Dr. Kalet León Monzón

Modeling the role of IL2 on CD4+T cell dynamics: On the effect of IL-2 modulating therapies (2)

Summer school Leeds (2009)

Crosstalk model of dominant tolerance to self The key role of IL-2

T

R

T

T

R

RRegulatory T cells inhibit the activation and proliferation of helper T cells by an interaction which requires their colocalized activation at the APCs.

Helper T cells produce a growth factor that Regulatory T cells use to proliferate and survive

Leon et al J. Theor. Biol. 2000 Leon et al J. Immunol. 2001

Leon et al 2002 pHD thesis

IL-2 is a good candidate growth factor

In vitroRegulatory cells do not express IL-2

Regulatory cells over-express CD25

Regulatory cells proliferate when stimulated in the presence of IL-2

Regulatory cells inhibit IL-2 mRNA expression in their targets

In co-cultures, regulatory T cells proliferate/survive by using the IL-2 produced by their targets. Moreover they consume this IL-2 starving the normal T cell

In vivoAnimals deficient in IL-2, CD25, IL-2Rb chains exhibit lympho-proliferative disorders

and autoimmunity

These autoimmune disorders, in receptor deficient mice, can be prevented by transfer of wild-type Tregs. But in Il-2 deficient mice, they can be prevented by the addition of Il-2 or

transfer of IL-2 producing cells.

In-vivo depletion of IL-2 induces autoinmunity in 10-20 day thymectomized BalB/c mice.

Our Master Modelling Plan

To extend the Cross-Regulation model to include IL2 dynamics

-Check in which conditions the extended models conserve the key dynamical features of the original cross-regulation model.

-Compare models of different hypotheses about the specific role of IL2 in T cell mediated suppression

To study the effect of IL2 modulating therapies currently used in clinical or preclinical studies

-Study the impact of IL2 depletion treatments.

-Study the impact of IL2 administration treatments.

Adding IL-2 in the cross-regulation model

Basic interactions for T cells

-E cells produce IL-2 upon activation at the APCs.

-E cells need both APCs and c cytokine signal to proliferate and survive in vivo.

-E cells use in vivo either IL2 or another homeostatic cytokine (denoted IL-) to proliferate and/or survive.

-R cells do not produce IL-2

-R cells need both APC and IL-2 signals to proliferate and survive in vivo.

-R cells inhibit E cell activation and production of IL2 by a mechanism requiring their co-localized activation at the APCs

-R cells constitutively over express the Alpha chain of IL2R (CD25), while E cells up regulate this molecule transiently after activation.

-The main sources of IL2 in vivo are the helper CD4+ FoxP3- T cells (our E cells)

-IL-2 concentration is reduced in vivo by both by renal elimination and cell internalization upon binding to the IL2 receptor

ER

APC

IL-2(+)

(+)IL- (-)

(-)

Postulates: regarding T cells Live Cycle

EN EA

R cells

Production of IL-2

EC

{IL-2, IL-

2x

Death

ThymusRNRA

Cognate APCs

RC

{IL-2

2x

Death

R cells live cycle E cells live cycle

Three functional states for E and R cells during their life cycle:

Naïve: T cells that have not interacted recently with APCs

Activated: T cells which have been activated by interaction with APCs.

Cycling: T cells irreversibly committed to cell division. They are activated T cells that have obtained enough cytokine-related signals

Postulates: regarding IL2 “life Cycle”

EN

R cells

Cognate APCs

EA

Production binding to IL2r Internalization

+degradation

Degradation/ elimination

IL2 interactions

unbinding of IL2r

F2IL TB2IL

Processes regarding IL2:

IL2 is produced by E cells upon activation on the APCs

IL2 is degraded by renal elimination while not bound to receptor.

IL2 is internalized and degraded by T cells, following its binding to receptors.

Postulates: regarding interactions with APCs

T cell get signals from APCs upon direct conjugation. Conjugation to APCs is assumed to be a fast process and taken to quasi-steady state equilibrium.

