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Development
ab T lymphocyte Development
Where do T cells come from?
1. Multipotent Lymphoid
Progenitors Migrate from the
Bone Marrow to the Thymus
2. In the Thymus, the
Lymphoid Progenitors
Differentiate to pre-T Cells
and are Educated to
Differentiate Self from Nonself
3. Positively Selected T Cells
Emigrate from the Thymus to
Mediate and Effect the
Cognate Immune Response
T Lymphocyte Maturation in the Thymus
The CD4 by CD8 FACS Plot as an Indicator
of Normal (and Abnormal) Thymocyte
Development
CD4
CD8
Ikaros controls commitment to Lymphoid lineage
(Ikarus DN)
Bone Marrow
Thymus
Notch 1 is required for T cell lineage
commitment
Control
Notch1-/-
CD4
Ratke et al, Immunity, 1999
How Notch Signaling Works
Ligands expressed
In the thymus
Reciprocal Regulation
Pax-5 induces the expression
Of B lineage genes (CD19, BLNK)
And represses the expression of
Notch
But really location controls the T cell versus B cell
Lineage Choice
So what controls homing of
CLPs to the thymus?
And what controls the
expression of these homing
receptors?
T Lymphocyte Maturation in the Thymus
As T
Lymphocytes
Develop, They
Migrate From
the Thymic
M/C junction
to the Cortex
to the Medulla
DN1 Cells in the Thymus Are Not
Committed to become T Lymphocytes
• TCR loci are in germline
configuration
• Cell can differentiate to
become a B lymphocyte,
Natural Killer cell, or
Dendritic cell (Michie et al, JI,
2000; Ikawa et al, JEM,
1999;Sanchez et al, JEM, 1994)
The DN2 Subset of Thymocytes are More
Committed, but Not Quite Exclusive
• TCR loci are in germline
configuration
• DN2 thymocytes may
give rise to dendritic
cell(Moore et al, 1995; Wu et al
1996; Ardavin et al, Nature 1993;
Shortman et al, Imm Rev. 1998)
• DN2 thymocytes no
longer differentiate to the
NK cell or B lymphocyte
lineages
DN3 Cells are Committed to the T Lineage
• Downregulation of
CD44 expression
• Upregulation of RAG
genes
• V-D-J recombination
of TCRb chain locus
• Expression of preTCRa chain
The pre-TCRa chain (DN3)
• Invariant Type I TM
protein
• Contains only one Ig
Domain
• Physically associated with
TCRb chain
• Signaling appears to be
ligand-independent
From Fundamental Immunology, 4th ed. (Paul)
– TCRb chain in this complex
lacking extracellular
domain is sufficient to
allow progression to DP
stage
Targeted Gene Mutants Unable to
Mature to the DP Stage
After Assembly of a Functional pre-TCR
• Shut down of TCRb rearrangement; TCRb
allelic exclusion
• Onset of proliferation/expansion
• Differentiation to DN4, CD8ISP, and then
DP
Three Models of ab vs. gd Lineage
Determination
• Earliest (fetal) waves of T cells are
mostly gd
• Later production is skewed toward ab
lineage
• Conflicting Data Supporting Each
Model
• Mature ab T cells have rearranged g
locus; gd’s have rearranged b locus
• Even DN4 cells (which have selected a
b chain) can become gd lymphocytes in
transfer experiments
From Fundamental Immunology, 4th ed. (Paul)
CXCR4
Notch
SCF
c-kit
IL-7
Jak3/
BMP
Smad
Wnt
Tcf/Lef
V(D)J Recombination in developing T cells
• b chain occurs first (DN3 stage) – VDJ
• a chain occurs second – VJ, no D region
• Allelic exclusion
• Combinatory diversity of the adaptive immune system
• This is a big problem
Fates Awaiting a DP Thymocyte
Default
(95%)
Positive Selection: Host MHC determines
CTL immune responsiveness (Bevan, Nature, 1977)
Balb/c H-2d/b BM -> H-2b/d
Balb/c H-2d/b BM -> H-2d
Balb/c H-2d/b BM -> H-2b
immunize with B10H-2d/b splenocytes
restim in vitro
look for MHC restricted cytotoxicity
H-2d/b BM -> H-2b/d
H-2d/b BM -> H-2d
H-2d/b BM -> H-2b
d(with peptides)
+++
+++
+
b(with peptides)
+++
+
+++
Evidence for MHC Restriction of CD4+ and CD8+
T Lymphocytes is Apparent in MHC KO Mice
CD8
CD4
(Grusby et al, PNAS, 1993)
HY TCR Male
HY TCR female
Negative Selection
HY TCR Tg is negatively selected by a male specific
antigen
(Kisielow et al, Nature, 1988)
Negative and Positively selecting peptides different
in there Kd
(Alam et al, Nature, 1996)
Model of Thymocyte Education
Signal Transduction in thymocyte
development
TCR Signal Transduction
But what are the down stream effectors?
