Le cellule T CD8+ giocano un ruolo cruciale nel controllo dei tumori del fegato come l’epatocarcinoma, tuttavia, abbiamo una conoscenza limitata delle precise interazioni a livello di singola cellula tra le cellule T CD8+ e le cellule parenchimali e non parenchimali del fegato. Precedenti studi del nostro laboratorio hanno dimostrato che, nel contesto in cui gli epatociti esprimono HBV, le cellule circolanti T CD8+ effettrici (Teff) svolgono l’attività di immuno sorveglianza riconoscendo l’antigene ed esercitando un’attività citotossica verso gli epatociti che esprimono il virus. E’ stato infatti dimostrato che queste cellule sono capaci di estendere protrusioni citoplasmatiche attraverso le fenestrature delle cellule endoteliali rimanendo all’interno del lume sinusoidale. In questo progetto studiamo quindi se un meccanismo simile è coinvolto nella capacità delle Teff di migrare, riconoscere l’antigene e svolgere le proprie funzioni effettrici nel contesto di epatocarcinoma. Il primo obbiettivo di questo studio è stato quello di creare un nuovo modello di epatocarcinoma spontaneo in cui solo le cellule neoplastiche esprimessero sia dei marker fluorescenti (ZsGreen e TdTomato) che l’oncogene SV40 large T-antigen (TAg). Siamo riusciti ad ottenere dei topi che sviluppassero epatocarcinomi spontanei ed altamente proliferanti sparsi in tutto il parenchima sano del fegato. Dopo aver differenziato in vitro le Teff specifiche per l’antigene TAg (TCR-I Teff), abbiamo trasferito queste cellule in topi in cui era presente il tumore e abbiamo osservato che solo alcuni topi rispondevano totalmente o parzialmente all’attività citotossica delle cellule trasferite, le quali erano in grado di eliminare il tumore. Questo effetto era correlato alla dimensione di ogni singola lesione tumorale. Infatti, usando un approccio matematico multi-parametrico abbiamo scoperto che il trasferimento delle cellule TCR-I Teff era efficace solo in lesioni neoformate con un volume < 10 mm3 (lesioni responder [R]). Invece, quando il volume della lesione era > 100 mm3, il tumore non rispondeva alla terapia cellulare e continuava a crescere (lesioni non-responder [NR]). Abbiamo quindi deciso di studiare i fattori in grado di conferire alle nostre cellule effettrici un’attività terapeutica nei R e quelli in grado di frenare la loro efficacia nelle lesioni NR. Il principale risultato che abbiamo ottenuto riguarda la differenza del fenotipo della vascolatura tra lesioni R e NR. I nostri dati supportano infatti l’ipotesi che alcune caratteristiche anatomiche, emodinamiche e ambientali acquisite dai tumori durante la loro crescita possano influenzarne la responsività alla terapia delle Teff. La natura innovativa del nostro lavoro chiarirà nuovi meccanismi con cui le Teff effettuano immuno sorveglianza ed esercitano le loro funzioni effettrici nel contesto di tumore epatico.