Cells in different states (naïve, activated, cycling) have different conjugation constants.

Regulatory cells suppress E cell activation or IL2 production by a mechanism dependent on co-localized activation on APCs.

EN

R

EA

EN

(-)

RA

EN

RN

RA

Postulates: regarding interactions with IL2

EA

RA

+ {IL2 or IL EC

EN

EN

{IL2+ RC

RN

RN

R and E cells in different functional states express different number of IL2 receptors on their surface. Binding of IL2 to these receptors is assumed to be a very fast process taken to quasi-steady state equilibrium

The mean number of Il2 receptors bound per cells is computed.

The fraction of cells differentiating into cycling cells is computed as a sigmoid function of this number 0 500 1000 1500 2000

0

0.2

0.4

0.6

0.8

1.0

SE

Bound receptors/ cell

Fra

ctio

n o

f ce

lls

diff

eren

tiat

ing

SR

Model Equations

)RIL+RIL+R(ILK

)EIL+E(ILKILKAs

REK=

dt

ILd

CCR

BAAR

BNNR

BRin

CCE

BAAE

BEin

id

)(sBTB

Nipi

222

22212

1

CRPAhR

Bh

R

hRBR

SC RKR

)(Il+)(S)(Il

K=dtRd

A

A

22

ARS

BN

RA

A RKRK=dt

Rd

FN

RdAhR

Bh

R

hRR

SCRp

BN

RAR

N RKR)(IL+)(S

)(SKα+RK+RK=

dt

RdA

22

CEPAhhE

Bh

E

hhEBE

SC EKE

(Ilα+)(Il+)(SIlα+(Il

K=dtEd

A

A

)2)()2

AES

)η(sBTB

NEA

A EKAs

REK=

dt

Ed

1

1

FN

EdAhhE

Bh

E

hEE

SC

)η(sBTB

NEAE

N EKE(Ilα+)(Il+)(S

)(SKαEE

PK+As

REK=

dt

EdA

)221

1E

ffec

tors

Cel

lsR

egul

ator

y C

ells

IL2

Plus algebraic relations to compute the number of cells bound to APCs and of Il2 bound to its receptor

Handling parameters

The models have 30 parameters, thus although we are interested in generic properties not dependent on specific parameter values, we try to find a good reference set of parameters based on existing experimental data.

The idea is to use this set of values as a reasonable reference to study model properties, but then study robustness of model behavior to parameter changes always around these reference values.

Thus we do a partial calibration parameterization of the model

Lymph node volume VNL=10 -6 L Personal communication

# of dendritic cells. APCS= 104 – 105 Personal communication

T cell division rates Cycling time (E=R) = ln2/tc1/2 ; tc1/2=5 h

are taken such that the mean doubling time is between 6-20 h

Stoll, Science, 2002. 296: 1873

(Confocal microsc)

T cell death rate Kd= ln2/t1/2 ; t1/2=7-30 días Surh et al, 2006

# of High affinity IL2 receptors

700 – 1000R up to 10 time more

Smith KA, Cell Res, 2006. 16: 11

Conjugation IL2 to IL2Rs alpha: Kon= 107 M-1s-1 ;

Koff= 10-1 s-1

Beta-Gamma: Kon= 105 M-1s-1 ;

Koff= 10-4 s-1

Wang H, J Exp Med, 1987, 166:1055

IL2/IL2R internalization rate

= ln2/t1/2 ; t1/2=15 min

Fallon, J Biol Chem, 2000. 275: 6790

Free IL2 degradation Rate = ln2/t1/2 ; t1/2=20 min Donohue et al, 1984

Most parameters values are bounded from existent dataMost parameters values are bounded from existent data

;K;KEAK R

A ;KES

RS

=KEPK R

P

K id

=KEinK R

in

Most parameters values are bounded from existent dataMost parameters values are bounded from existent data

