Terapia Cellulare Non Trapiantologica In Ematologia ed Oncologia Fraia Melchionda

Possibile Definizione Terapia Cellulare: Trattamento che prevede la ricostituzione, l’aumento di numero o di efficacia di una funzione biologica ottenuto mediante l’impiego di popolazioni cellulari.

Terapia Cellulare Correlata a Procedura Trapiantologica Trapianto di Celulle Staminali Ematopoietiche Allogeniche Donor Lymphocites Infusion ,, ,, Allo-EBV-CTL nel trattamento delle Post Tramplant Lymphoprolipherative Disorders (PTLD) in pz sottoposti a BMT; AutoEBV-CTL nelle PTLD post SOT Utilizzo di CTLp anti-leucemia (LAM) Terapia Cellulare anti-infettiva CTL anti CMV, BK e Aspergillo APC artificiali (CD3+/CD28+ beads) nel contesto di Auto-TMO (NHL, CML, MM)

Terapia Cellulare non Trapiantologica Strategie per stimolare/amplificare la risposta immunitaria antitumorale T cellulo mediata Ripopolazione Adottiva (T cell) T Linfociti (CTL, TIL, LAK, NK) Ottimizzazione della presentazione dell’antigene Aumento antigen up-take e processing Modulazione della maturazione delle DCs Fornire co-stimulazione Antige discovery e modificazione dei peptidi per incrementare la loro avidità Manipolazione della risposta immune Amplificazione T cell in vivo (es: IL2, IL7, IL15) Diminuzione dei meccanismi di immunosoppressione (es:. -TGF-b, a-IL-10, T Reg /CD4+25+) Terapia Genica Today I’d like to show you some of my experiments develop strategy to modulate t cell mediated response in the contest of DCs based vaccine. Using T cell active cytokines

La storia dell’immunoterapia M. Burnet e L. Thomas nel 1957 proposero il concetto di “Cancer Immunesurveilance” Le prime prove di tale ipotesi furono testate mediante l’utilizzo di ceppi murini per K.O. per alcuni geni dimostratisi essenziali per lo sviluppo di alcuni componenti fondamentali del sistema immunitario: Es.: RAG2-/- pertanto incapaci di formare T Ly, B ly, NKT; perforina-/-; IFN gamma-/- ecc. Questi animali sviluppano, con diverse caratteristiche, tumori spontanei o elevata percentuale dopo stimolazione con sostanze chimiche

Immunosorveglianza Tumor Immunology: Cancer Immunoediting Dunn & Schreiber Nature Immunology 2002 (3)11; 991-998 Imm

Possibili Meccanismi di Tumor-Escape Immunosorveglianza Possibili Meccanismi di Tumor-Escape Fattori del compartimento linfocitario: Scarso numero T cell help Insufficiente numero CTL anti-tumorali ,, avidita’ dei CTL per il tumore Tolleranza immunologica Down-regulation di TCR signaling Apoptosi CTL Inadeguata funzione T cell (citochine, lisi) Aumento di T cellule a carattere soppressivo (T reg, CD4+CD25+) Produzione di citochine immunosoppressive (IL-10) Fattori Tumorali Insufficiente/assente espressione dell’antigene Perdita di espressione MHC Assenza di molecole co-stimolatorie Produzione di citochine immunosoppressive (VEGF, IL-10,TGFb)

IMPLICAZIONI TERAPEUTICHE Possibili Effetti Indotti dalla Crescita Tumorale sul Compartimento T Cellulare IMPLICAZIONI TERAPEUTICHE TOLLERANZA - delezione clonale - anergia clonale Jenkins MK. JExpMed 1987;165 302 Pardoll D. Nature, 2001; 411, 1010 Ripopolazione con T cell Adottiva STIMOLAZIONE Ottimizzare presentazione dell’Ag Strategia DC based, peptide based, APC artificiali, ecc. NEGLETTA Ochsenbein A.F et Al Nature, 2001, 411: 1058 T- Cell Ag TCR SIGNAL # 1 SIGNAL # 2 ATTIVAZIONE/ INSUFFICIENTE ESPANSIONE Speiser D.E. et Al. J.Exp.Med. 1997; 186, 645-653 APC Cross priming Ag Our lab is interested in studying the role of immune system in tumor bearing hosts, This slide represents a schemaic view of the scenario we can have when a Tumor cell encounts a T cell Paradigms in tumor immunology emphasize the capacity of the Tumor to induce T cells to Tolerance. It’s also reported how tumor associate Antigens appear to be weak, tumor cell can have a lack of co-stimolatory molecules as well as low number of Professional APC in the tumor eviroment so tumor can lead to tolerance via clonal deletion and clonal anergy. Or immune system and tumor can not see each other, Neglecting any Immune response Alternatively professional APCs can present tumor Ags via cross priming and lead to some activation but an insufficient expansion to control tumor develop. It is important to define which is the prevalent mechanism because the therapeutic implications are pretty different in the clinical setting. (read pannels) We are studying in the lab. Murine models, to investigate those posibilities. Cellula Tumorale Terapie immunologiche Ottimizzare presentazione dell’Ag (DCs, vaccini) Amplificazione della risposta T cellulo mediata Terapia anti-soppressiva

