Biology subjects

Marotta, G.

Publications and source records attributed to Marotta, G..

5 recordsLinked to original sources

bioRxiv2024

Thrombospondins 1 and 4 undergo coordinated transport to multicore cytotoxic granules to regulate SMAP biogenesis and function in CTL-mediatedcytotoxicity

Supramolecular Attack Particles (SMAPs) are particulate entities, characterized by a cytotoxic core enriched in granzymes and perforin surrounded by a glycoproteic shell, released by CTLs and NK cells. Prior proteomic analysis identified thrombospondin-1 (TSP-1) and thrombospondin-4 (TSP-4) as putative components of SMAPs. While TSP-1 has been validated as a component of the SMAP shell and shown to contribute significantly to CTL-mediated killing, the expression and function of TSP-4 in CTLs, and its interplay with TSP-1 in SMAP biogenesis and function, has not been investigated as yet. Here we demonstrate that TSP-4 and TSP-1 have a complementary expression profile during in vitro human CD8+ T cell differentiation to CTLs and sequentially localize to lytic granules (LG), with TSP-4 being required for TSP-1 association with LGs. Correlative light microscopy identified the TSP-enriched LGs as the SMAP-containing multicore granules. We show by STED microscopy a heterogeneity among TSP-enriched LGs, the most abundant population being positive for both TSP-4 and TSP-1. We also show that TSP-1 and TSP-4 are co-released in association with SMAPs at immune synapses formed on planar supported lipid bilayers, as assessed by dSTORM imaging. Finally, we provide evidence that TSP-4 is required for CTL- and SMAP-mediated cell killing. Of note, we found that chronic lymphocytic leukemia (CLL) cell supernatants, which suppress CTL mediated killing, also suppress expression of TSP-4 as well as of cytolytic effectors and impair SMAP biogenesis. These results identify TSP-4 as a key player in SMAP structure and activity and suggest that SMAPs may be a new target for immune suppression by CLL.

immunology↗

bioRxiv2023

Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia

The tumor microenvironment (TME) plays a central role in the pathogenesis of chronic lymphocytic leukemia (CLL), contributing to disease progression and chemoresistance. Leukemic cells shape the TME into a pro-survival and immunosuppressive niche through contact-dependent and contact-independent interactions with the cellular components of the TME. Immune synapse (IS) formation is defective in CLL. Here we asked whether soluble factors released by CLL cells contribute to their protection from cytotoxic T cell (CTL)-mediated killing by interfering with this process. We found that healthy CTLs cultured in media conditioned by leukemic cells from CLL patients or E-TCL1 mice upregulate the exhaustion marker PD-1 and become unable to form functional ISs and kill target cells. These defects were more pronounced when media were conditioned by leukemic cells lacking p66Shc, a proapoptotic adaptor whose deficiency has been implicated in disease aggressiveness both in CLL and in the E-TCL1 mouse model. Multiplex ELISA assays showed that leukemic cells from E-TCL1 mice secrete abnormally elevated amounts of CCL22, CCL24, IL-9 and IL-10, which are further upregulated in the absence of p66Shc. Among these, IL-9 and IL-10 were also overexpressed in leukemic cells from CLL patients, where they inversely correlated with residual p66Shc. Using neutralizing antibodies or the recombinant cytokines we show that IL-9, but not IL-10, mediates both the enhancement in PD-1 expression and the suppression of effector functions in healthy CTLs. Our results demonstrate that IL-9 secreted by leukemic cells negatively modulates the anti-tumor immune abilities of CTLs, highlighting a new suppressive mechanism and a novel potential therapeutical target in CLL.

cancer biology↗

bioRxiv2023

Polo-like kinase 1 regulates immune synapse assembly and cytotoxic T cell function by driving microtubule dynamics

Elimination of virally infected or tumoral cells is mediated by cytotoxic T cells (CTL). Upon antigen recognition CTLs assemble a specialized signaling and secretory domain at the interface with their target, the immune synapse (IS). During IS formation CTLs acquire a transient polarity, marked by re-orientation of the centrosome and microtubule cytoskeleton toward the IS, thus directing the transport and delivery of the lytic granules to the target cell. Based on the implication of the kinase Aurora-A in CTL function we hypothesized that its substrate, the mitotic regulator Polo-like kinase 1 (PLK1), may participate in CTL IS assembly. We demonstrate that PLK1 is phosphorylated upon TCR triggering and polarizes to the IS. PLK1 silencing or inhibition results in impaired IS assembly and function, as witnessed by defective synaptic accumulation of TCRs as well as compromised centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing. This function is achieved by coupling early signaling to microtubule dynamics, a function pivotal for CTL-mediated cytotoxicity. These results identify PLK1 as a new player in CTL IS assembly and function. Summary statementThe mitotic kinase Polo-like kinase 1 promotes centrosome polarization to the immune synapse in cytotoxic T cells by coupling TCR signaling to microtubule dynamics