Conjugation constant T to APC

Kon= 10-13 L.s-1.cél-1

Koff=10 -3 s-1

Equal for R and E cells

Castellino F. Nature, 2006. 440: 890

García Z. PNAS 2007. 104(11): p. 4553-8

Constant for IL2 association and dissociation in the cell membrane to diferent receptor chains

IL2/ + :

Kon=10-4 s-1 ; Koff= 10-4

s-1

IL2/

Kon> 10 s-1 ; Koff= 10-1

s-1

Wang H, J Exp Med, 1987, 166:1055

IL2 production rate

Kpi> 10 3 molécs / h Yamazaki S, J Exp Med, 2003. 198: 235

Sensibility to IL2 derived signal

SE=SR=500; h=4 Smith KA, 2004 Med Immunol, 3: 3

Bound IL2 (molec./cell)x10-3

Bou

nd IL

2 (m

olec

./cel

l) \

Fre

e IL

2 (p

M)

Does the extended model does conserve the key dynamical features of the original

cross-regulation model ?

Bistability One stable state interpreted as tolerance and the other as immunity

Adaptive response to the quantity of APCsChange from tolerance to immunity with abrupt increase of APC numbersAdaptation the tolerant state upon slow increase of APC numbers.

Bistability is conserved: Two steady states Autoimmunity and Tolerance

The actual values of T cell numbers and IL2 concentration in the Tolerant and AutoImmune States are quite reasonable despite not direct fitting of the parameter values.

Other two types of steady state are observed: The trivial immune state (OS) where both E and R cell collapse to very small values; The reinforced tolerance state (TR) containing only expanded R cells, which could be attained when external source of IL2 is added.

Parameter Regions Autoimmunity IS Self-Tolerance TS

Time (weeks)

IL2 co

nc. (p

M)

0 2 4 6 8

IL2

ET

RT

104

103

102

101

100

10-1

Time (weeks)

Nu

mb

er o

f ce

lls

IL2

ET

RT

106

108

102

104

0 2 4 6 8

IL2

deg

rad

atio

n r

ate

Kd

i (h

-1)

IL adimen. Conc.200 300 500

10

103

104

1

102

OS, IS

OS IS

TS

TS, IS

Adaptive response to APC variation is also conserved

0 2 4 6 8 10

10

106

108

2

104

Time (weeks)

1010

ET

RT

10

103

-1

101

A

1 3 5 7

1

102

time

Nu

mb

er o

f ce

lls

Free

IL2 c

on

c. (pM

)

0 2 4 6 8 10

10

106

108

2

104

1010

Time (weeks)

IL2F

ET

RT

10

103

-1

101

1 3 5 7

1

102

time

A

Nu

mb

er o

f ce

lls

Free

IL2 c

on

c. (pM

)

IL2F

Bifurcation diagrams, with the number of APCs, show also a similar structure to those obtained with the original cross-regulation model.

Abrupt APC change Gradual APC change

Three necessary parameter conditions for a reasonable model behavior

R cells use IL2 more effectively at low concentrations

E cells have net growth rate faster than R cells in IL2 excess

E cells have to use a cytokine alternative to IL2 to proliferate

AR

RAE

E RIS>RIS //

Rd

RA

REd

EA

E KKKKKK //

0Ilα

The effect of knocking down IL in the modelIL

2 d

egra

dat

ion

rat

e K

di (h

-1)

IL adimen. Conc.

200 300 500

10

103

104

1

102

OS, IS

OS IS

TS

TS, IS

If Il is forced to go to zero, the system reaches a parameter regime where only autoimmune or a trivial steady state could be attained

This result would predict a dramatic effect on T cells populations of knocking down this hypothetical cytokine

Interestingly the only gamma chain family cytokine whose knockout has a profound effect on the size of T cell population is Il7. Thus the model would predict it to be the best candidate for our IL cytokine.