Terapia Cellulare non Trapiantologica Strategie per stimolare/amplificare la risposta immunitaria antitumorale T cellulo mediata Ripopolazione Adottiva (T cell) T Linfociti (CTL, TIL, LAK, NK) Ottimizzazione della presentazione dell’antigene Aumento antigen up-take e processing Modulazione della maturazione delle DCs Fornire co-stimulazione Antige discovery e modificazione dei peptidi per incrementare la loro avidità Manipolazione della risposta immune Amplificazione T cell in vivo (es: IL2, IL7, IL15) Diminuzione dei meccanismi di immunosoppressione (es:. -TGF-b, a-IL-10, T Reg /CD4+25+) Terapia Genica Today I’d like to show you some of my experiments develop strategy to modulate t cell mediated response in the contest of DCs based vaccine. Using T cell active cytokines

Cellule Effettrici ad Azione Citotossica CTL: T Linfociti che esercitano la loro attività citotossica mediante riconoscimento di cellule target (antigenicamente differenti) nel contesto del MHC classe I e II. TIL (Tumor Infiltrating Lymphocytes): popolazione eterogenea di linfociti (prevalentemente CD8+CD4+NK) localizzati nel contesto tumorale (es. melanoma, ca.ovarico) o linfonodi drenanti il tumore, aventi azione citotossica per lo più LAK-type, spesso non MHC-mediata). LAK (Lymphokine-Activated Killer): cellule con un’azione citotossica su target cellulari NK-resistenti (es. Daudi cell) questa è implementata da IL-2. Cellule NK: hanno una spontanea attività citotossica nei confronti di cellule target (es. cellula tumorale) con una modalità non-MHC ristretta.

Immunosorveglianza: Modello Murino MRD Impianto K7M2 Monitoraggio volume Tum. Volume: 0.8-1 cm3 Monitoraggio Recidiva Amputazione This is the model of K7M2 minimal residual disease developed by Chand Khanna In this model we induced tumor develop by implanting a fragment of K7M2 issue in the extremity, then we monitored for a specific tumor volume (0.8 cm3-1 cm3) At a pre-selected size (0.8 to 1 cm3 volume) we performed amputation of the extremity to create a setting of minimal residual disease and we monitored for develop of recurrency and metastasis

Osteosarcoma nell’Ospite SCID: Invariata la crescita tumorale primaria significativo incremento di recidive/metastasi P r i m a y T u o G w t h Death from Recurrent Disease 1 1 . Amputation size 8 . 7 5 6 p = . 8 In this experiment I induced tumor as just descreibed in 2 groups SCID and Balb/c. In the first graph you can see that the primary tumor growth is similar in groups but interstingly the percentage of survival is different, showing a significant survival advantage in the immunocompetetnt group. Looking at this results we can speculate an ability of the endogenous immune system to control MRD in this model. . 5 4 . 2 5 B a l b / c S C I D B a l b / c S C I D 2 . 1 2 3 4 5 6 7 2 5 5 7 5 1 1 2 5 1 5 D a y s a f t e r t u m o r i m p l a n t D a y s A f t e r I m p l a n t

