Myeloproliferative neoplasms represent a group of clonal hematopoietic disorders of which myelofibrosis (MF) is the most aggressive. MF is characterized by bone marrow (BM) fibrosis, inflammation, and a median survival of five years from diagnosis. In the context of myeloid neoplasms there is a growing recognition of the dysregulation of immune response and T cell function as significant contributors to disease progression and immune evasion. The abnormal clonal expansion of myeloid cells in myeloid disorders leads to the production of inflammatory cytokines and modifies the composition of bone marrow microenvironment. These alterations, combined with persistent antigen stimulation in the tumour microenvironment, can induce the differentiation of T effector cells into terminally exhausted T cells. These exhausted T cells exhibit impaired function in recognizing and eliminating malignant cells thereby compromising the immune surveillance mechanisms against the disease. We thus investigated T cell exhaustion in MF to assess the possibility of immune checkpoint inhibition as a new effective therapy to restore immune response against malignant cells. Peripheral blood mononuclear cells were isolated from 50 MF patients and 22 healthy donors (HD). Cytotoxic CD8+ T cells were characterized by multicolour flow cytometry to assess surface expression of inhibitory receptors (IRs) like PD-1, CTLA-4 and others. Results were correlated with patient’s clinical features. Residual cytokines production was assessed via intracellular flow cytometry staining. IRs-ligands expression was evaluated on monocytes and granulocytes by multiparametric flow cytometry. A co-culture system was established to investigate the immunosuppressive interplay between the dysfunctional T cells and cells from the neoplastic clone and to test anti-CTLA-4 inhibition in vitro. In the end, Patient-Derived-Xenograft (PDX) mice were established by transplanting CD34+ MF cells and infusing patient-derived expanded T cells in NSGS immunodeficient mice to test the effect of anti-CTLA-4 treatment in vivo. Increased expression of IRs like CTLA-4 was observed on cytotoxic CD8+ T cells from MF patients together with a reduced secretion of IFNɣ and TNFɑ. Correlation analysis evidenced a more severe disease in patients with higher IRs expression on cytotoxic T cells. IR-ligands like CD80 and CD86 were increased on MF granulocytes and monocytes highlighting a possible role for myeloid cells in suppressing T cells activation in MF patients. While cytotoxic T cells from HDs were effectively activated either alone or in co-culture with myeloid cells, the activation of cytotoxic T cells from MF patients was attenuated in the presence of myeloid cells and restored when T cells were cultured alone or treated with anti-CTLA-4. Moreover, neoplastic monocytes and immature granulocytes are depleted when co-cultured with CD8+ T cells only when treated with anti-CTLA-4 antibody. Anti-CTLA-4 treatment in vivo reduced myeloid engraftment, targeting preferentially granulocytes and monocytes, and led to T cells expansion in the spleen and in the BM. In conclusion, our data evidenced in MF patients the presence of an impaired population of cytotoxic T cells expressing multiple IRs and with reduced cytokine production. An immunosuppressive interplay between the myeloid neoplastic clone and T cells was reported and partially reverted by anti-CTLA-4 treatment in vitro. Moreover, anti-CTLA-4 in vivo treatment in PDX mice preferentially targeted malignant cells and expanded the lymphoid compartment. Taken together, this data highlights T cell exhaustion likely implicated in immune escape that could be targeted and potentially reverted by anti-CTLA-4 treatment.

