The Two-Signal Model of T-cell Activation

1
The Two-Signal Model of T-cell Activation
DC
T cell
CD4 or CD8
12Full activation
1
TCR
MHC
2
COSTIMULATION
2

• Experimental evidences for the two signal model
  of T cell activation
• Experimental evidences that CD28 is a
  costimulatory receptor
• Effects of CD28-mediated costimulation on T
  cells
• Mechanism of action
• The ligands for CD28 B7 molecules
• The complexity of the B7/CD28 system CD28 plays
  a role in
• promoting T cell tolerance!!

3
Marc Jenkins and Ronald Schwartz in the late 80s
The first definitive experimental demonstration
that TCR engagement alone was insufficient for T
cell activation.
Proliferative response of T cell clones (pigeon
cytochrome c peptide 81-104 presented by I-Ek) to
normal or ECDI(chemical crosslinker)-fixed
peptide-pulsed APCs
Jenkins M.K., and Schwartz R.H. J. Exp. Med.
165302-319, 1987.
ECDI-treated APCs fail to stimulate proliferation
by normal T cell clones Not the result of
extensive modification of the MHC class II
molecule ECDI treatment inactivated an accessory
(costimulatory) function of the APC
4
T cell clones stimulated in vitro by EDCI-treated
APCs were induced into a state of
unresponsiveness (Anergy).
Jenkins M.K., and Schwartz R.H. J. Exp. Med.
165302-319, 1987.
5
Cells with this costimulatory function
macrophages, activated B cells and dendritic
cells (professional antigen presenting cells).
Subsequently,it was shown that Naïve T cells
have similar requirement for these costimulatory
signals in order to produce IL-2 and progress
through the cell cycle.
6
This requirement for costimulation and the
restricted pattern of expression of the
costimulatory ligands to professional APCs, was
proposed to be a mechanism for maintenance of
peripheral T cell tolerance.
TCR
Costimulatory Receptors
Costimulatory Ligands
peptide/ MHC I
Parenchymal cell
7
The concept of costimulation was consistent with
the characteristics of a newly identified
molecule called CD28, which is expressed on naive
CD4 and CD8 T lymphocytes.
Artificial APC
T
C
R
A
g
/
M
H
C
CD28
Physiological APC
PROLIFERATION IL-2 PRODUCTION INDUCTION OF
ANERGY
T
C
R
A
g
/
M
H
C
CD28
Artificial APC
8
In vitro T cell responses in CD28 KO mice
Shahinian A. et al. Science 261609, 1993.
Adapted from Green J. et al. Immunity 1501, 1994.
9

• The importance of CD28-mediated costimulation has
  also been demonstrated in vivo.
• Blockade of CD28-mediated costimulation has been
  shown to
• Prolong graft survival
• Block primary antibody response
• Prevent or reduce in intensity experimentally,
  antigen-induced models of
• autoimmunity including arthritis,
  encephalomyelitis, myocarditis, thyroiditis and
• myasthenia gravis

10
Major effects of CD28-mediated costimulation in T
cells
Proliferation IL-2 (transcription, mRNA
stabilization) IL-2R up-regulation ?G1 cell cycle
kinases ?Cell cycle inhibitor p27Kip Survival
Bcl-xL Effector function ? CD40-L, OX-40, 41BB,
ICOS ? cytokines expression ? cytotoxic
molecules
The major effects of CD28-mediated costimulation
are to augment and sustain T cell responses
initiated by antigen receptor signal by promoting
T-cell survival and enabling cytokines to
initiate T cell clonal expansion and
differentiation.
11
T cells stimulated in the presence of signal 1
alone can respond only when a certain number of
TCR complexes are triggered (threshold 8000
TCR engaged molecules). In the presence of
costimulation through CD28, the number of TCR
complexes that should be engaged for the
induction of a response is decreased to 1500 or
less.
A B human T cells C T cell clones
Viola, A. and Lanzavecchia A. Science 273 104,
1996
CD28 engagement lowers the activation threshold,
thus making T cells more sensitive to antigenic
stimulation.
12
Mechanism
The conventional view CD28 delivers
intracellular signals that synergize distally
with the TCR signaling pathways, at the level of
JUN-kinase.
13
The new concept (based on the visualization of T
cell activation) Kupfer et al. have shown that T
cells polarize toward the APCs, thereby forming a
highly structured synapse (the immune
synapse). This process involves a redistribution
of surface and intracellular molecules. Some
molecules, such as TCR, CD4, src kinases are
enriched in a central zone, whereas others, such
as LFA-1 and CD45, remain outside.

