receptors leads to Jak-
mediated phosphorylation of the
receptor tail
• STAT can now bind and be activated
• STATs dimerize (homo- or
heterodimerization) and translocate to
the nucleus where they stimulate
transcription, changing gene expression
• Cellular response is different depending
on the combination of receptor, Jak and
STAT activated

78. Radtke,
Immunity
Volume
32,
Issue
1
2010
14
-­‐
27
Notch signaling impacts lymphocyte development and activation
How
Notch
signaling
works:
1. Export
of
Notch
to
the
plasma
membrane
• Furin-­‐like
protease
cleavage
• Glycosyla&on
by
the
fringe
glycosyltransferases
2. Binding
ligand
on
opposing
cell
membrane
• Jagged
• Delta
like
ligands
(Dll)
3. Cleavage
at
the
plasma
membrane
• ADAM
(A
Disintegrin
And
Metalloprotease)
• γ-­‐secretase
4. Release
of
Notch
intracellular
domain
into
the
cytoplasm
5. Nuclear
Transloca&on
6. Binds
to
CSL
transcrip&on
factor
and
recruits
coac&vatoràgene
transcrip&on
7. Proteosomal
degrada&on
1
2
3
4
5
6
7

79. Radtke,
Immunity
Volume
32,
Issue
1
2010
14
-­‐
27
Notch signaling impacts lymphocyte development and activation
Notch1
signaling
in
T
cell
development
• Maintains
a
stem
cell
popula&on
(HSC)
that
seeds
the
thymus
• Suppresses
B
lineage
development
in
DN
stage
• Supports
prolifera&on
at
the
DN4àDP
transi&on
due
to
Notch
signaling
that
supports
prolifera&on
and
survival.
Notch
in
peripheral
T
cells
• Expression
increases
and
Notch
intracellular
domain
is
seen
a€er
TCR
ac&va&on
• Occurs
even
without
APC
sources
of
Notch
ligand
• T
cells
don’t
seem
to
express
much
Notch
ligand
How
is
Notch
ac8va8on
happening??
• Jagged-­‐Notch
may
drive
Th2
differen&a&on

80. Radtke,
Immunity
Volume
32,
Issue
1
2010
14
-­‐
27
Notch signaling impacts lymphocyte development and activation
Impact
of
Notch
Signaling:
Regulates
lineage
commitment
during
development
through
transcrip&on
factor
ac&va&on
Drives
signaling
that
Promotes
• Prolifera&on
• Metabolism
• Survival
Using
pathways
that
influence
• Apoptosis
(p53,
PI3K/Akt)
• Cell
Cycling
(p27Kip1)
• Metabolism
(mTOR)

81. NF-κB Pathway
TNR/IL-1R/TLR

82. The history of Toll-like receptors — redefining innate immunity
Luke A. J. O'Neill,
Douglas Golenbock
& Andrew G. Bowie
Nature Reviews Immunology 13, 453–460 (2013) doi:10.1038/nri3446
TLRs & signaling
(Abbas Ch4)
MyD88, TRIF
ê
TRAFs
ê
Trxn factor
(NFkB, IRFs)

