MODEL D' Positive and negative selection happen at distinct stages of thymocytes development and in

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MODEL D. Positive and negative selection happen
at distinct stages of thymocytes development and
in different anatomical locations
The thymus (cortical) epithelium is responsible
for imprinting self on T cells Thymus transplants
show that nonlymphoid cells, or the radiation
resistant portion of the organ is responsible for
positive selection. Used irradiated mice
reconstituted with bone marrow stem cells and a
thymus graft 1970s doherty Perhaps positive and
negative selection induced by different cells
types, perhaps sequential according to order the
thymocytes are presented with these APCs in the
thymus Anatomical correlates
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UNOPPOSED POSITIVE SELECTION AND AUTOREACTIVITY
IN MICE EXPRESSING CLASS II MHC ONLY ON THYMUS
CORTEX NATURE 1996 p81-5Terri LauferLaurie
Glimcher

• Approach mouse with class II under keratin 14
  promotor as a trangene crossed to a class II-/-
  mouse to re-express class II only in cortical
  thymic epithelial cells K14 mouse

Mouse SP CD4 cells in the periphery
(spleen, ln)
Autoreactivity Prolif to self
Class II-/-
-
1- 4

K14
5-18
-
36-5
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C. Normal mouse has class II expressed in Cortex
Medulla
a. K14 MHC in cortical epith only (weaker)
d. 36-5 tg expresses on CM, but nowhere else in
the body
e. Ab-/-
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Conclusions

• Cortical epithelial cells can positively select,
  but cant negatively select.
• Medulary epithelial cells can negatively select
  (MTEC, specialized thymic APC in which Aire
  directs the expression of tissue specific
  antigens ?)
• Others experiments indicate the cells of
  hematopoetic origin are good negative selectors
  (thymic dendritic cells) (evidence that some
  antigens travel to thymus via BM derived APC
  (relevant to EAE) and negative selection purges
  reactive clones?
• Authors conclude positive selection of
  thymocytes occurs before, independently of, and
  in distinct anatomical sites from negative
  selection.

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Cathepsin L Critical Role in Ii Degradation and
CD4 T Cell Selection in the Thymus. Nakagawa
Rudensky, Science 1998 April 17 280 450-453.
Fig. 1. Normal expression of surface MHC class
II molecules and antigen presenting function of
ctsl/ SCs. (not shown)

Fig. 2. Reduced number of CD4 T cells in
CTSL-deficient mice. Fig. 3. Radioresistant
thymic epithelial cells are responsible for the
defect in development of CD4 T cells in ctsl/
mice. Fig. 4. Within the thymus, CTSL activity
was found in cTECs and thymocytes, but not in
BM-derived thymic DCs whereas CTSB was
ubiquitously expressed Fig. 5. Altered MHC class
II processing in thymic cortical epithelial
cells of ctsl/ mice.
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Mizuochi et al J Exp Med 1992 1751601
Cresswell summarizes implications of Rudensky
studies
They appear to use cathepsin S for the late
stages of invariant chain degradation
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Positive and negative selection happen at
distinct stages of thymocytes development and in
different anatomical locations (Laufer).
Therefore, difference in positive and negative
selection could reflect 1) differential
responses to differing populations of thymic APC
that either contain different peptides (rudensky)
or APC that express different costimulatory
molecules (ie CD30 needed for negative, but not
positive selection) Amakawa, Cell 1996. 2) T
cell at different stages of development respond
to TCR engagement differentially, possible if
really sequential.
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Perturbing TCR signal transducers and assessing
affects on negative and positive selection
familiar models re-invented/refined

• Strength of Signal Model of Thymocyte Development
  (the TCR is an on/off switch with high, medium,
  and low settings
• Direct extension of the Avidity Model
• Receptor occupancy translates into strength of
  signal distinguish high avidity from low avidity
  by counting receptors engaged and counting
  intensity of signal
• Negative selection induced by a strong signal
  (high avidity agonist)
• Positive selection induced by a weaker signal
  (lower avidity partial agonists/antagonists
• Insufficient signal leads to death by neglect
• Quantitative differences

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What are the predictions for each model regarding
how modulators of TCR signal transduction would
affect positive vs negative selection?See
assigned reading for a review including recent
signaling data.

• Positive and Negative Selection invoke distinct
  signaling pathways (the TCR is not a simple
  on/off switch
• recruitment of different transducers under
  different circumstances.
• Alternate signals result in distinct functional
  outcomes.
• Qualitative differences
• Kinetic proofreading an individual TCR can
  distinguish positively and negatively selecting
  ligands by reading the occupancy time
• Receptor could generate an ordered set of
  biochemical signals such that the earliest signal
  lead to positive selection and the later signals
  would induce negative selection.

