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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are needed to see this picture.

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