• helps maintaining the interaction between the
  TCR and the
• peptide/MHC complexes
• (low concentration and low affinity for the TCR)
• helps in the local accumulation of intracellular
  signaling complexes

14
Wulfing, C. and and Davies, M.M. Science
2822266, 1998. 1. Actine cytoskeleton reorients
toward the T cell-APC interface soon after the
start of T cell activation
Beads are attached to the surface of T cells and
monitor the movement of the cytoskeleton in the T
cells.
Figure 1. Bead movement toward the interface.
Single frames of a video microscopy experiment
(36) of the interaction of 5C.C7 T cells, loaded
with 4.5-µm anti-ICAM-1 (YN1) beads, with
peptide-loaded CH27 B cell lymphoma cells (35)
are shown. The CH27 cell is substantially larger
than the T cells. The T cell intracellular
calcium concentration is overlaid in a false
color scale from blue (low concentration) to red
(high concentration) to mark the onset of T cell
activation, set to time 000 min. Although the
beads, one of which is marked with an arrow, are
bound to the posterior end of the central T cell
before and at the time of its activation, they
can be seen to move toward the T cell-B cell
interface in subsequent frames.
15
Type of beads/treatment/APC Movement Movement
toward interface away interface No
mov n --------------------------------------------
--------------------------------------------------
----------------- Type of beads Anti-ICAM 74 0
26 81 Streptavidin (surface biotin) 83 7 10
29 Streptavidin (lipid biotin) 64 5 32 22 A
ntibody blocking anti-B7 8 0 92 24 anti-VCA
M 62 0 38 21 anti-CD48 75 0 25 20
Adapted from Wulfing, C. and and Davies, M.M.
Science 2822266, 1998.
2. CD28 engagement is necessary and sufficient to
induce the movement of the T cell actine
cytoskeleton toward the newly formed T cell-APC
interface. CD28 activates an actin-myosin-driven
transport process that delivers receptors and
signaling complexes to the TCR contact area.
16
Viola, Schroeder, Sakakibara and Lanzavecchia,
Science 1999 The plasma membrane is composed of
discrete lipid microdomains in which membrane
molecules are differentially partitioned (lipid
rafts). These rafts concentrate
glycophosphatidylinositol-linked proteins,
glycosphingolipids, as well as several molecules
involved in signal transduction such as Lck,
LAT, Ras, and G proteins. CD28 participates in
the organization of the immune synapse by
recruiting rafts into it. The recruitment of
rafts to the site of TCR engagement may represent
a general mechanism by which costimulation can
increase the signaling process. A new paradigm
is emerging that is built around the central
role of costimulation in contact formation
necessary for T cell activation.
CD28-induced raft redistribution to the site of
TCR engagement. Resting T cells were stained
with FITC-CTx and analyzed by confocal
microscopy. (A) Unstimulated resting T cells.
(B) T cells stimulated for 20 min with beads
coated with anti-CD3 alone (10 µg/ml) and (C)
stimulated for 20 min with beads coated with
anti-CD3 (10 µg/ml) plus anti-CD28. raft marker
(FITC)-labeled cholera toxin (CTx) B subunit,
which binds the GM1 glycosphingolipid
17
The ligands for CD28
1982, B7.1(CD80) Artificial APCs transfected
with the cDNA coding for B7.1 induce T cell
proliferation and IL-2 secretion. However, when
T cells are stimulated with professional
APCs anti-B7.1mAb does not block completely T
cell activation while anti-CD28 mAb does.
Yes, there is another ligand for CD28 B7.2
(CD86) (1993)
Artificial APCs transfected with the cDNA coding
for B7.1 or B7.2 induce similar T cell
proliferation and IL-2 production.
18
B7.1 and B7.2 Whats the difference?
