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Dr. Kalet León Monzón

Modeling the role of IL2 on CD4+T cell dynamics: On the effect of IL-2 modulating therapies (2). Dr. Kalet León Monzón. Summer school Leeds (2009). R. R. R. T. T. T. Crosstalk model of dominant tolerance to self The key role of IL-2.

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Dr. Kalet León Monzón

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  1. Modeling the role of IL2 on CD4+T cell dynamics: On the effect of IL-2 modulating therapies (2) Dr. Kalet León Monzón Summer school Leeds (2009)

  2. R R R T T T Crosstalk model of dominant tolerance to self The key role of IL-2 Regulatory 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

  3. In vitro Regulatory 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 vivo Animals deficient in IL-2, CD25, IL-2Rb chains exhibitlympho-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.

  4. 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.

  5. Adding IL-2 in the cross-regulation model Basic interactions for T cells IL-2 (+) • E cells produce IL-2 upon activation at the APCs. • E cells need both APCs and gc cytokine signal to • proliferate and survive in vivo. • E cells use in vivo either IL2 or another homeostatic • cytokine (denoted IL-a) 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 (+) (-) IL-a (-) R E APC

  6. Postulates: regarding T cells Live Cycle R cells Cognate APCs Production of IL-2 Death Death RA RN EN EA Thymus {IL-2, IL-a} {IL-2} 2x 2x RC EC E cells live cycle R 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

  7. T IL 2 IL 2 B F Postulates: regarding IL2 “life Cycle” IL2 interactions EN Cognate APCs Production binding to IL2r Internalization + degradation R cells unbinding of IL2r EA Degradation/ elimination 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.

  8. EN EN EN R RA Postulates: regarding interactions with APCs EA RN RA (-) 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.

  9. EN EN RA 1.0 0.8 Fraction of cells differentiating 0.6 SR 0.4 SE 0.2 0 0 500 1000 1500 2000 Bound receptors/ cell Postulates: regarding interactions with IL2 + {IL2 or ILa} EA EC + RN {IL2} RC 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

  10. E K P Model Equations - η(s 1 ) æ ö æ ö B h d E R (S ) ç ÷ ç ÷ G - × × - × + × × × - × E B E E F N T E = K E 1 + 2 E α K E K E ç ÷ ç ÷ E A N C S A d N × E h h h dt s A (S ) + (Il 2 ) + (Il α ) è ø è ø A E B - η(s 1 ) Effectors Cells æ ö B d E R ç ÷ × × - - × E B E A T = K E 1 K E ç ÷ A N S A × dt s A è ø æ ö E h h d E (Il 2 ) + ( Il α ) A ç ÷ × × - × E E C B = K E K E ç ÷ S A P C E h h h dt (S ) + (Il 2 ) + (Il α ) è ø A E B æ ö h d R (S ) ç ÷ G - × × × × × - × R B R R R F N R = K R + 2 K R + α K R K R ç ÷ R A N p C S A d N R h h dt (S ) + (IL 2 ) è ø A R B d R Regulatory Cells × - × R B R A = K R K R A N S A dt æ ö R h d R (Il 2 ) A ç ÷ × × - × R R C = K B R K R ç ÷ S A P C R h h dt (S ) + (Il 2 ) è ø A R B - 1 (s ) æ ö B R d IL 2 IL2 E E ç ÷ G + × × - - × - × - i B i E T = K E 1 K IL 2 K (IL 2 E + IL 2 E ) C A ç ÷ i p N d in B A B C × dt s A è ø R R R - × R K (IL 2 R + IL 2 R + IL 2 R ) N C A in B N B A B C Plus algebraic relations to compute the number of cells bound to APCs and of Il2 bound to its receptor

  11. 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

  12. R =K P E K R E R E ;K K ;K ;K P A A S S E R K =K in in i K d Most parameters values are bounded from existent data

  13. Most parameters values are bounded from existent data Bound IL2 (molec./cell) \ Free IL2 (pM) Bound IL2 (molec./cell)x10-3

  14. 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 APCs Change from tolerance to immunity with abrupt increase of APC numbers Adaptation the tolerant state upon slow increase of APC numbers.

  15. AutoimmunityIS Self-ToleranceTS 4 10 ET 6 8 4 2 10 10 10 10 3 10 RT IS OS IL2 IL2 degradation rate Kdi (h-1) Number of cells TS IL2 conc. (pM) 2 10 RT ET 3 2 4 0 1 -1 10 10 10 10 10 10 1 IL2 10 OS, IS TS, IS 200 300 500 ILa adimen. Conc. Time (weeks) Time (weeks) 0 0 2 2 4 4 6 6 8 8 Bistability is conserved: Two steady states Autoimmunity and Tolerance Parameter Regions 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.

  16. 10 10 10 2 ET 10 3 10 3 10 10 A RT IL2F 8 8 1 10 10 1 3 5 7 time ET 1 1 10 6 6 10 10 10 RT Free IL2 conc. (pM) Free IL2 conc. (pM) Number of cells Number of cells 4 4 10 10 -1 -1 2 IL2F 10 10 10 A 2 2 10 10 1 1 3 5 7 time 0 2 4 6 8 10 0 2 4 6 8 10 Time (weeks) Time (weeks) Adaptive response to APC variation is also conserved Gradual APC change Abrupt APC change Bifurcation diagrams, with the number of APCs, show also a similar structure to those obtained with the original cross-regulation model.