83. Nature Reviews | Immunology
Dectin 2
ITAM
HDM allergens
Malassezia sp.
CARD9
BCL-10 MALT1
IKK
IKK IKK
I B
p50p65
P
Proteasome
Nucleus
Cytoplasm
Fungal
hyphae
FcR
P
P
SYK
P
P
SYK
Mincle
Myeloid DC
Cysteinyl
leukotrienes
?
?
_ _+ _ _ +
p50p65
Tnf and Il6
FcRγ results in NF-κB activation10,58
, although a role
for the SYK-CARD9–BCL-10–MALT1 complex has
not yet been confirmed. Thus, dectin 2 triggering alone
might induce an adaptive immune response, which is
supported by data showing that dectin 2 recognition of
fungal hyphae from C. albicans, Trichophyton rubrum
and Microsporum audouinii leads to TLR-independent
production of the pro-inflammatory cytokines TNF and
IL-6 (REF. 10). Furthermore, dectin 2 recognition of house
dust mite allergens activates SYK through FcRγ to gen-
erate cysteinyl leukotrienes, an important mediator of
allergic inflammation in the lungs58
. Notably, recogni-
tion of Histoplasma capsulatum β-glucans by dectin 1
also leads to the production of leukotrienes59
, suggesting
that a common SYK-dependent pathway is involved in
leukotriene synthesis after CLR triggering.
The related CLR mincle also pairs with FcRγ and
induces gene transcription through the SYK–CARD9–
BCL-10–MALT1 complex12
. In macrophages, recogni-
tion of dead cells by mincle through the endogenous
ligand SAP130 (SIN3A-associated protein, 130 kDa)
mediates CXC-chemokine ligand 2 (CXCL2) and TNF
production in a SYK- and CARD9-dependent manner,
which induces neutrophils to migrate into damaged tis-
sues12
. Mincle also interacts with α-mannosyl PAMPs
expressed by the pathogenic fungus Malassezia spp. and
induces gene transcription and TNF production without
the involvement of TLRs, further suggesting that mincle,
similarly to dectin 2, couples FcRγ-signalling to NF-κB
activation18
. The similarities of mincle downstream sig-
nalling with the dectin 1 pathway suggest that both CLRs
couple SYK activation to NF-κB activation through the
CARD9–BCL-10–MALT1 complex.
In contrast to dectin 2 and mincle, BDCA2 does not
induce TLR-independent cytokine production even
though it also pairs with FcRγ. The unusual signal-
ling pathway induced by BDCA2 might be because it is
expressed only by pDCs, whereas dectin 2 and mincle
are expressed by myeloid-derived antigen presenting
cells (TABLE 1). A recent study has shown that lymphoid
and myeloid cells have differential requirements for
CARD proteins in BCL-10-mediated NF-κB activation32
,
which might explain why dectin 2 and mincle couple
FcRγ-signalling to NF-κB activation and BDCA2 does
Figure 4 | Signalling by dectin 2 and mincle leads to
cytokine expression. BothDC-associatedC-typelectin2
(dectin2)andmacrophage-inducibleC-typelectin(mincle)
pairwiththesignallingadaptormoleculeFcreceptor
γ-chain(FcRγ) throughthepresenceofapositivelycharged
aminoacidresidueintheirtransmembraneregions.The
phosphorylationoftheimmunoreceptortyrosine-based
activationmotifs(ITAMs)ofFcRγ followingC-typelectin
receptor(CLR)activationservestorecruitspleentyrosine
kinase(SYK)andinducessignallingpathwaysthatmodulate
cytokineexpression.Dectin2bindstopathogen-associated
molecularpatterns(PAMPs)expressedbyfungalhyphae,
andminclebindstoα-mannosylPAMPsonMalasseziaspp.
fungi.BothsignallingpathwaysleadtoToll-likereceptor
(TLR)-independentproductionofcytokinessuchastumour
necrosisfactor(TNF)andinterleukin-6(IL-6);dectin2
triggeringisknowntoresultinnuclearfactor-κB(NF-κB)
p50–p65activation,andmincletriggeringinducesaCARD9
(caspaserecruitmentdomainfamily,member9)-dependent
signallingpathway.Similaritieswiththedectin1signalling
REVIEWS
P
Nature Reviews | Immunology
Dectin 1
P P
SYKRas
PP
RAF1
CARD9
BCL-10 MALT1
IKK
IKK
IKK
NIK
IKK
I B
p50p65
RELB
RELBp100
p52
RELB
RELB
p52
RELBp65
P
RELBp65
P
RELBp65
P
Proteasome
p50p65
P Ser276
p50p65
P
p65
PCBP
Ac
Ac
Nucleus
Cytoplasm
p50p65
P
Ac
Ac
Il6 and Il10
p50
p50
p50REL
p50REL
REL
p65
P
Ac
Ac
Il12b
Il1b
Ccl17 and Ccl22
p50p65
P
Ac
Ac
Il12a
Il23p19
Fungia b
?
Or
Inactive NF- B
p50REL
Figure 3 | Dectin 1 signalling through SYK and RAF1 directs NF-κB-mediated cytokine expression. a | The binding
of fungi to DC-associated C-type lectin 1 (dectin 1) induces phosphorylation of the YxxL (in which x denotes any amino
acid) motif in its cytoplasmic domain. Spleen tyrosine kinase (SYK) is recruited to the two phosphorylated receptors, which
leads to the formation of a complex involving CARD9 (caspase recruitment domain family, member 9), B cell lymphoma 10
(BCL-10) and mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1); this induces the activation of
the IκB kinase (IKK) complex through an unknown pathway. IKKβ phosphorylates inhibitor of NF-κBα (IκBα), thereby
targeting it for proteasomal degradation. This results in the release of nuclear factor-κB (NF-κB; consisting of either
p65–p50 or REL–p50 dimers), which then translocates into the nucleus. SYK activation also leads to the activation of the
non-canonical NF-κB pathway that is mediated by NF-κB inducing kinase (NIK) and IKKα, which target p100 for proteolytic
processing to p52; this subsequently leads to nuclear translocation of RELB–p52 dimers. In a SYK-independent manner,
dectin 1 activation leads to the phosphorylation and activation of the serine/threonine protein kinase RAF1 by Ras
proteins, which leads to the phosphorylation of p65 at Ser276. Phosphorylated Ser276 serves as a binding site for the
histone acetyltransferases CREB-binding protein (CBP) or p300 (not depicted) to acetylate (Ac) p65 at different lysine
residues. Ser276-phosphorylated p65 also dimerizes with RELB to form inactive dimers that cannot bind DNA, and hence
attenuates the transcriptional activity of RELB. b | Binding of acetylated p65 to the Il10 (interleukin-10) enhancer and Il6
REVIEWS
PSYK
TLR9DNA
Endosome MYD88 MYD88
TLR9
DNA
Endo
BDCA2 DCIR
P P
P
BLNK
BTK PLC 2
?
Ca2+
mobilization
TLR pathway
SHP1
or SHP
Plasmacytoid DCa b
ITAM
FcR
P
P
ITIM
_ _+
Ifna, Ifnb,
Tnf and Il6
Ifna
Tnf
Cytoplasm
Nucleus
Similar to DC-SIGN, neither DCIR nor
been shown to induce immune respons
own, but instead modulate signalling
Figure 2 | Signalling by BDCA2, DCIR and M
protein (BDCA2) leads to the recruitment of sp
tyrosine-based activation motif (ITAM) of the p
theactivationofacomplexconsistingofBcell
which induces Ca2+
mobilization. The signalling
downregulation of Toll-like receptor 9 (TLR9)-in
(TNF) and interleukin-6 (IL-6) by plasmacytoid d
the recruitment of myeloid differentiation prim
TLR-induced cytokines. b | Activation of DC im
compartments, where TLR8 and TLR9 reside. T
(ITIM) recruits the phosphatases SH2-domain-c
the activation of an unidentified signalling pat
TNF production or TLR9-induced IFNα and TNF
c | Cross-linking of myeloid C-type lectin-like r
ITIM and the recruitment of SHP1 or SHP2. MIC
signal-regulated kinase (ERK). However, it is no
TLR4-induced IL-12 production. LPS, lipopolys
molecule; TRIF, TIR-domain-containing adapto
ITAM Signaling Pathways in the Innate Immune System: C-type Lectins
Nat
Rev
Immunol.
2009
Jul;9(7):465-­‐79.
hemITAM
Classically involved in recognition of pathogen surface carbohydrates
FcRgamma coupled ITAM signaling.
In some cases, ITAM signaling antagonizes TLR
signaling.

