Background: Toll-like receptor 9 (TLR9) agonists are known for their ability to activate innate immune cells and have been extensively tested for the treatment of different types of tumors. However, no satisfied results have been achieved so far. Indeed, to prevent uncontrolled and potentially harmful immune reactions, TLR9 activation induces several immunoregulatory mechanisms that, eventually, restrain the immune response. Among such processes, it has been observed that TLR9 triggering determines PD-1 receptor up-regulation on immune cells. PD-1 is reported to be expressed by adaptive and innate immune cells and to dampen immune system function upon binding to its two ligands (PD-L1 and -L2). By over-expressing such ligands, tumor cells exploit PD-1/PD-Ls axis to evade immune system attack. The blockade of PD-1 using specific monoclonal antibodies, disrupting PD-1/PD-Ls interaction, is able to restore the activity of the immune cells. However, only 30% of patients with advanced cancer benefit of this kind of therapy. Therefore, a combinatorial immunotherapeutic regimen based on the activation of the immune system, by TLR9 agonists administration, and on the abrogation of an immunosuppressive mechanism, utilizing anti-PD-1 antibody, represent a promising immunotherapeutic strategy in the treatment of cancer. Aim: The efficacy of the co-administration of these two immunotherapeutic drugs have been deeply investigated in several clinical and preclinical studies that mainly focused on the role played by the adaptive immune system. Considering that TLR9 agonists primarily targets innate immune cells and PD-1 can be also expressed by the innate immune system, the aim of the present study was to evaluate the contribution of the innate immune cells to the final outcome of this combinatorial therapy. Methods: Athymic mice, intraperitoneally xenografted with IGROV-1 human ovarian cancer cell line, received TLR9 agonist (CpG-ODN), anti-PD-1 antibody or their combination. In vivo macrophages depletion was achieved by liposomal-containing clodronate administration. Tumor immune contexture was evaluated by immunofluorescence analysis. In vitro studies were carried out by exposing RAW264.7 mouse macrophage cell line to CpG-ODN and/or anti-PD-1 antibody. The effect of the combinatorial treatment on macrophages was assessed by microarray profile and subsequent bioinformatic analysis, Real-time PCR, multiplex ELISA and 51Cr-release assays. To unravel the specific contribution of the antigen-binding site and the fragment crystallizable (Fc) domain of anti-PD-1 antibody, modified anti-PD-1 antibodies were utilized. The involvement of TRIM21 was investigated by pharmacological inhibition and silencing experiments. Results: Mice receiving CpG-ODN/anti-PD-1 antibody combination experienced an acceleration of tumor growth paralleled by a marked infiltration of CD206+ and IL-10+ macrophages compared to CpG-ODN-treated animals. The increased tumor progression observed in the combination group was completely abrogated by macrophage depletion. In vitro experiments showed that RAW264.7 cells stimulated with CpG-ODN and anti-PD-1 antibody exhibited a gene expression profile completely different to those exposed to each drug alone, acquiring a phenotype characterized by the up-regulation of both M1- and M2-related markers. Multiplex ELISA assays confirmed an augmented secretion of M1 and M2 cytokines, validating gene profile data. Functional assay showed that combination-primed macrophages were able to dampen NK cell cytotoxic activity compared to CpG-ODN- or anti-PD-1- antibody-treated cells. The use of engineered anti-PD-1 antibodies revealed that either the Fc domain and the antigen-binding site of anti-PD-1 antibody are necessary for determining the changes in gene expression profile on macrophage when combined with TLR9 agonists. Finally, TRIM21 was found to be involved in shaping macrophage phenotype by interacting with anti-PD-1 antibody Fc domain. Conclusions: Our results indicate that, when TLR9 signaling is activated, the co-administration of anti-PD-1 antibody induces in macrophages the acquisition of a “mixed” M1/M2 phenotype with enhanced immunosuppressive features, eventually negatively impacting on the immune response and promoting tumor growth. Since TLR9 stimulation and PD-1 blockade combinatorial immunotherapy is under investigation in different clinical trials, the impact of both agents on macrophages should be taken into consideration to avoid potentially harmful adverse effects, especially in tumors where the infiltration of macrophages is particularly abundant.