  17. Three necessary parameter conditions for a reasonable model behavior R cells use IL2 more effectively at low concentrations E R S / RI > S / RI A A E R E cells have net growth rate faster than R cells in IL2 excess × > × E E E R R R K K / K K K / K A d A d E cells have to use a cytokine alternative to IL2 to proliferate > Il α 0

  18. 4 10 3 10 IS OS IL2 degradation rate Kdi (h-1) TS 2 10 1 10 TS, IS OS, IS 200 300 500 ILa adimen. Conc. The effect of knocking down ILa in the model If Ila 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 ILa cytokine.

  19. IL-2 (+) R (+) R (-) IL-a (-) R R E APC T T T Including IL2 breaks locality and specificity of interactions in cross-regulation model In the extended models, some interactions are non-local (sharing of IL2) In the cross-regulation model, all the interactions are local near the APCs

  20. A1 <- E1,R1 <- E2,R2 A2 Implications for independence of concomitant responses to different self-antigen in a given lymph node In the extended models there should be strong interference between the immune reactions of different clones

  21. (+) (+) (-) IL-a (-) R E APC Comparing different hypotheses in the suppressive effect of R cells over E cells Extended model variant 2: R cells also inhibit E cell activation Extended model variant 1: R cells drive competition for IL2 IL-2 IL-2 (+) (+) (-) IL-a (-) R E APC Full coupling of responses to different antigens Bi-standard interactions between clones, but still independent responses

  22. But, another possible solution to be explored in the future is further locality on the use of IL2 inside a given lymph node A1 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 <- E1,R1 <- E2,R2 A2

  23. 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.

  24. IL2 depletion therapy can subvert the preexistent Autoimmune or Tolerant steady states 4 10 3 10 We model IL2 depletion treatment by an incerase in IL2 degradation rate IS OS IL2 degradation rate Kdi (h-1) TS 2 10 1 10 OS, IS TS, IS 200 300 500 ILa adimen. Conc. 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.

  25. 6 4 2 2 8 6 4 8 10 10 10 10 10 10 10 10 4 4 0 -1 1 3 2 0 3 1 -1 2 10 10 10 10 10 10 10 10 10 10 10 10 10 IS 5 10 4 10 3 TS 10 2 10 0 0 10 5 10 20 30 15 20 40 0 1 2 3 4 IL2 depletion can subvert the preexistent Autoimmune or Tolerant steady states 6 + ET IL2 Breaking Tolerance Depletion Dose Number of cells IL2 conc. (pM) RT 4 10 + - RT Reestablishing Tolerance Depletion Dose TS Number of cells 3 IL2 conc. (pM) 10 ET IS IL2 2 10 1 2 3 4 5 6 7 Duration of treatment (w) Time (weeks) This supports applications of IL2 depletion both in the treatment of autoimmune diseases and cancer, but these applications strongly depend on treatment schedules

  26. 5 10 IS 4 7.10 ΓT= 10-4 4 5.10 DosS ΓT= 10-6 ΓT= 10-8 4 3.10 TS 4 2.10 0 1 2 3 4 Duration of treatment (weeks) 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. Setoguchi et al. JEM 2005, 201(5):723–735

  27. Anti-IL2 MAbs can induce tumor regression 5 d 2 d 1 mg AcM IL-2 iv 5x105 IU/Kg sc IL-2 F10 F10 irradiated Montero et al. 2005 unpublished results

  28. IL2 injection therapy can not subvert the preexistent Autoimmune or Tolerant steady states 8 10 RT, IS 6 10 We model IL2 injection treatment by an increase in the external source of IL2 (Gi) 4 10 Γi 2 10 TS, IS OS, IS 0.1 1 10 100 1000 10000 Ilα

  29. 5 5 9 9 10 10 10 10 4 4 10 10 RT 7 7 10 10 3 3 10 10 Number of cells IL2 conc. (pM) 2 2 5 5 10 10 10 10 ET 10 10 3 3 10 10 IL2 1 1 -1 -1 10 10 10 10 0 2 4 6 8 10 12 14 Time (weeks) Therapy with IL2 can only reinforce the preexistent Steady State Reinforcement of Tolerance Reinforcement of Autoimmunity ET Number of cells IL2 IL2 conc. (pM) RT 0 2 4 6 8 10 12 14 Time (weeks) 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.

  30. IL2 administration can reinforce tolerance promoting allograft acceptance 2 daily doses of IL-2, sc tail base Check growth of allogenic tumor 2*105 F10 sc fd Incidence of MB16F10 tumors 100 PBS 3 80 IL-2 5*10 U/kg 4 IL-2 5*10 U/kg 60 5 IL-2 5*10 U/kg Incidencia tumoral (%) 6 IL-2 5*10 U/kg 40 20 0 0 5 10 15 20 25 30 35 40 45 Días Montero et al Ann. N.Y. Acad. Sci. 1107: 239–250 (2007).