Ruolo dell’Immuno-ricostituzione nella Prevenzione di Metastasi K7M2 Osteosarcoma MRD Ruolo dell’Immuno-ricostituzione nella Prevenzione di Metastasi SCID Monitoraggia volume K7M2 Impianto Amputazione Monitoraggio Di Recidiva LN early Immuno-ricostituzione CD4+ e CD8+ 25x 106 cells = 10 % T cell repertoire Tumor Burden LN late Immuno-ricostituzione CD4+ e CD8+ 25x 106 cells = 10 % T cell repertoire MRD In this second experiment I applied the same model to SCID mice, I treated one group called “late” at the time of amputation (so during a MRD condition) with high dose Lymphocytes Current model in tumor immunology suggests that Tumor induced tolerance occurs early during the course of tumor progression and significantly precede the onset of the more generalized immunosuppression that frequently accompanies advanced tumor burdens. These results suggest that T cell anergy to tumor antigens may impose a significant barrier to therapeutic tumor vaccine strategies. In this light we tested a second group of mice that I immunereconstituted early at the time of tumor implant so intruduced in the system during the phase of aggressive tumor growth potentially able to induce tolerance/anergy vs tumor Ags

T Cell Immuno-Ricostituzione Puo’ Prevenire Recidiva Tumorale y T u m o r G r o w t h Death From Recurrent Disease 1 . 1 s c i d p= 0.005 vs. SCID Esposizione di cellule T alla Crescita Tumorale Primaria Non Enficia la Capacita’ di Controllo di Recidiva s c i d L N e a r l y 8 L N l a t e L N e a r l y . 7 5 6 . 5 p= 0.002 vs. SCID 4 . 2 5 2 . Again primary tumor growth is similar in scid and reconstituted mice but the survival significantly different . There are not statistical differences between early and late reconstituted mice supporting the hypothesis that tumor induced tolerance didn’t occur during the primary tumor growth in this model. 1 2 3 4 5 6 7 5 1 1 5 2 D a y s A f t e r I m p l a n t D a y s A f t e r I m p l a n t

Produzione di IFNg dopo stimolazione in vitro con cellule K7M2 Evidenza di Priming Immunologico in Animali con Progressiva Crescita di Osteosarcoma Produzione di IFNg dopo stimolazione in vitro con cellule K7M2 Naive T. Bearing 2 4 6 8 1 p= 0.0092 Tum. Bear.+ DCs Tum. Bear.+ Lisato tum.- DC Naive+ Tum. Lysate DC 2 4 6 8 1 p= 0.03 Produzione di IFNg dopo stimolazione in vitro con DC pulsate Lisato Tumorale Based some elegant experiment in the litterature, I investigate the production of Ifng based on the presumption that IFNg production is one of the central feature of effector cells. To do this I set up an elispot assay in the first graph I tested the IFNg production by mice with bulky tumor and naïve splenociytes in response to irradiated K7M2 cell, and you see how there is a significant production of IFNg. Tumor bearing and naïve splenocites in response to BM derived DCs pulsed in vitro with Tumor Lysate and the IFNg production is maintained

Terapia Cellulare non Trapiantologica PNAS 2004

Terapia Cellulare non Trapiantologica Strategie per stimolare/amplificare la risposta immunitaria antitumorale T cellulo mediata Ripopolazione Adottiva (T cell) T Linfociti (CTL, TIL, LAK, NK) Ottimizzazione della presentazione dell’antigene Aumento antigen up-take e processing Modulazione della maturazione delle DCs Fornire co-stimulazione Antige discovery e modificazione dei peptidi per incrementare la loro avidità Manipolazione della risposta immune Amplificazione T cell in vivo (es: IL2, IL7, IL15) Diminuzione dei meccanismi di immunosoppressione (es:. -TGF-b, a-IL-10, T Reg /CD4+25+) Terapia Genica Today I’d like to show you some of my experiments develop strategy to modulate t cell mediated response in the contest of DCs based vaccine. Using T cell active cytokines

Ottimizzazione di Strategie Vaccinali Mediante l’Utilizzo di DC Cellule Dendritiche (DC): biologia Ottimizzazione di Strategie Vaccinali Mediante l’Utilizzo di DC Le DC sono cellule specializzate alla presentazione dell’Ag avendo la capacita’ di iniziare una risposta immune primaria, ivi inclusa la tolleranza immunologica In considerazione dell’importanza delle Dc nel controllo della risposta immunitaria lo studio della loro biologiga e’ essenziale per lo sviluppo di strategie immunoterapiche Copyright  2000-2003 Cell Systems Initiative, University of Washington