La mielofibrosi (MF) è la più aggressiva tra le neoplasie mieloproliferative, è caratterizzata da una progressiva fibrosi midollare, infiammazione e una sopravvivenza media di cinque anni dalla diagnosi. Sempre più evidenze riconoscono una deregolazione della risposta immunitaria nelle neoplasie mieloidi e un’alterazione funzionale delle cellule T, fattori che contribuiscono alla progressione della malattia. L’espansione clonale delle cellule mieloidi porta alla produzione di citochine infiammatorie e altera la composizione del microambiente midollare che, insieme a una stimolazione antigenica persistente nel microambiente tumorale, induce il differenziamento delle cellule T effettrici in cellule T esauste. Le cellule T esauste mostrano una funzione compromessa nel riconoscere ed eliminare le cellule maligne. Abbiamo quindi indagato l'esaurimento delle cellule T nella MF per valutare l’efficacia della terapia con inibitori dei checkpoint immunitari nel ripristinare la risposta immunitaria contro le cellule maligne. Le cellule mononucleate sono state isolate da 50 pazienti con MF e 22 donatori sani (HD). Le cellule T citotossiche CD8+ sono state caratterizzate mediante citofluorimetria multiparametrica per valutare l'espressione di recettori inibitori (IR) tra cui PD-1 e CTLA-4. I risultati sono stati correlati con le caratteristiche cliniche dei pazienti. La produzione residua di citochine da parte delle cellule T e l'espressione dei ligandi dei IR è stata valutata su monociti e granulociti mediante analisi citofluorimetrica. È stato sviluppato un sistema di co-coltura per indagare l'interazione immunosoppressiva tra le cellule T disfunzionali e le cellule del clone neoplastico e per testare l'inibizione con anti-CTLA-4. Infine, topi PDX (xenotrapianto derivante dal paziente) sono stati generati trapiantando cellule CD34+ di MF e infondendo cellule T dello stesso paziente in topi immunodeficienti NSGS per testare l'effetto di anti-CTLA-4 in vivo. Le cellule T CD8+ di MF hanno mostrato un’aumentata espressione di IR come CTLA-4 e una ridotta secrezione di IFNɣ e TNFɑ. L'analisi di correlazione ha evidenziato una malattia più grave nei pazienti con una maggiore espressione di IR sulle cellule T CD8+. I ligandi dei IR, come CD80 e CD86, sono risultati aumentati su granulociti e monociti di MF, evidenziando un possibile ruolo delle cellule mieloidi nell'inibire l'attività delle cellule T. Mentre le cellule T CD8+ dei HD sono state efficacemente attivate sia da sole che in co-coltura con le cellule mieloidi, l'attivazione delle cellule T citotossiche dei pazienti con MF è risultata attenuata in presenza di cellule mieloidi e ripristinata quando le cellule T sono state coltivate da sole o trattate con anti-CTLA-4. I monociti e i granulociti immaturi del clone neoplastico sono stati eliminati quando coltivati insieme alle cellule T CD8+ e trattati con anticorpo anti-CTLA-4. Il trattamento con anti-CTLA-4 in vivo ha ridotto l’attecchimento delle cellule mieloidi, ha bersagliato i granulociti e i monociti maligni e ha espanso le cellule T. I nostri dati hanno evidenziato nei pazienti con MF la presenza di una popolazione compromessa di cellule T citotossiche con aumentati livelli di IR e con ridotta produzione di citochine. È stata identificata un'interazione immunosoppressiva tra le cellule mieloidi del clone neoplastiche e le cellule T, che può essere parzialmente revertita dal trattamento con anti-CTLA-4 in vitro. Inoltre, il trattamento con anti-CTLA-4 in vivo nei topi PDX ha bersagliato preferenzialmente le cellule maligne e ha espanso il compartimento linfoide. Questi dati evidenziano uno stato di esaurimento funzionale probabilmente implicato nell’evasione immunitaria che potrebbe essere revertito dal trattamento con anti-CTLA-4.

LE CELLULE T CITOTOSSICHE DI PAZIENTI CON MIELOFIBROSI MOSTRANO CARATTERISTICHE DI ESAURIMENTO PRECOCE BERSAGLIABILI DALL’INIBIZIONE DI CTLA-4 / Lara Tavernari , 2024 May 22. 36. ciclo, Anno Accademico 2022/2023.