Introduzione: Gli agonisti del recettore Toll-like 9 (TLR9) sono noti per la loro capacità di attivare le cellule del sistema immunitario innato e sono stati ampiamente studiati per il trattamento di diversi tipi di tumore. Tuttavia, ad oggi non sono stati raggiunti risultati soddisfacenti. Infatti, per prevenire reazioni immunitarie incontrollate e potenzialmente dannose, l’attivazione del TLR9 induce diversi meccanismi immunoregolatori che, in ultima istanza, possono inibire la risposta immunitaria. A questo proposito, è stato osservato che la stimolazione del TLR9 provoca un aumento dell’espressione del recettore PD-1 sulle cellule immuni. PD-1 è espresso dalle cellule del sistema immunitario sia innato sia adattativo e, in seguito al legame con i suoi due ligandi (PD-L1 e –L2), provoca lo spegnimento dell’attività del sistema immunitario. Per sfuggire alla risposta immunitaria, le cellule tumorali possono sfruttare l’asse PD-1/PD-Ls grazie all’over-espressione dei ligandi di PD-1. Il blocco di PD-1 mediante specifici anticorpi monoclonali, che impediscono l’interazione tra PD-1 e i suoi ligandi, può ripristinare l’attività delle cellule immunitarie. Tuttavia, solamente il 30% dei pazienti con tumore in stadio avanzato trae beneficio da questo tipo di terapia. Di conseguenza, una coterapia basata sull’attivazione del sistema immunitario mediante agonisti del TLR9, e sul blocco di un meccanismo immunosoppressivo, utilizzando un anticorpo anti-PD-1, rappresenta una promettente strategia immunoterapeutica per il trattamento del cancro. Scopo: L’efficacia della co-somministrazione di questi due farmaci è stata largamente studiata in molteplici studi preclinici e clinici, che si sono focalizzati principalmente sul ruolo del sistema immunitario adattativo. Poichè le cellule immunitarie innate, che possono esprimere PD-1, rappresentano i principali target degli agonisti del TLR9, lo scopo di questo studio è stato la valutazione del ruolo delle cellule del sistema immunitario innato nel determinare gli effetti della co-terapia. Metodi: Un agonista del TLR9 (CpG-ODN) e un anticorpo anti-PD-1 sono stati somministrati, singolarmente o in combinazione, a topi atimici xenotrapiantati intraperitonealmente con cellule IGROV-1 del carcinoma ovarico umano. I macrofagi sono stati depletati in vivo mediante clodronato incapsulato nei liposomi. Il microambiente tumorale è stato valutato con analisi di immunofluorescenza. Sono stati effettuati studi in vitro trattando la linea cellulare di macrofagi murini RAW264.7 con CpG-ODN e/o anticorpo anti-PD-1. L’effetto della combinazione sui macrofagi è stato valutato con analisi del profilo di espressione genica seguite da analisi bioinformatiche, Real-time PCR, multiplex ELISA e saggi di rilascio del 51Cr. Per investigare il ruolo specifico del sito di legame con l’antigene e del dominio del frammento cristallizzabile (Fc) dell’anticorpo anti-PD-1, sono stati utilizzati anticorpi anti-PD-1 ingegnerizzati. Il coinvolgimento del TRIM21 è stato valutato attraverso l’inibizione farmacologica ed esperimenti di silenziamento. Risultati: I topi che hanno ricevuto la combinazione CpG-ODN/anticorpo anti-PD-1 hanno mostrato un aumento della crescita tumorale e una marcata infiltrazione di macrofagi CD206+ e IL-10+ rispetto agli animali trattati con solo CpG-ODN. La deplezione dei macrofagi ha completamente annullato l’aumentata progressione tumorale osservata nel gruppo della combinazione. Gli esperimenti in vitro hanno evidenziato come le cellule RAW264.7 incubate sia con CpG-ODN sia con anticorpo anti-PD-1 esibiscano un profilo di espressione genica completamente differente da quelle trattate con il singolo farmaco, sviluppando un fenotipo caratterizzato dall’up-regolazione di markers associati sia a M1 sia a M2. I saggi multiplex ELISA hanno evidenziato un’aumentata secrezione di citochine relative sia a M1 sia a M2, validando i dati di espressione genica. I saggi funzionali hanno mostrato che i macrofagi stimolati con la combinazione sono in grado di ridurre l’attività citotossica delle cellule NK rispetto ai macrofagi incubati con CpG-ODN o anticorpo anti-PD-1. L’utilizzo di anticorpi anti-PD-1 ingegnerizzati ha rivelato che sia il riconoscimento del dominio Fc sia il sito di legame con l’antigene dell’anticorpo anti-PD-1 sono necessari per determinare I cambiamenti del profilo di espressione genica dei macrofagi osservati nella combinazione. Infine, si è dimostrato che il TRIM21 è coinvolto nelle modificazioni del fenotipo dei macrofagi mediante l’interazione con il dominio Fc dell’anticorpo anti-PD-1. Conclusioni: I risultati di questo studio mostrano che, quando nei macrofagi viene attivato il signaling del TLR9, la co-somministrazione con anticorpo anti-PD-1 induce in queste cellule immunitarie l’acquisizione di un fenotipo misto M1/M2 caratterizzato da funzionalità immunosoppressiva, in grado di compromettere la risposta immunitaria e promuovere la crescita tumorale. Poichè l’associazione di agonisti del TLR9 con anticorpi anti-PD-1 è tuttora investigata in diversi studi clinici, l’impatto di questi agenti faramacologici sui macrofagi dovrebbe essere preso in considerazione per evitare reazioni avverse potenzialmente pericolose, soprattutto nei tumori ad elevata infiltrazione di macrofagi.