Le Sentinelle del Sistema Immunitario Cellule Dendritiche: biologia … Le Sentinelle del Sistema Immunitario M. Jefford et al. Lancet 2003 M. Jefford, E Maraskovsky, J cebon and I D davis Lancet oncology june 2001 vol2 DC precursors leave the circulation and maintain a sentinel role, predominantly around sites of potential antigen entry (eg mucosal surfaces). On antigen uptake, and in the setting of proinflammatory signals, DC undergo a phenotypic change, becoming mature. Mature DC upregulate MHC and costimulatory molecules necessary for efficient presentation of antigen to T cells, and become sensitive to chemical signals (chemokines). They then migrate to the local lymph nodes, and acquire the ability to stimulate T cells. In the lymph nodes, DC present antigen to specific T cells (see Figure 3), resulting in T-cell activation and proliferation. Effector T cells later migrate to the site of inflammation or antigen entry. DCs are derived from bone marrow APCs. They are found throughut the body, but are concentrated in areas of potential antigen entry, especially around epithial and mucosal surfaces where they capture and process ag. DCs are aslo found in the thymus where they have a role in establishing tolerance to self reactive Ag, and also in 2ndary lymphoid organs where they present Ag specific to T cells DC migrate from blood into the tissues, where they act as ‘sentinels’, sampling the local environment for foreign as well as autoantigens (Figure 2). Immature DC are efficientphagocytic cells, but are inefficient at presenting antigens to T cells. After encountering antigens at sites of mucosal disruption or inflammation, DC undergo phenotypic and functional changes, resulting in a ‘mature’ stage. Mature DC have increased surface expression of major histocompatibility complex (MHC) antigens of class I and class II, and costimulatory molecules (required for efficient presentation of peptides and stimulation of T cells). DC achieve a much higher density of peptide/MHC complexes on their cell surface than other APC, such as B cells and monocytes. Mature DC are less phagocytic than immature ones, but are potent T-cell stimulators. They also become migratory and travel to the local lymph nodes, where they present antigens in association with MHC to specific T cells.1 Several studies have shown that DC generated in vitro must be matured to migrate optimally and to stimulate T cells efficiently.1 DCs originate in the bone marrow and circulate via the bloodstream to most tissues of the body. These rare cells serve as "sentinels of the immune response," continuously scanning the environment for antigens in the form of microbes, allergens, toxins, virally infected cells, and tumor cells. Various processes can be employed which allow the DC to capture the antigen. Under the influence of additional environmental factors, the DC can then traffic from the peripheral tissue to lymphoid tissues via the lymphatics and the peripheral circulation. They also undergo a maturation process which equips them with a potent capacity to present antigen (Ag) to the very small fraction of naïve T cells which express the appropriate antigen receptor. Effective DC/T cell interactions result in activation and expansion of antigen-specific T cells. This slide illustrates such a process in the context of a developing immune response against a tumor.

La Processazione dell’Antigene MHC-II peptide CD4 endocitosi MHC-II Endosoma/lisosomi Cross-presentation TAP MHC-I Golgi Proteosoma peptidi citosol Patogeno Essistono diversi pathway di processazione dell’Ag per le molecole MHC clesse I o II ristrette MHC class I presentano peptidi primariamente derivati da sintesi endogena di proteine in seguito a stimolazione (self o patogeno). Queste proteine sono degradate in peptidi dal sist. Proteosoma e trasposrtate con transporters of antigen-processing (TAP) molecules nel reticolo endoplasmatico. Per essere caricati su MHC class I molecules. Diversamente molecole MHC class II presentano proteine che sono di origine esogena attraverso l’endocitosi. Tali proteine sono degradate dal sistema endosomiale/lisosomiale e caricate sul molecole MHC II. Inoltre Antigeni catturati mediante endocitosi possono essere cross-presentati attraverso la via TAP dipendente ed avere inoltre accesso alla presentazione MHC I (CD8) questa via alternativa e’ tutt’oggi ancora in via di studio piu’ approfondito Modified by William R. Heath et al. Nature Reviews Immunology 2001 CROSS-PRESENTATION IN VIRAL IMMUNITY AND SELF-TOLERANCE William R. Heath1 & Francis R. Carbone2 about the authors MHC-I peptide CD8 DC