Which downstream pathways contribute to
thymocyte selection?
Do Positive and Negative Selection Signals Activate the
Same Signal Transduction Pathways?
Disruption of the ERK Pathway Interferes With
Positive but not Negative Selection (an effect on
differentiation and proliferation?
• Also:
ERK1
ERK1
– DN Mek1
• Alberola-Ila et al, 1995
• Alberola-Ila et al, 1996
– DN p21ras
• Swan et al, 1995
• Alberola-Ila et al, 1996
– DN Raf
• O’Shea et al, 1996
– RasGRP
• Dower et al, 2000
– Indirect (ERK Induction
from CD3d chain)
Pages et al 1999 Science 286:1376
• Werlen et al, 2000
• Delgado et al, 2000
Calcineurin B is required for positive
selection
Neilson et al, 2004
Cnb1deficient and Control Thymocytes are
Equally Sensitive to TCR Crosslinking and
Peptide Complexed with MHC
%PI-AV-DP thymocytes relative to control
MCC-Mediated Cell Death dose response
100
Cnbf/
Lck-Cre + Cnbf/
80
60
40
20
0
-12
-11
-10
-9
-8
-7
-6
-5
-4
concentration MCC peptide [2 X 10 X M]
Neilson et al, 2004
Which downstream pathways contribute to
thymocyte selection?
(positive selection)
(positive selection)
Vav GEF is required for positive
selection
Active Rac1 changes positive to
negative selection
Female
Female/active Rac1
(Gomez et al, Immunity, 2001)
Data = Explanation?
• Synapse during negative selection (Richie et al, Immunity, 2002)
• Synapse formation in peripheral T cells requires
Vav/actin dynamics (Holsinger et al, Curr. Bio, 1998; Wulfing et al, PNAS,
2000)
• Positive selection likely does not form a synapse (Peter Ebert
unpublished)
• Therefore, lacking Vav/Rac etc. changes negative to
positive selection via an effect on synapse formation
and subsequent signaling
Which downstream pathways contribute to
thymocyte selection?
(positive selection)
(Modulator
(positive selection)
Both)
Negative Selection is Impaired in DP Thymocytes Deficient
in the Pro-Apoptotic Bcl-2 Family Member Bim
Bouillet et al 2002 Nature 618:108
Three Models for the CD4 vs CD8 Choice
From Fundamental Immunology, 4th ed. (Paul)
Modulation of lck activity can alter CD4/8 lineage
commitment (based on the fact that the cytoplasmic tail of CD4 binds to
more lck than CD8)
Dn-lck
Act-lck
Other studies indicate that the duration of signal controls
CD4 versus CD8 with CD4 requiring longer
(Yasutomo et al, Nature, 2000)
Regulatory T cells may develop
in the Thymus
HA specific TCR Transgenic mouse
Mouse expressing HA peptide
TCR Tg
CD25
2D model
Hypothetical 3D model of thymocyte
development
What a DP Thymocyte Needs to
Progress to the SP Stage
•
•
•
•
•
•
Everything it Needed to Become DP
Functional TCRa chain rearrangement
CD4 and MHC II (To be a CD4+ cell)
CD8, MHC I and TAP (To be a CD8+ cell)
ERK signaling
Calcineurin signaling
T Lymphocyte Maturation in the Thymus
T Lymphocyte Maturation after leaving
the Thymus
Some evidence that cells that have
recently left the thymus may have
special “status” in the periphery in
regards to tolerance induction or
the ability to homeostatically
proliferate
Antigen presentation and MHC
expression control thymocyte selection
•DC
•Medullary Epithelial cells
•Cortical Epithelial cells
•Endothelial cells
DO CERTAIN CELL TYPE CONTROL
DIFFERENT T CELL DEVELOMENTAL FATES?
-Positive selection
-Negative selection
-Receptor editing
Aire-/- mice develop autoimmunity because Aire Mediates
Ectopic Gene Expression in Medullary Stroma
Ohashi 2002 Science, 298:1348
The Transcription Factor Autoimmune
Regulator (Aire) Mediates Ectopic Gene
Expression in the Thymic Medullary Stroma
Ohashi 2002 Science, 298:1348
Take Home Messages
• Notch steers CLP’s to T lymphocyte Fate
• TCR chains undergo V(D)J recombination to generate
diversity; they also exhibit allelic exclusion
– TCRb chain is selected with an invariant pTa chain at the DN3
stage
– TCRa chain is selected with pre-existing TCRb chain at the DP
stage
• DP Thymocytes Undergo Positive and Negative Selection
to Generate a Population of Mature T Lymphocytes that
can Recognize Self MHC with Intermediate Affinity
• Recent Evidence Indicates that Positive and Negative
Selection Are Mediated by Distinct Pathways
• APC in the thymus may present self antigen that effect
negative selection
T Lymphocyte Development and Function is
Controlled at Various Stages of Development
by Signals through the T Cell Receptor
Signaling 1983