  31. 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).

  32. Chain a IL-2 Chain b Chain g Could the particular interface of the IL2 being recognized by IL2 blocking MAbs make a difference? Dimerization of the b and g chain is responsible for signaling MAbs, could block the IL2 binding to the a chain of the receptor without affecting the binding to the b and g chains or vice versa MAbs blocking binding to b and/or g chain will fully inhibit signaling, but those just blocking binding to a chain will allow it Thus we study these classes of MAbs being used alone or in preformed complexes with IL2

  33. Modeling therapies with IL-2 and/or anti-IL2 MAbs in a virtual mouse Vs=2.5 mL Lymph Node Vn=1.0 mL #T=107 BLOOD IL-2 #APC=1 105 (+) (+) (-) IL-a (-) R E Therapies of interest APC 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 Renal and/or fecal elimination of molecules Model parameters are calibrated from independent publicly available Data

  34. 8 4 6 2 8 2 4 6 10 10 10 10 10 10 10 10 3 1 2 2 4 3 1 0 -1 0 -1 4 10 10 10 10 10 10 10 10 10 10 10 10 1 mg 2.5 mg 1.0 mg 1.7 mg ET RT RT RT 3 mg IL2 Number of cells IL2 conc. (pM) ET ET ET RT IL2 IL2 IL2 RT ET IL2 Time (weeks) Time (weeks) Time (weeks) 0 0 0 5 5 5 10 10 10 15 15 15 20 20 20 MAbs blocking either a or b interfaces of IL2 similarly break a preexistent tolerant state 5 mg ET Face b IL2 Number of cells IL2 conc. (pM) RT KD=10-10 M t1/2=3 days Face a 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.

  35. 4 8 2 4 6 2 8 6 10 10 10 10 10 10 10 10 2 4 4 3 3 1 -1 1 0 -1 0 2 10 10 10 10 10 10 10 10 10 10 10 10 RT IL2 conc. (pM) Number of cells ET IL2 0 0 10 10 20 20 30 30 40 40 Time (weeks) MAbs blocking b, but not a, interfaceof IL2 can reverse autoimmunity, reestablishing tolerance Face b MAb Face a MAb + - + - ET KD=10-10 M t1/2=3 days IL2 IL2 conc. (pM) Number of cells RT Dos=20 mg/d Time (weeks) Despite achieving similar reduction of IL2 concentration, these MAbs behave in a qualitatively different way. Interface b MAbs reduce the actual IL2 related signal more, triggering a larger reduction of E cell number that ultimately allows the reestablishment of tolerance.

  36. What about injecting Immune-complexes? MAbs + IL2

  37. 4 2 8 6 4 6 8 2 10 10 10 10 10 10 10 10 3 4 2 1 0 -1 5 3 0 1 2 4 -1 -1 1 3 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 5 mg/d 1 week 20 mg/d 1 week 20 mg/d 1 week RT Complexes RT Number of cells Conc. (pM) IL2 conc. (pM) Number of cells IL2 conc. (pM) ET IL2 ET IL2 IL2 Time (weeks) 0 0 0 5 5 5 10 10 10 15 15 15 20 20 20 Time (weeks) Time (weeks) Both types of Immunocomplexes reinforce tolerant state. Availability of Free IL-2 signal Interface a MAb +IL2 Interface b MAb +Il2 Both types of immunocomplexes appear to expand regulatory T cell activity in the model, although interface b derived complexes appear quantitatively more efficient at low doses However injection of interface a 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.

  38. 8 4 6 8 2 2 4 6 10 10 10 10 10 10 10 10 4 2 1 3 0 -1 -1 2 3 0 4 1 10 10 10 10 10 10 10 10 10 10 10 10 0 0 10 10 20 20 30 30 40 40 50 50 60 60 70 70 Immunocomplex with interface b but notinterface a anti- IL2 MAbs can reestablish Tolerant State. Face b MAb +IL2 Face a MAb +IL2 0.3 mg/d 50week 0.3 mg/d 50week ET ET IL2 conc. (pM) Number of cells IL2 conc. (pM) IL2 Number of cells IL2 RT RT Time (weeks) Time (weeks) Despite both types of immunocomplexes appearing to expand regulatory T cell activity in the model, only interface b derived complexes appear capable of reversing the autoimmune state.

  39. Conclusions through the Modeling relationship Postulates, Axioms Experimental Observations Model Predictions

  40. Model Predictions and explanations It explains: • 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. It predicts: • That IL7 is the likely gamma chain family cytokine that helper cells use as an alternative to Il2 to growth in vivo • That Il2 depletion treatments can be used to reestablish tolerance in an autoimmune individual. (interface b Mabs) • 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. • 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) • 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.

  41. Acknowledgements CIM Lic. Tania Carmenate M.Sc. Karina García Lic. Ileana Gonzalez Lic. Jose Antonio Dr. Enrique Montero Dr. Agustín Lage IGC (Portugal) Dr. Jorge Carneiro Financial support IGC and CIM

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