Download - The Importance of “Seeing” Self Thymic selection Lymphocyte survival: A red queen hypothesis
The Importance of “Seeing” Self
Thymic selectionLymphocyte survival: A red queen hypothesis
Enhancing foreign antigen recognition
MCC: moth cyt. C88-103
A D L L A L Y K Q A T K 99
Ehrich et al. (1993)J Exp Med 178: 713.
TCR CDR 3 Loops Exhibit HighConformational Mobility and Heterogeneity
Willcox, et al. ‘99, Garcia et al. ‘98
Comparison of Peptide-MHC Epitope Contributionto 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 selectionLymphocyte survival: A red queen hypothesis
Necessities of life. .
Necessities of life.
The presence of the transcription factor LKLF is essential for the survival of naïve T cells, the cells that respond to foreign antigens.
Running to stay in place.
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) (M) (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 red
Monk et al. Nature 395:82 ‘98
Bilayers as artificial APCs
MHC + p
ICAM-1
PC
glass
• Defined, uniform surface for imaging• Mobile lipids and lipid-linked glycoproteins• Control molecular composition and
protein density• Molecules can be fluorescently
labeled prior to incorporation• Extent of membrane interaction can be
determined
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 Agonist 16.6 900 12.1 352 770
T102S 1 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
. ------------------------------------------------------------------------Ligand Relative activity Classification KA (mM1) kon (M1 s1) t1/2 (s) Density(molec./µm2) Total no. of molec.------------------------------------------------------------------------Cytochrome system MCC88-103 100 Agonist 16.6 900 12.1 352T102S 1 Weak agonist 4.2 1,500 1.92 193 310T102G <0.001 Antagonist 0.66 3,400 0.14 53 50K99A 0 Null ND ND ND <10 <10
(s)KA (mM-1) kon (M-1s-1)0 50 100 0 10000 20000 0 5 10 15
050
100150200250
050
100150200250
050
100150200250
Half-lifeMH
C-p
eptid
e de
nsity
in c
SM
AC
MHC-peptide density isdetermined by off-rate when MHC-peptide dose is fixed
Krummel et al. Science 289: 1349 ‘00
Clustering of CD3
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
1
2
3a
3b
4
5
5a
6
APC
Stage Morphology Synapse
T IntegrinTCR/CD3CD4
TCR engagement
TCR micro clusteringCD4 co-cluster
CentralizationCD4, CD45 excluded
Internalization of TCR
Kummel and Davis, Current Opinion of Immunology ‘02
Polarizes T cell secretory apparatusSustained TCR signaling
T Cells are important but their functions are unclear
k/o mice fare worse or differently in several infection models.
Function-the importance of location IEL vs. IEL
Antigen recognitionTargets
Innate vs. Adaptive
IELsmay be able to deal with a broad range of pathological situation quickly, despite the diversity of TCRs
IELs 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, IEL are ready to act, with no requirement for new gene synthesis.
IELs 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
TCR recognizes antigens directly, with no antigen processing and presentation requirement.
Implication-Cells and pathogens can be recognized directly, responses can be initiated.
Challenge-To identify such ligands and to find a normal population of T cells recognizing them.
G8- Balb/c nude immunized with B10.BR spleen cells
KN6- Double negative thymocyte from C57B/6
T10/T22 has been found twice!
.
A population of T cells recognize T22
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1:100-1:500 peripheral T cells recognize T22
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T22 HLA-A2
Anti- TCR
Tetr
amer
T22 tetramer
Crowley et al. Science 287: 314, 00
T cell Status of immune system Frequency1
T10/T22- specific T cells ~ 1/250
MHC/peptide specific T cells(not primed) ~ 1/1.000,000
MHC/peptide specific T cells(Immunized; effector phase) ~1/2-1/100
Comparison of ligand affinities of and TCR
TCR Type Ligand Kd (M) kon (M-1s-1) koff (s-1) t1/2
G8 T10 /T22 0.1 65,000 0.008 88
2C p2Ca/Ld 3.3 8,300 0.027 26
2B4 MCC/IEk 40.0 1,600 0.060 12T102S/IEk 240.0 1,500 0.360 2
LBK5 IEk > 240.0 N.D. 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) G(kcal/mole)
T10/m2m 43 1.5
peptide/MHC 65-72 >5
FcRn (pH 6) 62 (pH 8) 51
Immunoregulatory role for T10/T22 specific T cells?
activation
cytotoxicity?cytokine release?Others?Crowley et al. Science 287:314 ‘00
lymphocyte Tcell
T22T10 TCR
Expression of multiple class I MHC subclasses in distinct regions of the mature CNS.
Huh et al. Science 290:2155, 00