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Transcript
The Importance of “Seeing” Self
Thymic selection
Lymphocyte survival: A red queen hypothesis
Enhancing foreign antigen recognition
MCC: moth cyt. C
88-103
ADLLALYKQATK
99
Ehrich et al. (1993)
J Exp Med 178: 713.
TCR CDR 3 Loops Exhibit High
Conformational Mobility and Heterogeneity
Willcox, et al. ‘99, Garcia et al. ‘98
Comparison of Peptide-MHC Epitope Contribution
to TCR Stability and Association
Stability
Association
TCR may dock on the MHC first, and the CDR3s can adjust to different peptide.
A proof of principal:
Altered peptide ligand and differential signaling
Thymic selection
Lymphocyte survival: A red queen hypothesis
Necessities of life.
.
Running to stay in place.
The presence of the transcription factor LKLF is essential for the
survival of naïve T cells, the cells that respond to foreign antigens.
Initiation of TCR Signaling
Multivalent engagement of peptide/MHC
ligand
The need for membrane reorganization and
the formation of specialized junction
(SMAC or immunological synapse)
Ligand
KD
kon
koff
t1/2 (s)
(mM)
(M-1s-1)
(s-1)
-----------------------------------------------------------------------MCC/2B4
40
900
0.06
12.1
Ld/2C
3.3
8300
0.027
26
IgM
0.011
110,000
0.0012
580
IgG
0.001
270.000
0.00025 2800
CD80/CD28Ig
2.5
>600,000
>1.6
<1.6
Davis, SJ and van der Merwe, A.
(1996) Imm Today 4:177.
Talin-green, PKC q-red
Monk et al. Nature 395:82 ‘98
Bilayers as artificial APCs
• Defined, uniform surface for imaging
• Mobile lipids and lipid-linked glycoproteins
• Control molecular composition and MHC + p
protein density
• Molecules can be fluorescently
labeled prior to incorporation
• Extent of membrane interaction
determined
glass
ICAM-1
can be
PC
2B4 T cells with IE/MCC
IE-green, ICAM-1-red
Grakoui et al. Science 285: 221
Ligand
Relative Classification
KA
kon
t1/2 (s) Density
Total number
activity
(mM-1) (M-1s-1 )
(molec./µm2) of molecules
-----------------------------------------------------------------------MCC
100
T102S
1
Agonist
16.6
900
12.1
352
770
Weak agonist
4.2
1,500
1.92
193
310
T102G
<0.001
Antagonist
0.66
3,400
0.14
53
50
K99A
0
Null
ND
ND
ND
<10
<10
MHC-peptide density in cSMAC
MHC-peptide density is
determined by off-rate when MHCpeptide dose is fixed
250
250
250
200
200
200
150
150
150
100
100
100
50
50
50
0
0
50
100
KA (mM-1 )
0
0
10000
20000
kon (M-1 s-1 )
0
0
5
10
15
Half-life (s)
Clustering of CD3 z
Krummel et al. Science 289: 1349 ‘00
Wulfing et al. (2002) Nat Immunol 3:42-47.
“Null” peptide MCC 99A can form dense clusters in
the presence of trace amounts of MCC/ I-Ek
complexes
Inhibitory peptide (MCC 99E)/IE complexes do not
Synapse Formation
• Synapse formation appears to be driven by
costimulation and involves the active
transport of membrane/cytoskelatal
associated molecules/rafts to the interface
(Wuelfing et al. 1998, Viola et al. 1999,
Wuelfing, Irvine et al., unpublished). It is
sensitive to PI3Kinase and myosin motor
inhibition.
Costimulation dependant bead movement.
Wuelfing et al.Science 1998
Stage
1
2
3a
3b
4
5
Morphology
T
Synapse
Kummel and Davis, Current
Opinion of Immunology ‘02
Integrin
TCR/CD3
CD4
APC
TCR engagement
TCR micro clustering
CD4 co-cluster
Centralization
CD4, CD45 excluded
Internalization of TCR
Polarizes T cell secretory apparatus
Sustained TCR signaling
gd T Cells are important but their
functions are unclear
gd k/o mice fare worse or differently in several
infection models.
Function-the importance of location
abIEL vs. gd IEL
Antigen recognition
Targets
Innate vs. Adaptive
gdIELs may be able to deal with a broad range of pathological
situation quickly, despite the diversity of TCRs
gdIELs are constitutively transcribing cytolytic, NK activating and
inhibitory genes. Activation of cytotoxic function, however, is likely
to occur through the overcoming of inhibitory receptor signals. Thus,
gd IEL are ready to act, with no requirement for new gene synthesis.
gdIELs could be "fired" either through general activatory surface
molecules, or through the T cell receptor, with potentially different
outcomes in each case.
Fahrer AM et al. P.N.A.S. 98:10261 (2001).
CDR3 Length Distribution of Immune Receptor Chains
Rock et al. J. Exp. Med.
179:323 ‘94
gd TCR recognizes antigens directly, with no
antigen processing and presentation requirement.
ImplicationCells and pathogens can be recognized directly,
responses can be initiated.
ChallengeTo identify such ligands and to find a normal
population of gd T cells recognizing them.
T10/T22 has been found twice!
G8- Balb/c nude immunized with B10.BR
spleen cells
KN6- Double negative thymocyte from C57B/6
.
A population of gdT cells recognize T22
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
T22
HLA-A2
QuickTime™ and a
Photo - JPEG decompressor
are needed to see this picture.
T22 tetramer
1:100-1:500 peripheral gd T cells
recognize T22
Anti-gd TCR
Crowley et al. Science 287: 314, 00
T cell Status of immune system
Frequency1
T10/T22- specific gd T cells
~ 1/250
MHC/peptide specific ab T cells
(not primed)
~ 1/1.000,000
MHC/peptide specific ab T cells
(Immunized; effector phase)
~1/2-1/100
Comparison of ligand affinities of ab and gdTCR
TCR Type Ligand
Kd (mM) kon (M-1s-1) koff (s-1) t1/2
G8
gd
T10 /T22
0.1
65,000
0.008
88
2C
ab
p2Ca/Ld
3.3
8,300
0.027
26
2B4
ab
MCC/IEk
40.0
1,600
0.060
12
T102S/IEk
240.0
1,500
0.360
2
> 240.0
N.D.
LBK5 gd
IEk
N.D.
(N.D. = not determined)
T10/T22 on cell surface are likely to have a shorter
half-life than classical MHC molecules
Tm(oC) DG(kcal/mole)
T10/mb2m
43
1.5
peptide/MHC
65-72
>5
FcRn (pH 6)
(pH 8)
62
51
Immunoregulatory role for T10/T22
specific gd T cells?
lymphocyte
gd Tcell
activation
T22
T10
gd TCR
cytotoxicity?
cytokine release?
Others?
Crowley et al. Science 287:314
‘00
Expression of multiple class I MHC subclasses in
distinct regions of the mature CNS.
Huh et al. Science 290:2155, 00