LE CELLULE T CITOTOSSICHE DI PAZIENTI CON MIELOFIBROSI MOSTRANO CARATTERISTICHE DI ESAURIMENTO PRECOCE BERSAGLIABILI DALL’INIBIZIONE DI CTLA-4

TAVERNARI, LARA
2024

Abstract

Myeloproliferative neoplasms represent a group of clonal hematopoietic disorders of which myelofibrosis (MF) is the most aggressive. MF is characterized by bone marrow (BM) fibrosis, inflammation, and a median survival of five years from diagnosis. In the context of myeloid neoplasms there is a growing recognition of the dysregulation of immune response and T cell function as significant contributors to disease progression and immune evasion. The abnormal clonal expansion of myeloid cells in myeloid disorders leads to the production of inflammatory cytokines and modifies the composition of bone marrow microenvironment. These alterations, combined with persistent antigen stimulation in the tumour microenvironment, can induce the differentiation of T effector cells into terminally exhausted T cells. These exhausted T cells exhibit impaired function in recognizing and eliminating malignant cells thereby compromising the immune surveillance mechanisms against the disease. We thus investigated T cell exhaustion in MF to assess the possibility of immune checkpoint inhibition as a new effective therapy to restore immune response against malignant cells. Peripheral blood mononuclear cells were isolated from 50 MF patients and 22 healthy donors (HD). Cytotoxic CD8+ T cells were characterized by multicolour flow cytometry to assess surface expression of inhibitory receptors (IRs) like PD-1, CTLA-4 and others. Results were correlated with patient’s clinical features. Residual cytokines production was assessed via intracellular flow cytometry staining. IRs-ligands expression was evaluated on monocytes and granulocytes by multiparametric flow cytometry. A co-culture system was established to investigate the immunosuppressive interplay between the dysfunctional T cells and cells from the neoplastic clone and to test anti-CTLA-4 inhibition in vitro. In the end, Patient-Derived-Xenograft (PDX) mice were established by transplanting CD34+ MF cells and infusing patient-derived expanded T cells in NSGS immunodeficient mice to test the effect of anti-CTLA-4 treatment in vivo. Increased expression of IRs like CTLA-4 was observed on cytotoxic CD8+ T cells from MF patients together with a reduced secretion of IFNɣ and TNFɑ. Correlation analysis evidenced a more severe disease in patients with higher IRs expression on cytotoxic T cells. IR-ligands like CD80 and CD86 were increased on MF granulocytes and monocytes highlighting a possible role for myeloid cells in suppressing T cells activation in MF patients. While cytotoxic T cells from HDs were effectively activated either alone or in co-culture with myeloid cells, the activation of cytotoxic T cells from MF patients was attenuated in the presence of myeloid cells and restored when T cells were cultured alone or treated with anti-CTLA-4. Moreover, neoplastic monocytes and immature granulocytes are depleted when co-cultured with CD8+ T cells only when treated with anti-CTLA-4 antibody. Anti-CTLA-4 treatment in vivo reduced myeloid engraftment, targeting preferentially granulocytes and monocytes, and led to T cells expansion in the spleen and in the BM. In conclusion, our data evidenced in MF patients the presence of an impaired population of cytotoxic T cells expressing multiple IRs and with reduced cytokine production. An immunosuppressive interplay between the myeloid neoplastic clone and T cells was reported and partially reverted by anti-CTLA-4 treatment in vitro. Moreover, anti-CTLA-4 in vivo treatment in PDX mice preferentially targeted malignant cells and expanded the lymphoid compartment. Taken together, this data highlights T cell exhaustion likely implicated in immune escape that could be targeted and potentially reverted by anti-CTLA-4 treatment.
CYTOTOXIC T CELLS FROM MYELOFIBROSIS PATIENTS SHOW EARLY EXHAUSTED FEATURES TARGETABLE BY CTLA-4 INHIBITION
22-mag-2024
MANFREDINI, Rossella
RONTAUROLI, SEBASTIANO
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