Immature vs Mature DCs Importanza della maturazione, effetti sulla risposta immunitaria Bacterial products Cytokines T cells DC Immature DC Mature Danger signals Uptake antigene Endocitosi Processazione antigene via MHC class I/II La maturazione delle Dc e’ un punto di estrema criticita’ infatti la maturazione e’ un processo che gradualmente trasforma la DC da una cellula specializzata nel uptake e nella processazione dell’antigene a un cellula specializzata nella stimolazione di cellule T. La maturazioen delle DC e’ stimolata da patogeni, molecole pro-infiammatorie, danno tissutale (Danger signals) In vitro, CD4+ T cells repetitively stimulated with allogeneic immature DC1 differentiate into IL-10 producing regulatory cells which inhibit the proliferation of alloreactive T cells [34]. Injection of immature DC1 pulsed with influenza matrix peptide (MP) and KLH into healthy volunteers, induced MP-specific IL-10 producing CD8+ T cells and silenced MP-specific CD8+ T cell effector function [35]. ( Fig. 3) Fig. 3. Modulation of immunity by DCs. In their immature state, human DCs induce the differentiation of antigen-specific CD4+ and CD8+ regulatory T cells. CD4+ regulatory T cells produce interleukin (IL)-10, express CD25 and CTLA-4 and inhibit the proliferation of CD4+ helper cells through contact-dependent inhibition. CD8+ regulatory T cells produce IL-10 and lack cytolytic capacity. By contrast, mature DCs induce the development of helper CD4+ T cells and cytolytic CD8+ T cells. Abbreviations: Ag, antigen; CTLA-4, cytotoxic T-lymphocyte associated protein-4; DC, dendritic cell; IFNsmall gamma, Greek, interferon small gamma, Greek; Th1, T helper cells 1. Thus a full understanding of the phenotypic and functional maturation of DC is important for exploiting the full potential of these potent APC in immunization strategies Costimolazione high CD40, CD54, Class II Migrazione vs linf. Presentazione del Ag Stimolazione cell T Sviluppo cellule CD4+ CD8 T antigene specifiche (CTL)

Terapia Cellulare non Trapiantologica Possibili Fonti di Antigeni Tumorali nella strategia Vaccinale DC-based DCs DCs Estratti proteici cRNA mRNA rProteine Corpi apoptotici cDNA Tumor cells Tumor Antigens (TA) Peptidi Tumorali L’esasequenza di TA e la restrizione MHC e’ necessaria Loading diretto dei Peptidi su MHC DC Proteine tumorali (es. corpi apoptotici) Possono essere usate quando TA sono sconosciuti Un numero variabile di epitopi TA possono essere presentati Possibile disfunzione nel meccacismo di presentazione Peptidi DCs Cellule di fusione DCs Peptidi sintetici (modificati)

Terapia Cellulare non Trapiantologica Classificazione degli Antigeni Tumorali Tumore specifici bcr/abl protein (CML, ALL) Idiotipo immunoglobuline (NHL, MM) Pml/Rar alfa protein (APL) Shared Tumor-specific Ag (cancer testis Ag, o Melanoma associate) MAGE BAGE GAGE Antigeni di differenziazione (espressi anche dai normali tessuti) Melan A Mart-1 Proteinase-3 WT-1 Oncogene/tumor suppression Ag P53 Ras mutated protein Virus Associated Ag EBV

Terapia Cellulare non Trapiantologica L’attivita’ di Antigen discovery nei tumori dell’adulto implicazioni nei tumori pediatrici NBL GAGE-1 (80%) MAGE-1 (50-80%) EWS GAGE-1 XAGE-1,2 RMS XAGE-1,2 Sarcomas GAGE-2 (25%) NY-ESO-1(80%) MAGE Glioblastoma GAGE-3,6 (70%) MAGE-1 Her-2 gp100 Medulloblastoma GAGE-3,6 (45%) Head & Neck PRAME ALL t(8;21) PRAME