Including IL2 breaks locality and specificity of interactions in cross-

regulation model

ER

APC

IL-2(+)

(+)IL- (-)

(-)T

R

TT

RR

In the cross-regulation model, all the interactions are local near the APCs

In the extended models, some interactions are non-local (sharing of IL2)

Implications for independence of concomitant responses to different self-

antigen in a given lymph node

A1

A2

<- E1,R1

<- E2,R2

In the extended models there should be strong

interference between the immune reactions of

different clones

Comparing different hypotheses in the suppressive effect of R cells over E cells

IL-2

ER

APC

(+)(+)

IL- (-)

(-)

ER

APC

IL-2(+)

(+)IL- (-)

(-)

Extended model variant 1:R cells drive competition for IL2

Full coupling of responses to different

antigens

Extended model variant 2:R cells also inhibit E cell activation

Bi-standard interactions between clones, but still independent responses

But, another possible solution to be explored in the future is further locality on the use of

IL2 inside a given lymph node

A1

A2

<- E1,R1

<- E2,R2

But since the suppression mechanism relies on competition of E and R cells for IL2, the lymph node will have to be subdivided in sub-domains presenting different antigens

The extended model can be used to study the effect of IL2 modulation therapies. i.e Therapies that deplete IL2 and Therapies that inject IL2

Let´s study whether these treatments can trigger a switch of steady states in the models: Tolerance to immunity and viceversa.

IL2

deg

rad

atio

n r

ate

Kd

i (h

-1)

IL adimen. Conc.

200 300 500

10

103

104

1

102

OS, IS

OS IS

TS

TS, IS

IL2 depletion therapy can subvert the preexistent Autoimmune or Tolerant steady states

We model IL2 depletion treatment by an incerase in

IL2 degradation rate

Treatments that deplete IL2 are predicted to both break preexistent tolerance by taking the system to autoimmune state or to reset a previously autoimmune system back into the tolerance state.

IL2 depletion can subvert the preexistent Autoimmune or Tolerant steady states

Breaking Tolerance

Reestablishing Tolerance

This supports applications of IL2 depletion both in the treatment of autoimmune diseases and cancer, but these applications strongly depend on treatment schedules

0 5 10 15 20

106

108

102

104

104

103

102

101

100

10-1

IL2 co

nc. (p

M)N

um

ber

of

cells

IL2

ET

RT

+

0 10 20 30 40

106

108

102

104

104

103

102

101

100

10-1

IL2 co

nc. (p

M)N

um

ber

of

cells

IL2

ET

RT

Time (weeks)

+ -

Dep

leti

on

Do

seD

eple

tio

n D

ose

104

102

1 2 3 4 5 6 7

103

IS

TS

6

1 2 3 40

104

10

102

105

103

IS

TS

Duration of treatment (w)

IL2 Blocking MAbs have been shown to induce autoimmunity, but in adult thymectomized mice.

The model explains the thymus dependence of this effect by showing a decrease in the minimal dose of depletion treatment, which is required to change state, as the thymus output is decreased.

5.104

105

2.104

7.104

3.104

1 2 3 40

Do

sS

IS

TS

ΓT= 10-4

ΓT= 10-6

ΓT= 10-8

Duration of treatment

(weeks)

Setoguchi et al. JEM 2005, 201(5):723–735

Anti-IL2 MAbs can induce tumor regression

1 mg AcM IL-2 iv

5x105 IU/Kg sc IL-2

5 d2 d

F10 F10 irradiated

Montero et al. 2005 unpublished results

IL2 injection therapy can not subvert the preexistent Autoimmune or Tolerant steady states

We model IL2 injection treatment by an increase in the

external source of IL2 (i) Γi

0.1 1 10 100 1000 10000

10

106

108

2

104

Ilα

TS, IS

RT, IS

OS, IS

Therapy with IL2 can only reinforce the preexistent Steady State

Reinforcement of Tolerance Reinforcement of Autoimmunity

This might explain the limited success of IL2 therapy in Cancer. It would be predicted to result just on those patients with a preexistent but insufficient immune response.

It will predict that, despite the role of IL2 as homeostatic factor for Tregs, it might not be used as therapy for autoimmune diseases.