IMPAIRMENT OF TOLL-LIKE RECEPTOR 9 AGONIST ANTI-TUMOR ACTIVITY BY ANTI-PD-1 ANTIBODY: ROLE OF MACROPHAGES / S.l. Indino ; tutor: M. Sommariva ; coordinatore: C. Sforza. Dipartimento di Scienze Biomediche per la Salute, 2023 Jan 27. 35. ciclo, Anno Accademico 2022.
IMPAIRMENT OF TOLL-LIKE RECEPTOR 9 AGONIST ANTI-TUMOR ACTIVITY BY ANTI-PD-1 ANTIBODY: ROLE OF MACROPHAGES
S.L. Indino
2023
Abstract
Background: Toll-like receptor 9 (TLR9) agonists are known for their ability to activate innate immune cells and have been extensively tested for the treatment of different types of tumors. However, no satisfied results have been achieved so far. Indeed, to prevent uncontrolled and potentially harmful immune reactions, TLR9 activation induces several immunoregulatory mechanisms that, eventually, restrain the immune response. Among such processes, it has been observed that TLR9 triggering determines PD-1 receptor up-regulation on immune cells. PD-1 is reported to be expressed by adaptive and innate immune cells and to dampen immune system function upon binding to its two ligands (PD-L1 and -L2). By over-expressing such ligands, tumor cells exploit PD-1/PD-Ls axis to evade immune system attack. The blockade of PD-1 using specific monoclonal antibodies, disrupting PD-1/PD-Ls interaction, is able to restore the activity of the immune cells. However, only 30% of patients with advanced cancer benefit of this kind of therapy. Therefore, a combinatorial immunotherapeutic regimen based on the activation of the immune system, by TLR9 agonists administration, and on the abrogation of an immunosuppressive mechanism, utilizing anti-PD-1 antibody, represent a promising immunotherapeutic strategy in the treatment of cancer. Aim: The efficacy of the co-administration of these two immunotherapeutic drugs have been deeply investigated in several clinical and preclinical studies that mainly focused on the role played by the adaptive immune system. Considering that TLR9 agonists primarily targets innate immune cells and PD-1 can be also expressed by the innate immune system, the aim of the present study was to evaluate the contribution of the innate immune cells to the final outcome of this combinatorial therapy. Methods: Athymic mice, intraperitoneally xenografted with IGROV-1 human ovarian cancer cell line, received TLR9 agonist (CpG-ODN), anti-PD-1 antibody or their combination. In vivo macrophages depletion was achieved by liposomal-containing clodronate administration. Tumor immune contexture was evaluated by immunofluorescence analysis. In vitro studies were carried out by exposing RAW264.7 mouse macrophage cell line to CpG-ODN and/or anti-PD-1 antibody. The effect of the combinatorial treatment on macrophages was assessed by microarray profile and subsequent bioinformatic analysis, Real-time PCR, multiplex ELISA and 51Cr-release assays. To unravel the specific contribution of the antigen-binding site and the fragment crystallizable (Fc) domain of anti-PD-1 antibody, modified anti-PD-1 antibodies were utilized. The involvement of TRIM21 was investigated by pharmacological inhibition and silencing experiments. Results: Mice receiving CpG-ODN/anti-PD-1 antibody combination experienced an acceleration of tumor growth paralleled by a marked infiltration of CD206+ and IL-10+ macrophages compared to CpG-ODN-treated animals. The increased tumor progression observed in the combination group was completely abrogated by macrophage depletion. In vitro experiments showed that RAW264.7 cells stimulated with CpG-ODN and anti-PD-1 antibody exhibited a gene expression profile completely different to those exposed to each drug alone, acquiring a phenotype characterized by the up-regulation of both M1- and M2-related markers. Multiplex ELISA assays confirmed an augmented secretion of M1 and M2 cytokines, validating gene profile data. Functional assay showed that combination-primed macrophages were able to dampen NK cell cytotoxic activity compared to CpG-ODN- or anti-PD-1- antibody-treated cells. The use of engineered anti-PD-1 antibodies revealed that either the Fc domain and the antigen-binding site of anti-PD-1 antibody are necessary for determining the changes in gene expression profile on macrophage when combined with TLR9 agonists. Finally, TRIM21 was found to be involved in shaping macrophage phenotype by interacting with anti-PD-1 antibody Fc domain. Conclusions: Our results indicate that, when TLR9 signaling is activated, the co-administration of anti-PD-1 antibody induces in macrophages the acquisition of a “mixed” M1/M2 phenotype with enhanced immunosuppressive features, eventually negatively impacting on the immune response and promoting tumor growth. Since TLR9 stimulation and PD-1 blockade combinatorial immunotherapy is under investigation in different clinical trials, the impact of both agents on macrophages should be taken into consideration to avoid potentially harmful adverse effects, especially in tumors where the infiltration of macrophages is particularly abundant.File | Dimensione | Formato | |
---|---|---|---|
phd_unimi_R12623.pdf
Open Access dal 05/06/2024
Descrizione: Tesi di Dottorato in Medicina Traslazionale
Tipologia:
Altro
Dimensione
3.76 MB
Formato
Adobe PDF
|
3.76 MB | Adobe PDF | Visualizza/Apri |
Pubblicazioni consigliate
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.