Terapia Cellulare: Strategia Vaccinale Selezione? Utilizzo di adiuvanti? MRD/low tumor burden Immmuno Riconstituzione Post alte dosi CT Quando? Quanto? Figure 3 | Strategy for immunization with autologous peptide-pulsed DCs. Peripheral blood leukocytes obtained by a leukopheresis procedure are elutriated to purify a monocyte fraction. The monocytes are cultured with granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 5 days to induce differentiation into immature dendritic cells (DCs; CD83 negative (CD83neg), CD80 low (CD80lo) and CD86 low (CD86lo)). These are then matured by addition of CD40 ligand (CD154), or some other maturing agent — for example, tumour-necrosis factor-alpha (TNF-alpha) or monocyte-conditioned medium. The mature DCs express high levels of CD83 (CD83hi), CD80 (CD80hi) and CD86 (CD86hi), and major histocompatibility complex (MHC) molecules. These are then coated with the appropriate vaccine peptide by incubation with peptide for 2 hours, washed, and infused back into the patient as an autologous DC vaccine to stimulate a T-cell response. PBMC, peripheral blood mononuclear cells. Nature Reviews Immunology 1, 209-219 (2001); doi:10.1038/35105075 printable pdf [3105K] STRATEGIES FOR DESIGNING AND OPTIMIZING NEW GENERATION VACCINES Jay A. Berzofsky, Jeffrey D. Ahlers & Igor M. Belyakov about the authors

Terapia Cellulare non Trapiantologica Fonti alternative di APC CD40 Activated B cell (CD40-B cell): Linfociti B attivati ex-vivo mediante stimolazione con CD40. Tale trattamento media la up-regolazione di molecole co-stimolatorie ed incremento della presentazione dell’Ag. Conferendo alla cellula caratteristiche che assomigliano a quelle tipiche della DC. ( espressione Rec. Chemochine, produzione di citochine, ecc.) Possono essere facilmente ottenute ed espanse in vitro, pertanto rappresentano una potenziale alternativa fonte di APC autologhe. APC artificiali: CD3/CD28 stimolazione mediante anticorpi coniugati con microbiglie. Mediano l’attivazione e l’espansione policlonale di popolazioni T cellulari (NHL, CML) Von Bergwelt et al. Cancer Res 2004; Lapointe R. Cancer Res 2004

Terapia cellulare non Trapiantologica Studi in ambito pediatrico Expression of MHC class I, MHC class II and cancer germline antigens in neuroblastoma (Wolfl et al Cancer Immunol Immunother 2004) Neuroblastona and DC function (Redlinger RE et al Semin Pedatr Surg 2004) Results of phase 1 study utilizing monocyte-derived Dendritic cells pulsed with tumor RNA in children and young adults with brain tumor (DA Caruso et al. Neuro-Oncol 2004) Restored immune response to an MHC-II restricted antigen in tumor bearing hosts after elimination of regulatory T cell. (Nicholl et al. J ped. Surg 2004) Characterization of CD34+progenitor-derived DC pulsed with tumor cell lysate for a vaccination strategy in children with malignant solid tumor and a poor prognosis (Ackermann et al Klin Pediatr 2004) Functional and molecular characterization of interleukin-2 transgenic Ewing tumor cell for in vivo immunotherapy (Burdach et al Ped Blood Cancer 2004) Pilot trial of tumor specific peptide vaccination and continous infusion of IL2 in patients with recurrent Ewing sarcoma and Alveolar Rhabdomiosarcoma (Dagher R Med. Ped. Oncol. 2002)

Terapia Cellulare non Trapiantologica Strategie per stimolare/amplificare la risposta immunitaria antitumorale T cellulo mediata Ripopolazione Adottiva (T cell) T Linfociti (CTL, TIL, LAK, NK) Ottimizzazione della presentazione dell’antigene Aumento antigen up-take e processing Modulazione della maturazione delle DCs Fornire co-stimulazione Antige discovery e modificazione dei peptidi per incrementare la loro avidità Manipolazione della risposta immune Amplificazione T cell in vivo (es: IL2, IL7, IL15) Diminuzione dei meccanismi di immunosoppressione (es:. -TGF-b, a-IL-10, T Reg /CD4+25+) Terapia Genica Today I’d like to show you some of my experiments develop strategy to modulate t cell mediated response in the contest of DCs based vaccine. Using T cell active cytokines

Effetti IL-2 mediati Potenzialmente Utili Nel contesto di Immunoterapia T Cell trophic factor Proliferation of activated T cell Differentiation of CTL Type 1 Cytokines Activation-Induced Cell Death (AICD) Synergize for production of other cytokines (e.g. IFN, TNF) IL-15 Therapy IL-2 Therapy Substitute IL15 in NK cell differentiation (via IL2/15Rc) NKpre NK cell  Innate Immunity CD56- CD56+ Expand NK cells (low dose)  Innate Immunity NK Activate NK cell (high dose) NK Lysis NK effector function Tumor  Innate Immunity

