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 that the kinase Aurora A has a role in CTL function, we hypothesized that its substrate, the mitotic regulator Polo-like kinase 1 (PLK1), might 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 T cell receptors (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.

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 the immune synapse (IS). We hypothesized that SARS-CoV-2 may target lytic IS assembly to escape elimination. We show that human CD8+ T cells upregulate the expression of ACE2, the Spike receptor, during differentiation to CTLs. CTL preincubation 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 polarization to the IS, resulting in impaired target cell killing and cytokine production. These defects were reversed by anti-Spike antibodies interfering with ACE2 binding and reproduced by ACE2 engagement by angiotensin II or anti-ACE2 antibodies, but not by the ACE2 product Ang (1-7). IS defects were also observed ex vivo in CTLs from COVID-19 patients. 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 elimination of infected cells.

Regulation of Selective B Cell Autophagy by the Pro-oxidant Adaptor p66SHC.

p66SHC is a pro-oxidant member of the SHC family of protein adaptors that acts as a negative regulator of cell survival. In lymphocytes p66SHC exploits both its adaptor and its reactive oxygen species (ROS)-elevating function to antagonize mitogenic and survival signaling and promote apoptosis. As a result, p66SHC deficiency leads to the abnormal expansion of peripheral T and B cells and lupus-like autoimmunity. Additionally, a defect in p66SHC expression is a hallmark of B cell chronic lymphocytic leukemia, where it contributes to the accumulation of long-lived neoplastic cells. We have recently provided evidence that p66SHC exerts a further layer of control on B cell homeostasis by acting as a new mitochondrial LC3-II receptor to promote the autophagic demise of dysfunctional mitochondria. Here we discuss this finding in the context of the autophagic control of B cell homeostasis, development, and differentiation in health and disease.

The intraflagellar transport protein IFT20 controls lysosome biogenesis by regulating the post-Golgi transport of acid hydrolases.

The assembly and function of the primary cilium depends on multimolecular intraflagellar transport (IFT) complexes that shuttle their cargo along the axonemal microtubules through their interaction with molecular motors. The IFT system has been moreover recently implicated in a reciprocal interplay between autophagy and ciliogenesis. We have previously reported that IFT20 and other components of the IFT complexes participate in the assembly of the immune synapse in the non-ciliated T cell, suggesting that other cellular processes regulated by the IFT system in ciliated cells, including autophagy, may be shared by cells lacking a cilium. Starting from the observation of a defect in autophagic clearance and an accumulation of lipid droplets in IFT20-deficient T cells, we show that IFT20 is required for lysosome biogenesis and function by controlling the lysosomal targeting of acid hydrolases. This function involves its ability to regulate the retrograde traffic of the cation-independent mannose-6-phosphate receptor (CI-MPR) to the trans-Golgi network, which is achieved by coupling recycling CI-MPRs to the microtubule motor dynein. Consistent with the lysosomal defect, an upregulation of the TFEB-dependent expression of the lysosomal gene network can be observed in IFT20-deficient cells, which is associated with defective tonic T-cell antigen receptor signaling and mTOR activity. We additionally show that the lysosome-related function of IFT20 extends to non-ciliated cells other than T cells, as well as to ciliated cells. Our findings provide the first evidence that a component of the IFT system that controls ciliogenesis is implicated in the biogenesis of lysosomes.

The pro-oxidant adaptor p66SHC promotes B cell mitophagy by disrupting mitochondrial integrity and recruiting LC3-II.