84. Fc Receptors

85. Important Functions of Fc receptors

86. Mast Cell Signaling: the cornerstone of
allergic inflammation
Gilfillan, 2006
Nat. Rev. Immunol

87. The ITAM signaling pathway is similar

88. ITIM-containing Receptors
Recruit PHOSPHATASES
• SHP1, SHP2 – SH2 containing tyrosine
phosphatases
• SHIP – SH2 containing inositol phosphatase

89. ITAM/ITIM crosstalk in B cells
Secreted IgG can form
complex with antigen
Crosslinks BCR and FcγRIIB
FcγRIIB recruits SHIP that
hydrolyses a phosphate on
PIP3 and terminates signaling
Proposed control mechanism
to stop antibody production

90. Signal Transduction Dynamics
The
linear
pathways
for
signal
transduc&on
presented
in
textbooks
can
be
misleading.
• Cross-­‐talk
amongst
signaling
pathways
influences
signal
outputs.
• Dynamical
nature
of
signal
varia&on
with
&me
is
important.
For
instance,
human
immature
Dendri&c
Cells
exhibit
spontaneous
oscilla&ons
of
intracellular
Ca2+
in
the
res&ng
state.
These
are
lost
as
the
cell
matures.
400
800
1200
1600
0 200 400 600
Ca2+
iIntensity(AU)
Time (sec)
DIC Fluo-4
+ ionophore

91. Signal Transduction Dynamics
Dolmetsch, et al. Nature 392, 933-936(30 April 1998)
• T cells also exhibit Ca2+ oscillations and spikes during activation
• Ca2+ clamp method can reproduce arbitrary oscillation amplitudes
and periods (left)
At low levels of Ca2+, oscillatory [Ca2+]i
increases the number of cells expressing
an NFAT reporter.