ET

Time (weeks)IL

2 con

c. (pM

)Nu

mb

er o

f ce

lls

0 2 4 6 8 10 12 1410

107

109

105

103

RT

102

104

1

10-1

105

10

103

IL2

ET

Time (weeks)

IL2 co

nc. (p

M)N

um

ber

of

cells

0 2 4 6 8 10 12 1410

107

109

105

103 R

T

102

104

1

10-1

105

10

103

IL2

IL2 administration can reinforce tolerance promoting allograft acceptance

2*105 F10 sc fd

Check growth of allogenic tumor

2 daily doses of IL-2, sc tail base

Incidence of MB16F10 tumors

0 5 10 15 20 25 30 35 40 450

20

40

60

80

100PBS

IL-2 5*103 U/kg

IL-2 5*104 U/kg

IL-2 5*105 U/kg

IL-2 5*106 U/kg

Días

Inci

den

cia

tum

ora

l (%

)

Montero et al Ann. N.Y. Acad. Sci. 1107: 239–250 (2007).

IL2 administration can reinforce tolerance preventing EAE induction

Interestingly this tolerizing effect of IL2 or Il2+MAbs is only observed when it is administered before the onset the disease.

Montero et al Ann. N.Y. Acad. Sci. 1107: 239–250 (2007).

Could the particular interface of the IL2 being recognized by IL2 blocking MAbs make a difference?

Chain

Chain

Chain

IL-2

Dimerization of the and chain is responsible for signaling

MAbs, could block the IL2 binding to the chain of the receptor without affecting the binding to the and chains or vice versa

MAbs blocking binding to and/or chain will fully inhibit signaling, but those just blocking binding to chain will allow it

Thus we study these classes of MAbs being used alone or in preformed complexes with IL2

Modeling therapies with IL-2 and/or anti-IL2 MAbs in a virtual mouse

BLOOD

Vs=2.5 mL

ER

APC

IL-2(+)

(+)IL- (-)

(-)

As many repeated units of Lymph nodes as necessary to guaranteed a T cell repertoire with 108 cells, 107 of which might be autoreactive T cells that participate in

the modeled dynamics

Vn=1.0 LLymph Node

Renal and/or fecal elimination of molecules

#T=107

#APC=1 105

The

rapi

es o

f in

tere

st

Model parameters are calibrated from independent publicly available Data

MAbs blocking either or interfaces of IL2 similarly break a preexistent tolerant state

106

108

102

104

Nu

mb

er o

f ce

lls

IL2

ET

RT

5 mg 104

103

102

101

100

10-1

IL2 co

nc. (p

M)

Face

Face

IL2

ET

RT

3 mg

IL2

ET

RT

1 mg

0 5 10 15 20

106

108

102

104

Nu

mb

er o

f ce

lls

IL2

ET

RT

Time (weeks)

2.5 mg

0 5 10 15 20

IL2

ET

RT

Time (weeks)

1.7 mg

0 5 10 15 20

104

103

102

101

100

10-1

IL2 co

nc. (p

M)IL2

ET

RT

Time (weeks)

1.0 mg

The difference between these MAbs is just quantitative. Affinity for IL2, half life and even the particular Kon and Koff values strongly influence the stringency of the effect.

KD=10-10 M

t1/2=3 days

MAbs blocking but notinterfaceof IL2 can reverse autoimmunity, reestablishing tolerance

0 10 20 30 40

106

108

102

104

104

103

102

101

100

10-1

IL2 co

nc. (p

M)

Nu

mb

er o

f ce

lls

IL2

ET

RT

Time (weeks)

+ -

Face MAb

0 10 20 30 40

106

108

102

104

104

103

102

101

100

10-1

IL2 co

nc. (p

M)

Nu

mb

er o

f ce

lls

IL2 ET

RT

Time (weeks)

Face MAb

Despite achieving similar reduction of IL2 concentration, these MAbs behave in a qualitatively different way.

