Specificity, Diversity, and Self-tolerance

of T-Cell Receptors by Thymic Selection

Andrej Kosmrlj Abhishek Jha

Mehran Kardar

Eric Huseby Arup K. Chakraborty

Antigen Presenting Cell

T Cell

T Cells orchestrate adaptive immunity, via

Receptors that recognize short peptides from pathogen proteins

TCR

pMHC

Self-tolerant, i.e. weakly binding to endogenous pMHC, to avoid autoimmune diseases, e.g.

Multiple sclerosis, diabetes, …

Diverse, to recognize an evolving landscape of pathogens.

Specific/degenerate, to lock on specific pathogen, and tolerate its mutants.

T Cells Receptors (TCRs) must be:

pMHC

n*= 5.3

What is the shortest peptide length that ensures self-tolerance?

Nu

mb

er o

f d

isti

nct

hu

man

pep

tid

es

Length of peptide, n

In principle all human peptides can be recognized from sequences of length 5-6.What is the physical mechanism that enables recognition of self-peptides?

T Cells are designed in the Thymuswhere a diverse repertoire of thymocytes is culled by encounters with self pMHC.

Positive selection TCR must bind sufficiently strongly to at least one self pMHC (implicated in MHC restriction, and sensitivity).

Negative selection TCR must not bind any self pMHC too strongly (deleting autoimmune TCR).

( )∑=

=

=10

1

,N

iii JLUE

TCR {L}

pMHC {J}

conserved variable

Miyazawa-Jernigan

Surviving T cells: E > EP for at least one peptide, E<EN for all peptides

Model for TCR selection in the Thymus

MHC peptide

E

+

--

✓

Diversity

The selection process biases the composition of amino-acids in mature TCRs:Negative selection leads to a slight preference for weak amino-acids.

STRONG WEAK

Frequency of TCR amino-acids

from known TCR-pMHC crystal structures

Fre

qu

ency

in T

CR

/Fre

qu

ency

in p

rote

ome

(MJ used for ordering)(Thermophiles used for ordering, Shakhnovich et al., PLOS, 2007)

Eric S. Huseby et al, Cell (2005); Nature Immunol. (2007),

compared the T cells of normal mice, with mice genetically engineered to present only one type of peptide in their thymus.

T cells selected in the thymus are challenged with an antigenic peptide, and reactive T cells identified.

Does a reactive T cell remain reactive upon mutating the peptide’s amino acids?

If mutations to an amino acid destroy reactivity with at least half the T cells,

the amino-acid is labeled a “hot spot”.

• Main results:

– Single peptide selection: few hot spots – cross-reactive T cells

– Many peptide selection: many hot spots – specific T cells

Specificity

• Specificity to antigen peptide:– Single peptide: mutations don’t matter – cross-reactive T-cells– Many peptides: mutations destroy reactivity – specific T-cells

† Hot-spots are defined as locations along the sequence, where mutations of a peptide amino acid destroy reactivity with more than half the reactive T cells

freq

uenc

y

Numerical results for hot-spots† mirror the experimental situation

Frustration during negative selection constrains TCR sequences

T C R

One peptide

Ep < E < EN

selected

Frustration during negative selection constrains TCR sequences

pii iN EjlUE >>∑ ),(T C R

One peptide

selected

Frustration during negative selection constrains TCR sequences

pii iN EjlUE >>∑ ),(T C R

One peptide

selected

E > ENnegatively selected

Many peptides

Optimizing interactions with one peptide can lead to “bad” interactions with another – FRUSTRATION.

Positive selection does not involve frustration.

T C R

Specificity, Diversity, and Self-tolerance

of T-Cell Receptors by Thymic Selection

Andrej Kosmrlj Abhishek Jha

Mehran Kardar

Eric Huseby Arup K. Chakraborty

[ ][ ]

),(),(

)exp(exp~)(1

))),((1ln(exp

))),((1ln(exp~

)()),((1()(

10

1

1

1

αα

α jlUjlE

EbE

EjlEM

EjlE

jpEjlElP

I

N

M

jN

M

jN

e

∑

∑

∏

=

=

=

=

=

Δ−ΔΘ−

−Θ−=

⎥⎦

⎤⎢⎣

⎡−Θ−

−Θ−=

rr

rr

rr

rrrr

Frustration during negative selection constrains TCR sequences

Frustration during negative selection constrains TCR sequences

Solution:

negative selection lead to choice of TCR amino acids which minimize interactions with other amino acids in En peptides.

Prob. that a TCR is selected

[ ] )(),(exp

..)..)...(..)((

exp)(

2,201,202221131211

20

1

10

1

jpjlbUh

hhhhhhh

hMlP

iij

jij

i

escape

=

+++++++=

⎥⎦

⎤⎢⎣

⎡−= ∑

==π

r

strong weak

COMPUTATIONAL RESULTSF

req

uen

cy i

n T

CR

/Fre

qu

ency

in

pro

teo

me

Robust to variations in potential

AMINO ACID FREQUENCIES FROM TCR-PMHC CRYSTAL STRUCTURES

(Thermophiles used for ordering, Shakhnovich et al., PLOS, 2007)

Fre

qu

ency

in

TC

R/F

req

uen

cy i

n p

rote

om

e

STRONG WEAK

Distribution of single site contact energies for selected T cell-antigenic peptide interactions

Increased number of moderate interactions

Decreased number of strong interactions

Selection with many peptides: TCR sequences contain amino acids that interact with Ag peptide amino acids moderately

strong weak

Modest interactions lead to specificity:

Weak multivalent interactions stabilize the interface, making each interaction important for recognition

Selection against one peptide – only few important sites

T C R

B3K 506 TCRC57BL/6 derived

MHC + peptide specific

YAe62.8 TCRIAb-SP derived

MHC + peptide degenerate

How much Free Energy of Binding is due to each amino acid for specific versus degenerate TCRs?