Expression Primarily limited to APCs B7.1 is
absent from unstimulated cells. B7.2 is
constitutively expressed at low levels on
unstimulated DCs and blood monocytes. Activation
of APCs induces B7 expression. In general, the
induction of B7.2 expression follows faster
kinetics and usually reaches higher values than
does B7.1 induction. In vivo, anti B7.2 mAb was
shown to be more efficient than anti-B7.1 mAb to
block the expansion of Ag-specific T cells. B7.1
KO vs B7.2 KO When immunized intravenously
without adjuvant, B7.2-deficient mice failed to
form germinal centers, whereas B7.1-deficient
mice gave Ab responses comparable with wild-type
mice. B7.2 is probably the major initial ligand
for CD28 while B7.1 which is expressed later
sustains T cell activation.
19
The differentiation of CD4 T cells into Th1 or
Th2 subsets has profound effects on the outcome
of immune responses Th1 INFg, eradication of
intracellular pathogens, autoimune disease
. Th2IL-4, IL-5, IL-10 eradication of
extracellular parasites, allergic reactions.
EAE Th1-dependent autoimmune disease anti-B7.1
mAb treatment reduced the incidence of disease of
murine EAE, anti B7.2 mAb made the disease worse
(Kuchroo et al., 1995). It has been suggested
that this differential effect of the B7 mAbs
reflected the abilitiy of B7.1 and B7.2 to
direct T cell differentiation along the Th1 or
Th2 pathway, respectively. B7.1 ? Th1
differentiation B7.2 ? Th2 differentiation
20
Production of cytokines when T cells are primed
in the absence of B7.1 or B7.2.
Schweitzer N.A. et al. J.Immunol. 1582713, 1997.
T cells TCR Tg CD4 specific for an OVA peptide
presented by I-Ad
2 days
4 days
IFNg IL-4
T cells WT APC pulsed with OVA peptide
T cells WT APC
T cells B7.1 KO APC pulsed with OVA peptide
T cells WT APC
IFNg IL-4
T cells B7.2 KO APC pulsed with OVA peptide
IFNg IL-4
T cells WT APC
21
IFNg (pg/ml)
IL-4 (pg/ml)
Adapted from Schweitzer N.A. et al. J.Immunol.
1582713, 1997.
When T cells are primed under optimal signal 1,
neither B7.1 or B7.2 plays an obligatory role in
priming for production of a particular cytokine.
These data do not support a simple
Th1/Th2-determining role for either B7.1 or B7.2.
When T cells are primed under suboptimal signal
1, both B7.1 and B7.2 are important for the
induction of Th1 or Th2 cytokines synthesis, with
B7.2 having a greater influence compared with
B7.1.
The influence of B7.1 or B7.2 on T cell
differentiation is determined mainly by their
distinct temporal kinetics and level of
expression, rather than distinct signaling
capacity of the respective B7.1 and B7.2
molecules.
22
The B7/CD28 pathway is much more complicated than
expected
Salomon, B., et al. Immunity 12431-440, 2000
NOD mice develop diabetes at 10 weeks of age.
This is the result of a T cell-dependent
autoimmune process against the pancreatic beta
cells. B7-/- NOD
More severe diabetes!!
23
Increased incidence and early onset of diabetes
in B7 -/- NOD mice
24
In the NOD model, Th1 cytokines such as IFNg are
pathogenic while Th2 cytokines, like IL-4 could
block disease development. CD28 -/- NOD mice
have impaired Th2 differentiation. Hypothesis
Imbalance in Th1/Th2 differentiation might be a
possible mechanism for exacerbation of diabetes
in NOD B7-/- mice. T cells from B7-deficient
mice were stimulated with anti-CD3 mAbs, and
supernatants were harvested to determine the
levels of IL-4, IL-5, and IFNg production. The
production of all cytokines was
significantly reduced as compared to cytokines
produced by T cells from wild-type mice. Isotype
analysis of antibodies to glutamic acid
decarboxylase (GAD), a potent autoantigen in
autoimmune diabetes, showed that the levels of