Interface MAbs reduce the actual IL2 related signal more, triggering a larger reduction of E cell number that ultimately allows the reestablishment of tolerance.

+ -

KD=10-10 M

t1/2=3 days

Dos=20 g/d

What about injecting Immune-complexes?

MAbs + IL2

Both types of Immunocomplexes reinforce tolerant state.

Both types of immunocomplexes appear to expand regulatory T cell activity in the model, although interface derived complexes appear quantitatively more efficient at low doses

However injection of interface MAbs +IL2 provokes a very large and sustained increase in the availability of IL2 signal, that could explain its reported effect over MP CD8+ cells.

0 5 10 15 20

106

108

102

104

104

103

102

101

100

10-1

IL2 co

nc. (p

M)N

um

ber

of

cells

IL2

ET

RT

Time (weeks)

5 g/d 1 week

Interface MAb +IL2

Interface MAb +Il2

0 5 10 15 20

106

108

102

104

104

103

102

101

100

10-1

IL2 co

nc. (p

M)N

um

ber

of

cells

IL2

ET

RT

Time (weeks)

20 g/d 1 week

Availability of Free IL-2 signal

0 5 10 15 20

105

103

101

10-1

Co

nc.

(p

M)

IL2

Complexes

Time (weeks)

20 g/d 1 week

Immunocomplex with interface but notinterface anti- IL2 MAbs can reestablish Tolerant State.

106

108

102

104

104

103

102

101

100

10-1

IL2 co

nc. (p

M)

Nu

mb

er o

f ce

lls

IL2

ET

RT

Time (weeks)

0 10 20 30 40 50 60 70

0.3 mg/d 50week

Face MAb +IL2 Face MAb +IL2

106

108

102

104

104

103

102

101

100

10-1

IL2 co

nc. (p

M)

Nu

mb

er o

f ce

lls

IL2

ET

RT

Time (weeks)

0 10 20 30 40 50 60 70

0.3 mg/d 50week

Despite both types of immunocomplexes appearing to expand regulatory T cell activity in the model, only interface derived complexes appear capable of reversing the autoimmune state.

Conclusions through the Modeling relationship

ExperimentalObservations

Model

Postulates, Axioms

Predictions

),,,( 432111 NNNNF

dt

Nd

),,,( 432122 NNNNF

dt

Nd

),,,( 432133 NNNNF

dt

Nd

Model Predictions and explanationsIt explains:

It predicts:

• The Cross-regulation model is compatible with IL2 being a growth factor of both E and R cells.

• That in the absence of IL7 the immune system could be either in a steady state with a few E and R cells sustained by thymic output or in an autoimmune steady state.

• That IL2 depletion can induce autoimmunity and that the absence of thymus could favor this effect.

• That IL2 depletion treatments could favor tumor rejection or potentiate cancer vaccines.

• That IL2 injection treatment could be effective in a subset of cancer patients or can be good adjuvants for some vaccines.

• That Il2 injection treatment could reinforce tolerance and promote allograft acceptance if applied before graft implant.

1. That IL7 is the likely gamma chain family cytokine that helper cells use as an alternative to Il2 to growth in vivo

2. That Il2 depletion treatments can be used to reestablish tolerance in an autoimmune individual. (interface Mabs)

3. That IL2 treatment could only reinforce a preexistent immune state, thus is could be used to adjuvate vaccines, but applying it preferentially after the vaccine and not before.

4. That Il2 can not be used as a therapy to autoimmune diseases, despite being the main growth factors for Tregs. (The same goes for immunocomplexes)

5. That the lack of success of Il2 based therapies on cancer is a consequence of the heterogeneity of the preexistent immune response of patients to tumors.

Acknowledgements

CIMLic. Tania CarmenateM.Sc. Karina García Lic. Ileana GonzalezLic. Jose Antonio Dr. Enrique MonteroDr. Agustín Lage

IGC (Portugal)Dr. Jorge Carneiro

Financial support

IGC and CIM