P-1EP-1E P8KP5KP3KP2Q

IAb + 3K KDΔΔG

Mutation μM kcal/molWT 7 0.0P-1 A 26 0.7P2 A 278 2.2P3 A > 550 > 2.6P5 A > 550 > 2.6P8 A 92 1.5

IAb + 3K KDΔΔG

Mutation μM kcal/molWT 8 0.0P-1 A 9 0.0P2 A 56 1.1P3 A 62 1.2P5 A > 550 > 2.5P8 A 7 -0.1

TCR sequences are specific, but diverse

One type of sequences are selected – ones with a predominance of weak amino acids

+

+

-

+

+

-+

+

++

+

-

TCR sequences are cross-reactive (degerate)

Several antigenic peptides composed of sufficient number of strong amino acids can interact productively with a given TCR

+

+

+

-

ADAPTIVE IMMUNITY IN HEALTH AND DISEASEFlexible system to combat

diverse pathogens

Mis-regulation leads to autoimmune diseases

Multiple Sclerosis

The challenge: develop principlesprinciples that govern the emergence of an immune response or autoimmunity and design design rulesrules for therapies

The problem: underlying mechanisms characterized by cooperative dynamic cooperative dynamic processesprocesses involving many componentsmany components and a spectrum of length/time scalesspectrum of length/time scales

Diabetes

dendriticcells

cytokines

chemokines

B7

B7 CD28+

CTLA4 -

TCRpMHC

B7

B7 CD28+

CTLA4 -

TCRpMHC

tissue

T CELLS RECOGNIZE SHORT PEPTIDES DERIVED FROM PATHOGEN’S PROTEINS

mixture of self (En) and antigenic (Ag/agonist)

As few as 3 Ag molecules in a sea of 30,000 En can activate a T cell (Nature, 2002)

How does this exquisite sensory apparatus work without frequent “noise”-induced autoimmune responses?

Extraordinary Sensitivity of T cells for Antigen

T cell sensitivity to Ag pMHC is predicated upon degenerate weak interactions with En pMHC. With Mark Davis’ lab. (Nature Imm., 2004; Nature, 2005; PNAS, 2007; unpublished)

INFLUENCE OF PARAMETERS IN MODEL

Parameters: Ec, EN, Ep

Ec-EN is large (conserved TCR-MHC interactions are very weak)

Ec Ep EN

Small gap

EcEp EN

Large gap

Positive selection limiting; These are the TCRs that are not positively selected (MHC restriction); one or many types of

peptides lead to similar consequences

Ec-EN is small (conserved TCR-MHC interactions are very strong)

Ec EpEN

Small gap

EcEp EN

Large gap

Negative selection very easy; For many types of peptides almost all T cells are negatively selected.

Theory/computation Experiments

statistical physics genetics biochemistry imaging

Life Sciences

Physical Sciences

Engineering sciences

chemical kinetics

An approach at the intersection of disciplines

THYMIC SELECTION THRESHOLDS

Sharp boundary separates positive and negative selectors MOLECULAR MECHANISM (w/A.Weiss’ lab.)

strongest weakest

Palmer lab, Nature (2006)

A MEMBRANE-PROXIMAL SIGNALING MODULE IMPLICATED (with Jayajit Das, Ashok

Prasad; Jeroen Roose, Art Weiss@UCSF)

A positive feedback loop results in digital signaling and a sharp threshold Cell, PNAS, in review (2008)

SHORT PEPTIDES ARE SUFFICIENT TO RENDER T CELLS SELF-TOLERANT AND REACTIVE TO FOREIGN

SHORT PEPTIDES ARE SUFFICIENT TO RENDER T CELLS SELF-TOLERANT AND REACTIVE TO FOREIGN

Longer peptides (length = n) will enable sampling of more distinct peptides ~ 20n

SHORT PEPTIDES ARE SUFFICIENT TO RENDER T CELLS SELF-TOLERANT AND REACTIVE TO FOREIGN

5 10 15 20 25101

102

103

104

105

106

107

108

# Unique peptides of length

n

Length of peptide, n

Condition for plateau:

20ln

ln ; 20

NnNn ==

Total # of unique peptides of size, nTotal size of human proteins

n=5.3

Group members:J. Das, A. Prasad, M. Artomov,

C. Govern, H. Zheng, A. Jha, J. Locasale, K. Fowler,M. Wolfson, A. Prabhakar, M. Yang, F. Liang, A. Kosmrlj.

Collaborators: M. Davis, A. Shaw, P. Allen, A. Weiss, J. Roose, H. Ploegh M. Dustin, M. Kardar, A Perelson, J. Chen, U. von Andrian, H. Eisen, V. Kuchroo, E. Palmer, E. Huseby.

ACKNOWLEDGMENTS

Immune Response Consortium

Funding: NIH

Is there an optimal peptide length?

0 5 10 15 20 250.00

0.01

0.02

0.03

0.04

0.05Constant Negative Selection Threshold ΔE=ΔE E

Neg−E

Neg=0.4

Length of Peptide n

Fraction of Reactive T Cells

Most TCR negatively selected

IMPORTANCE OF PEPTIDE IN ALLO REACTIVITY DEPENDS UPON THE ALLO MHC

Modest change Bigger change

Theoretical models and experimental tests (Nature Imm. (2004); Nature (2005); PNAS (2007); unpublished)

T cell sensitivity to Ag pMHC is predicated upon degenerate weak interactions with En pMHC that are tuned in the thymus.