the Th1-dependent IgG2b isotype were severely
decreased in these mice. The levels of
GAD-specific Th2-dependent, IgG1 isotype gG1
antibodies were significantly reduced in five
out of nine mice, but they were comparable to
wild-type mice in the remaining four
animals. Thus, a selective decrease in Th2
cytokine production could not explain the
increased disease severity in this B7-deficient
NOD strain.
25
Virtually all islets isolated from 9-week-old
prediabetic male and female B7-deficient mice
were highly infiltrated and scored as severe
insulitis. In contrast, in wild-type NOD mice,
less than 10 of the islets from female and
only 3 of the islets from male mice displayed a
severe insulitis. A kinetic analysis of islet
infiltration in B7-deficient mice was carried out
by scoring the intensity of insulitis at
different time points in young prediabetic
animalsA rapid increase in the levels of islet
infiltration was observed by 9 weeks of age. The
increased islet infiltration was not progressive
as in control NOD mice but occurred rapidly
between 7 and 9 weeks of age. T The accelerated
kinetics of islet infiltration in B7-deficient
mice suggested that a regulatory process that
normally modulates the autoreactivity in the
pancreas is lacking in B7 deficient NOD mice.
26
Previous studies have demonstrated the existence
of regulatory CD4CD25 T cells that can prevent
or limit autoimmunity. In the absence of this
regulatory subset, normal mice develop several
types of autoimmune disease spontaneously. In
most mouse strains, this subset represents 710
of the CD4 T cell subset. In NOD mice, these
cells are reduced in number, representing only
56 of CD4 T cells. The possibility that B7
expression might affect the regulatory CD4CD25
T cells in NOD mice was examined. A profound
decrease of CD4CD25 T cells was observed in the
B7-/- NOD mice. Less than 1 of CD4 T cells
expressed CD25 as compared to 5.4 in littermate
B7/ controls. Thus, B7-/- mice are deficient in
the immunoregulatory cells present in wild-type
NOD mice, supporting a potential role for B7
engagement in CD4CD25 regulatory T cell
development or homeostasis.
Profound absence of these cells also in
CD28-deficient mice suggesting that the B7/CD28
interaction was critical for the homeostasis of
these cells.
To determine if B7/CD28 costimulation is required
continuously for the homeostasis of this T cell
subset or is required for their
development. Blockade of B7 stimulation in WT NOD
mice resulted in decreased number of these cells
and in exacerbated diabetes.
27
To determine whether the CD4 CD25 T cells
control diabetes in CD28-deficient mice Transfer
of CD28-/- T cells into NOD.SCID. When CD4
CD25 are co-transferred, no diabetes. They also
examined whether the transfer of a limited number
of regulatory T cells directly into whole
prediabetic CD28-deficient NOD mice could alter
the progression of disease in these highly
susceptible animals. All the CD28-deficient NOD
mice either injected with PBS or with CD4CD25-
NOD T cells became diabetic by 11 weeks of age.
In contrast, in four out of the six
CD28-deficient mice, the transfer of CD4CD25
NOD T cells significantly delayed the development
of diabetes. In two animals, the disease did not
develop through 15 weeks of age. Together, these
studies suggest that the deficit of CD4CD25
regulatory T cells is responsible for the
exacerbation of diabetes of the B7/CD28-deficient
NOD mice.
28
Thus, in addition to a role in initial activation
of T cells, B7/CD28 interactions are important in
promoting T cell tolerance because they have a
critical role in the homeostasis of CD4 CD25
regulatory T cells which play an essential role
in regulating self-tolerance.