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Positive and Negative selection rely on TCR
transducers/costimulators to a different extent.

• Alberola-Ila et al., J Exp. Med. 184 9-18,
  1996. Positive, but not,
• negative, selection is blocked in mice expressing
  dominant-negative
• Ras and Mek-1 transgenes simultaneously.
• Backstrom et al Science, 1998 281835-8.
  Transgenic mice expressing
• an a/b TCR lacking the a-CPM (or CD3d also
  Science,281 1998),
• thymocytes are deficient in positive, but not
  negative, selection
• (and sustained but not transient ERK.
• RasGrp -/- affects positive, but not negative
  selection (sustained ERK?)
• Nature Immunology1, 317, 2000, Immunity 17,
  617, 2002.
• Grb-2 haplo-insufficiency (/-) affects negative
  but not positive selection
• Nature Immunology 2, 29, 2001 (transient ERK
  regulator?)

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• Not simply strength of signal
• Instead quality of signal
• Transient vs sustained ERK
• Multiple pathways control
• ERK activation
• Allows for independent
• regulation of signal intensity
• and duration
• (and localization/substrate
• specificity?)

TMEC
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Costimulation, Peripheral T cell Activation and
Tolerance InductionMay 2, 2005

• Reading
• Arlene Sharpe the B7-CD28 Superfamily
• Nature Reviews Immunology 12, 116-126 (2002)
• B7 Family Revisited Annual Review of Immunology
  2005 23515-48
• PARTS OF CH 8 RELEVENT TO COSTIMULATION

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Only professional APC should express
co-stimulatory ligands Provides a model for
peripheral tolerance to class I or class II
restricted antigen Perhaps signal 1 better
thought of as engaging receptor 1
TCR Signal 2 as engaging receptor
2costimulator
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Potential outcomes of interactions between T
cells and APCs as predicted by Bretcher and Cohn,
plus one

• Activation of the TCR in the presenceof a
  costimulator results in T cell clonal expansion
  and max induction of functions such as lymphokine
  production
• The interaction of the TCR with Ag/MHC in the
  absence of co-stimulator ligand leads to
  functional paralysis (anergy) or to death by
  apoptosis
• When resting T cells encounter the costimulatory
  ligand alone , predict no affect.
• (Activated T cells may be helped by costimulatory
  ligand alone, but are not totally dependent on
  it..ie not anergized in its absence.)

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T cell response of B10A mice to pigeon cytochrome
c
(anergy)
Mark Jenkins and Ron Schwartz, 1980s Add back of
allo-APC separated TCR cell engagement from
costimulator engagment and allowed for
characterization of molecular basis of
costimulation (separating it from TCR) However,
in vivo, ag/MHC and costim ligand function best
(exclusively) when they are on same cell
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CD28 costimulation by B7

• Jim Allison showed that anti-CD28 could
  substitute for allo-APC
• B7 transfected CHO cells could function as APC to
  rescue from anergy (anti-CD3 could function
  instead of fixed APC).
• Fab fragments of CD28 could block B7/CD28
  activation and induce anergy
• CD28 cloned
• CTLA-4 cloned by homology
• B7 family members shown to be ligands

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Induced on professional APC activated Macs, B
cells (by LPS,IL-2.IL-4,BCR,CD40, MHCII mature
dendritic cells
Expressed on activated T cells Binds B7s better
than CD28 CTLA4-Ig fusion protein used as
a B7/CD28 blocking
reagent
Constitutive expression on resting and activated
T cells
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Microbial substance induce costimulatory activity
in macsadjuvents (killed bacteria)-
immunologists dirty little secret
Innate and adaptive immune systems
Cooperate Innate sytem senses danger Toll
receptor engagemnt induces B7 expression
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CD28 knockouts have a proliferation defect,
partially rescued by exogenous IL-2 More severely
effected when look for an in vivo sustained
response to many challenges. After initial
expansion in response to some viruses, cells
crash (due to lack of survival)
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Langerhans cells can take up antigen in the
skin and migrate to lymphoid organs where they
present it to T cells. Langerhans cells can
ingest antigen by several means, but have no co-
stimulatory activity. In the presence of
infection, they take up antigen locally in the
skin and then migrate to the lymph nodes. There
they differentiate into dendritic cells that can
no longer ingest antigen but now have
co-stimulatory activity.
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CD28 acts functions by at least least 3 ways