Effetti IL-7 mediati Potenzialmente Utili Nel contesto di Immunoterapia Induce Type 1 cytokines  Memory T cell Induce Other Cytokines (e.g. IL-2, IFN)  Programmed Cell death Partially substitute IL-15 in the generation of memory CD8+ pool Up-regulation bcl-2 family molecules Broaden Immune response  Lytic Activity (CTLs, NK) IL-7 Therapy Induce Lak cells Recruit Low Affinity clones Expansion of existing or vaccine-induced Immune response GM-CSF + IL7 Generate Monocytes derived DCs Expand High affinity CD8+ clones Beside the known IL-7 effects on lymphocytes and T cell homeostasis. IL-7 could also potentially enhance T cell responses following vaccine administration through several mechanisms: Direct on mature T cells by: First Il-7 co-stimulate for T cell activation by enhancing proliferation and cytokines production, expecially in a setting of sub-optimal TCR triggering. So not only expanding high affinity CD8 clones and potentially CD4 clones and also broaden the immune response recuiting low affinity clones, which could be important for anti-tumor immunity (tumor Antigens are usually low affinity Ags) Even if Il-7 is not considered to play a role in type 1 vs type 2 T cell differentiation, IL-7 tends to induce type 1 immune response up-regulating IFNg and IL-2 production and also synergizing with IL-12 It is known its capacity to diminish programmed cell death on mature T cell partially by up-regualting bcl-2 family molecules and potentially through the up-regulation of the T cell survival factor lung Kruppel-like factor. Il-7 in les extent than Il2 can also induce the lytic activity of Citotoxic Tly, NK and NKt cells diminishing TGF production which is known to be one mechanism of tumor-induced suppression But we know IL-7 doesn’t have only effects on T cells but also It can have a role in the development and function of DC population leading together with GMcsf to the generation of monocites derived DCs. Expand High Affinity CD4+ clones DCs mono Ag Presenatation ? TGF production Tumor cell TGF production  Tumor Induced Suppression

Effetti IL-15 mediati Potenzialmente Utili Nel contesto di Immunoterapia Increase Memory CD8+ T cell Critical in the generation and Maintenance of Memory CD8+ pool  Innate Immunity NK Type 1 Cytokines Maintenance Expansion of NK cells Synergize for production of other cytokikens (e.g. IFN, TNF) IL-15 Therapy IL-15 Therapy NKpre NK cell  Innate Immunity CD56- CD56+ NK cell Differentiation Recruit Low Affinity CD8+ clones Broaden Immune response Expand High Affinity CD8+ clones No effect on naïve CD4+ T cell (low expression IL2/15R) Expansion of existing or vaccine-induced Immune response

Terapia Cellulare non Trapiantologica Strategie per stimolare/amplificare la risposta immunitaria antitumorale T cellulo mediata Ripopolazione Adottiva (T cell) T Linfociti (CTL, TIL, LAK, NK) Ottimizzazione della presentazione dell’antigene Aumento antigen up-take e processing Modulazione della maturazione delle DCs Fornire co-stimulazione Antige discovery e modificazione dei peptidi per incrementare la loro avidità Manipolazione della risposta immune Amplificazione T cell in vivo (es: IL2, IL7, IL15) Diminuzione dei meccanismi di immunosoppressione (es:. -TGF-b, a-IL-10, T Reg /CD4+25+) Terapia Genica Today I’d like to show you some of my experiments develop strategy to modulate t cell mediated response in the contest of DCs based vaccine. Using T cell active cytokines

Possibile Definizione Terapia Genica: Trattamento che si propone di correggere il/i difetti molecolari causa di una determinata patologia mediante introduzione nelle cellule somatiche del gene mancante o difettivo.

Terapia Genica e Vaccinoterapia Inserzione di geni in grado di ottimizzare la risposta immunitaria nel contesto di vaccini antitumorali es: Gene hIL-2 in TIL (vettori retrovirali) TCR antigene specifico (es. Anti-gp100) Gene per hIL-12 in DC Gene per GM-CSF in cellule tumorali Molecole co-stimolatorie (es. CD80) in cellule tumorali

Vaccinoterapia DCs Citochine Adoptive T cell Antigene specifici Proteine GM-CSF Terapia genica APC artificiali