92. Signal Transduction Dynamics
Dolmetsch, et al. Nature 392, 933-936(30 April 1998)
These three transcription factors respond similarly to continuous Ca2+ amplitude,
but have different behavior depending upon oscillation frequency.
Thus, different transcriptional programs could be controlled by Ca2+ signal
dynamics.
Encoding oscillations is usually a matter of some negative feedback in the signaling
system operating with a delay. For instance, the IP3R releases store Ca2+, but is
also inhibited by the rise in cytosolic Ca2+.
How would the cell decode oscillatory signals?

93. Signal Transduction Dynamics
TF-A: high Ca2+ affinity, short half-life
TF-B: low Ca2+ affinity, long half-life
time
%maxsignal
Calcium, TF-A, TF-B
TF-A gets activated by very little Ca2+
and decays rapidly. Its signal will be
oscillatory unless the period of Ca2+
oscillation is shorter than the half life of
TF-A.
TF-B only gets activated by high levels of Ca2+ (at the peak of the oscillation),
but it degrades slowly. This causes its signal to be persistent.
With this system, you could have regimes where only TF-A or both were
activated and TF-A could be periodic or persistent. This would be controlled by
the amplitude and period of the oscillatory stimulus.

94. Extra
slides
on
BCR
signaling
(if
there
is
Ame)

95. BCR signaling
• Antigen binding domain is
a surface-expressed
immunoglobulin with the
same antigen specificity
that the B cell will secrete
• Signaling occurs through
associated Igα and Igβ
ITAM containing proteins
• Multi-subunit receptor with
variable antigen receptor
+ invariant signaling
molecules

96. Signaling through BCR

97. Nature Reviews Immunology 13, 475–486 (2013) doi:10.1038/nri3469
B
cell
Ac&va&on
at
APC
contacts
involves
microclusters
and
cytoskeletal
dynamics
• BCR
ac&va&on
and
microcluster
forma&on
• B
cell
ac&n
mediated
spreading
on
APC,
integrin
ac&va&on
• Centripetal
mo&on
of
microclusters
on
MT
• Lysosome
recritment
• Contact
site
contrac&on
How
is
an&gen
acquired?
• Soluble
an&gens
may
diffuse
into
secondary
lymphoid
organs
and
be
acquired
from
fluid
phase.
• APCs
like
subcapsular
macs
and
follicular
DCs
present
large
par&culate
an&gens
How
do
B
cells
obtain
an8gens
from
surfaces?

98. Nature Reviews Immunology 13, 475–486 (2013) doi:10.1038/nri3469
B cells polarize lysosomes toward an APC contact
MTOC Lysosome
Laser ablation of a
single lysosome does
not impact overall
lysosome polarization to
the synapse
Ablation of the MTOC
causes the lysosomes to
disperse.
What is the purpose of
this with respect to
antigen acquisition?

99. Macrophages contacting LDL aggregates also target lysosomes to extracellular
contact sites for the purpose of degradation and material extraction
Abigail S. Haka et al. Mol. Biol. Cell 2009;20:4932-4940
All LDL aggr.
Extracellular
LDL aggr.
Lyso contents
released
Acidification of extracellular
contacts with agLDL due to
lysosomal release

100. Mechanisms
of
intercellular
exchange
of
proteins
FEBS Letters
Volume 583, Issue 11, pages 1792-1799, 14 MAR 2009 DOI: 10.1016/j.febslet.2009.03.014
http://onlinelibrary.wiley.com/doi/10.1016/j.febslet.2009.03.014/full#feb2s0014579309001872-fig2
B cells have also been proposed to
gather Ag from APC by
“trogocytosis”.
The B cell exerts force on the APC
membrane sufficient to rip out the
antigen and some accompanying
membrane.
Other mechanisms of protein
transfer are also known, though
their contribution to B cell Ag
gathering is less clear.