• CD28 signals increase mRNA stability (lt2hrs)
• lymphokine message (IL-2 and others)
• IL-2 receptor a chain mRNA
• CD40L mRNA
• CD28 signals increase IL-2 mRNA transcription
  (lt6hrs)
• IL-3, GMCSF, IFNg, (NFkB, AP-1 and NFAT and
  others)
• CD28 signals increase cells survival (protect
  apoptosis)
• Increase Bcl-x protein levels, increase NFkB
  activation

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Costimulation potentiates TCR signaling

• Doesnt increase the number of TCR engaged (as
  measured by TCR downregulation)
• Rather potentiates engagement
• Fewer TCR engagements required to get maximal
  functional outcome
• PTK substrates stay phosphorylated longer
• Also Lanzavecchia CD28 shortens the duration of
  TCR engagement required for functional outcome
• Lanzavecchia Science July 5, 1996 and again
  Science Jan 29, 1999.

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Science, Vol 283, Issue 5402, 680-682 , 29
January 1999Antonella Viola, Susanne
Schroeder, Yoichi Sakakibara, Antonio
Lanzavecchia also july 5, 1996 same authors (use
ag and APC)

• CD28 doesnt increase the number of TCRs engaged
  (measured by TCR downregulation)
• Rather potentiates engagement
• Fewer TCR engagements required for proliferation
• Closed figures with anti-CD28 open without

PTK substrates stay phosphorylated longer? More
about signaling wednesday PP1lck inhibitor
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CTLA-4 is a negative regulator of T cell
activation

• B7 has 100 fold greater affinity for CTLA-4 thank
  for CD28
• CTLA-4 is primarily intra-cellular in resting T
  cells
• 24-48 hrs post stimulation CTLA-4 is transiently
  expressed at the cell surface
• CTLA-4 is directly targeted to the synapse
  through an unknown pathway (exocytic)
• The balance between CD28 positive and CTLA-4
  negative signaling determines the activation
  threshold. Just begininging to understand what
  conditions normally favor CTLA-4 expression over
  CD28.

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T cells from CTLA4 hyper-proliferate wt solid
CTLA4-/- open (48hrs in vitro)
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Elevated Ig levels in ctla-/ mice closed triangle
vs wt (open squares) (secondary to T cell
hyper-activity)
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Molecular Basis of T Cell Inactivation by CTLA-4
Kyung-Mi Lee, Ellen Chuang, . Jeffrey A.
Bluestone Science 282, 2263, 1998
Fig. 1. Anti-CD3-induced LAT and TCR tyrosine
phosphorylation is inhibited by anti-CTLA-4
engagement. Activated T cells were admixed with
293 cells (transiently expressing a
membrane-bound single-chain mAb to CD3 in the
presence or absence of a membrane-bound
single-chain mAb to CTLA-4. The cells were
incubated at 37C for the time indicated and then
subjected to lysis in buffer (LB).
Immunoprecipitates (IPs) were prepared with mAb
FB2 to phosphotyrosine (pTyr), rabbit antiserum
to LAT (3), or mAb H146-968 to TCR. IPs separated
on a reducing SDS-12 polyacrylamide gel were
transferred to a polyvinylidene difluoride
membrane and subsequently immunoblotted (IB) with
mAb 4G10 to pTyr. Bound proteins were detected by
chemiluminescence. CTLA-4 cross-linking resulted
in a consistent but incomplete reduction of
tyrosine phosphorylated p23TCR and LAT.
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Fig. 4. SHP-2 associates with CTLA-4-TCR
complexes and regulates binding of TCR to CTLA-4.
(A) Equal numbers of activated T cells were lysed
and subjected to IP with mAbs to CTLA-4
(UC10-4F10), rabbit antiserum against SHP-2, and
mAb to TCR (H146). IPs were analyzed by SDS-PAGE
and immunoblotted with a rabbit antiserum to
SHP-2 , goat polyclonal antibodies to CTLA-4
(Q20, Santa Cruz, Santa Cruz, CA), or rabbit
antiserum to TCR (Ab 387). (B) 293 cells were
transiently transfected with either WT CTLA-4 or
mutated CTLA-4 (Y201F/Y218F) and TCR in the
presence or absence of p56lck or SHP-2. CTLA-4
IPs prepared from lysates (400 µg per sample) or
whole cell lysates (35 µg per lane) were
electrophoresed and immunoblotted with rabbit
antibody to SHP-2 or antiserum to TCR (Ab 387)
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Many T cell surface proteins act as
costimulators (in some capacities) Some
overlapping/distinct, Functions Some
overlapping/distinct expression
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