immunology↗

bioRxiv2023

Human colorectal cancer: upregulation of the adaptor protein Rai in TILs leads to cell dysfunction by sustaining GSK-3 activation and PD-1 expression

BackgroundThe immunosuppressive tumor microenvironment (TME) of colorectal cancer (CRC) is a major hurdle for immune checkpoint inhibitor-based therapies. Hence characterization of the signaling pathways driving T cell exhaustion within TME is a critical need for the discovery of novel therapeutic targets and the development of effective therapies. We previously showed that i) the adaptor protein Rai is a negative regulator of T cell receptor signaling and T helper 1 (Th1)/Th17 cell differentiation; and ii) Rai deficiency is implicated in the hyperactive phenotype of T cells in autoimmune diseases. MethodsThe expression level of Rai was measured by qRT-PCR in paired peripheral blood T cells and T cells infiltrating tumor tissue and the normal adjacent tissue in CRC patients. The impact of HIF-1 on Rai expression was evaluated in T cells exposed to hypoxia and by performing chromatin immunoprecipitation assays and RNA interference assays. The mechanism by which upregulation of Rai in T cells promotes T cell exhaustion were evaluated by flow cytometric, qRT-PCR and western blot analyses. ResultsWe show that Rai is a novel HIF-1-responsive gene that is upregulated in tumor infiltrating lymphocytes of CRC patients compared to patient-matched circulating T cells. Rai upregulation in T cells promoted PD-1 expression and impaired antigen-dependent degranulation of CD8+ T cells by inhibiting phospho-inactivation of glycogen synthase kinase (GSK)-3, a central regulator of PD- 1 expression and T cell-mediated anti-tumor immunity. ConclusionsOur data identify Rai as a hitherto unknown regulator of the TME-induced exhausted phenotype of human T cells. What is already known on this topicO_LITumor hypoxia contributes to establish an immunosuppressive microenvironment that unleashes T cell function and limits the efficacy of immunotherapy in CRC. C_LIO_LIThe molecular mechanisms underlying the impact of hypoxia on the signaling pathways controlling PD-1 expression are unknown. C_LI What this study addsO_LIA Rai/Akt/GSK-3 axis regulates PD-1 expression following TCR/CD28 co-stimulation C_LIO_LIThis study uncovers the signaling pathway controlling hypoxia-dependent T cell exhaustion. C_LI How this study might affect research, practice or policyRai expression levels in TILs of CRC patients might be explored as a potential new biomarker of T cell exhaustion and a predictive biomarker for anti-PD-1 response.

cancer biology↗

bioRxiv2022

SARS-CoV-2 Spike protein suppresses CTL-mediated killing by inhibiting immune synapse assembly

CTL-mediated killing of virally infected or malignant cells is orchestrated at a specialized intercellular junction, the immune synapse (IS). We hypothesized that SARS-CoV-2 may target IS assembly in CTLs to escape killing. We show that primary human CD8+ T cells strongly upregulate the expression of ACE2, the Spike protein receptor, during differentiation to CTLs. CTL pre-incubation with the Wuhan or Omicron Spike variants inhibits IS assembly and function, as shown by defective synaptic accumulation of TCRs and tyrosine phosphoproteins as well as defective centrosome and lytic granule polarisation to the IS, resulting in impaired target cell killing. These defects were reversed by anti-Spike antibodies that interfere with ACE2 binding and were reproduced by ACE2 engagement with Angiotensin-II or an anti-ACE2 antibody, but not by the ACE2 product Ang (1-7). These results highlight a new strategy of immune evasion by SARS-CoV-2 based on the Spike-dependent, ACE2-mediated targeting of the lytic IS to prevent the elimination of infected cells. Summary statementWe report a new mechanism of immune evasion by SARS-CoV-2 based on direct disabling CTLs to form immune synapses through Spike protein binding to ACE2. This mechanism could contribute to the failure of the immune system to control SARS-CoV-2 infection.

immunology↗