CD8+ T cells play a crucial role in controlling liver tumours, such as hepatocellular carcinoma (HCC) however we have only limited knowledge of the precise dynamics of their interactions with hepatic parenchymal and non-parenchymal cells at the single-cell level. Previous work from our laboratory, demonstrated that in the context of HBV-expressing hepatocytes circulating effector CD8+ T cells (Teff) perform their immune surveillance function recognizing the antigen and kill virus-expressing hepatocytes extending cytoplasmic protrusions through endothelial fenestrations while still within liver sinusoids. Here we dissected whether similar or different mechanisms govern the capacity of Teff to home, migrate, recognize the antigen, and exert effector function within HCC. The first effort to dissect the project was the establishment of a new murine model of spontaneous HCC in which just the transformed hepatocytes express a nominal antigen, the oncogene SV40 large T antigen (TAg) and a fluorescent protein. We were able to obtain mice that develop spontaneous HCC lesions, highly proliferating and spread in a normal liver parenchyma. After the adoptive transfer of in vitro differentiated TAg-specific Teff in tumor-bearing mice, we observed that just some mice respond to the cytotoxic activity of the transferred cells, eliminating partially or completely the tumor, while in other mice the adoptive transferred cells have no beneficial effect on the tumor elimination. Thus, using a mathematical approach, we managed to pick the lesion volume as the fundamental parameter to predict the fate of each single HCC lesion: we called “responders” (R) the HCC lesions that are responsive to the cytotoxic activity of the TAg-specific Teff, with a single lesion volume <10 mm3, while we called “non-responders” (NR) the lesions that are not responding and have a single HCC lesion volume > 100mm3. We then studied the determinants that confer the therapeutic activity to the TAg-specific Teff in R and the ones that dampen the therapeutic activity in the NR lesions. Our main finding was that the vessel phenotype was completely different in R and NR. Our data support the hypothesis that some anatomic, hemodynamic, and environmental features acquired by each individual HCC lesion, during their growth, can influence their responsiveness to the Teff killing. The innovative nature of our work will elucidate new mechanisms whereby Teff fail to exert their immune function and cytotoxic activity in tumorigenic liver.

Investigation of intratumoral cd8+ t cell spatiotemporal localization and function / Bianca Partini - : . , 2023 Jan 18. ((35. ciclo, Anno Accademico 2021/2022.

Investigation of intratumoral cd8+ t cell spatiotemporal localization and function

PARTINI, BIANCA
2023-01-18

Abstract

CD8+ T cells play a crucial role in controlling liver tumours, such as hepatocellular carcinoma (HCC) however we have only limited knowledge of the precise dynamics of their interactions with hepatic parenchymal and non-parenchymal cells at the single-cell level. Previous work from our laboratory, demonstrated that in the context of HBV-expressing hepatocytes circulating effector CD8+ T cells (Teff) perform their immune surveillance function recognizing the antigen and kill virus-expressing hepatocytes extending cytoplasmic protrusions through endothelial fenestrations while still within liver sinusoids. Here we dissected whether similar or different mechanisms govern the capacity of Teff to home, migrate, recognize the antigen, and exert effector function within HCC. The first effort to dissect the project was the establishment of a new murine model of spontaneous HCC in which just the transformed hepatocytes express a nominal antigen, the oncogene SV40 large T antigen (TAg) and a fluorescent protein. We were able to obtain mice that develop spontaneous HCC lesions, highly proliferating and spread in a normal liver parenchyma. After the adoptive transfer of in vitro differentiated TAg-specific Teff in tumor-bearing mice, we observed that just some mice respond to the cytotoxic activity of the transferred cells, eliminating partially or completely the tumor, while in other mice the adoptive transferred cells have no beneficial effect on the tumor elimination. Thus, using a mathematical approach, we managed to pick the lesion volume as the fundamental parameter to predict the fate of each single HCC lesion: we called “responders” (R) the HCC lesions that are responsive to the cytotoxic activity of the TAg-specific Teff, with a single lesion volume <10 mm3, while we called “non-responders” (NR) the lesions that are not responding and have a single HCC lesion volume > 100mm3. We then studied the determinants that confer the therapeutic activity to the TAg-specific Teff in R and the ones that dampen the therapeutic activity in the NR lesions. Our main finding was that the vessel phenotype was completely different in R and NR. Our data support the hypothesis that some anatomic, hemodynamic, and environmental features acquired by each individual HCC lesion, during their growth, can influence their responsiveness to the Teff killing. The innovative nature of our work will elucidate new mechanisms whereby Teff fail to exert their immune function and cytotoxic activity in tumorigenic liver.
18-gen-2023
IANNACONE, MATTEO
Investigation of intratumoral cd8+ t cell spatiotemporal localization and function / Bianca Partini - : . , 2023 Jan 18. ((35. ciclo, Anno Accademico 2021/2022.
Doctoral Thesis
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Descrizione: PhD thesis Bianca Partini
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11768/136738
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