Macroautophagy/autophagy has emerged as a central process in lymphocyte homeostasis, activation and differentiation. Based on our finding that the p66 isoform of SHC1 (p66SHC) pro-apoptotic ROS-elevating SHC family adaptor inhibits MTOR signaling in these cells, here we investigated the role of p66SHC in B-cell autophagy. We show that p66SHC disrupts mitochondrial function through its CYCS (cytochrome c, somatic) binding domain, thereby impairing ATP production, which results in AMPK activation and enhanced autophagic flux. While p66SHC binding to CYCS is sufficient for triggering apoptosis, p66SHC-mediated autophagy additionally depends on its ability to interact with membrane-associated LC3-II through a specific binding motif within its N terminus. Importantly, p66SHC also has an impact on mitochondria homeostasis by inducing mitochondrial depolarization, protein ubiquitination at the outer mitochondrial membrane, and local recruitment of active AMPK. These events initiate mitophagy, whose full execution relies on the role of p66SHC as an LC3-II receptor which brings phagophore membranes to mitochondria. Importantly, p66SHC also promotes hypoxia-induced mitophagy in B cells. Moreover, p66SHC deficiency enhances B cell differentiation to plasma cells, which is controlled by intracellular ROS levels and the hypoxic germinal center environment. The results identify mitochondrial p66SHC as a novel regulator of autophagy and mitophagy in B cells and implicate p66SHC-mediated coordination of autophagy and apoptosis in B cell survival and differentiation. Abbreviations: ACTB: actin beta; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; ATG: autophagy-related; CYCS: cytochrome c, somatic; CLQ: chloroquine; COX: cyclooxygenase; CTR: control; GFP: green fluorescent protein; HIFIA/Hif alpha: hypoxia inducible factor 1 subunit alpha; IMS: intermembrane space; LIR: LC3 interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR/mTOR: mechanistic target of rapamycin kinase; OA: oligomycin and antimycin A; OMM: outer mitochondrial membrane; PHB: prohibitin; PBS: phosphate-buffered saline; PINK1: PTEN induced putative kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SHC: src Homology 2 domain-containing transforming protein; TMRM: tetramethylrhodamine, methyl ester; TOMM: translocase of outer mitochondrial membrane; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.

Compartmentalized Cyclic AMP Production by the Bordetella pertussis and Bacillus anthracis Adenylate Cyclase Toxins Differentially Affects the Immune Synapse in T Lymphocytes.

A central feature of the immune synapse (IS) is the tight compartmentalization of membrane receptors and signaling mediators that is functional for its ability to coordinate T cell activation. Second messengers centrally implicated in this process, such as Ca2+ and diacyl glycerol, also undergo compartmentalization at the IS. Current evidence suggests a more complex scenario for cyclic AMP (cAMP), which acts both as positive and as negative regulator of T-cell antigen receptor (TCR) signaling and which, as such, must be subjected to a tight spatiotemporal control to allow for signaling at the IS. Here, we have used two bacterial adenylate cyclase toxins that produce cAMP at different subcellular localizations as the result of their distinct routes of cell invasion, namely Bordetella pertussis CyaA and Bacillus anthracis edema toxin (ET), to address the ability of the T cell to confine cAMP to the site of production and to address the impact of compartmentalized cAMP production on IS assembly and function. We show that CyaA, which produces cAMP close to the synaptic membrane, affects IS stability by modulating not only the distribution of LFA-1 and its partners talin and L-plastin, as previously partly reported but also by promoting the sustained synaptic accumulation of the A-kinase adaptor protein ezrin and protein kinase A while suppressing the ß-arrestin-mediated recruitment of phosphodiesterase 4B. These effects are dependent on the catalytic activity of the toxin and can be reproduced by treatment with a non-hydrolyzable cAMP analog. Remarkably, none of these effects are elicited by ET, which produces cAMP at a perinuclear localization, despite its ability to suppress TCR signaling and T cell activation through its cAMP-elevating activity. These results show that the IS responds solely to local elevations of cAMP and provide evidence that potent compartmentalization mechanisms are operational in T cells to contain cAMP close to the site of production, even when produced at supraphysiological levels.

Burkitt lymphoma beyond MYC translocation: N-MYC and DNA methyltransferases dysregulation.

BACKGROUND: The oncogenic transcription factor MYC is pathologically activated in many human malignancies. A paradigm for MYC dysregulation is offered by Burkitt lymphoma, where chromosomal translocations leading to Immunoglobulin gene-MYC fusion are the crucial initiating oncogenic events. However, Burkitt lymphoma cases with no detectable MYC rearrangement but maintaining MYC expression have been identified and alternative mechanisms can be involved in MYC dysregulation in these cases. METHODS: We studied the microRNA profile of MYC translocation-positive and MYC translocation-negative Burkitt lymphoma cases in order to uncover possible differences at the molecular level. Data was validated at the mRNA and protein level by quantitative Real-Time polymerase chain reaction and immunohistochemistry, respectively. RESULTS: We identified four microRNAs differentially expressed between the two groups. The impact of these microRNAs on the expression of selected genes was then investigated. Interestingly, in MYC translocation-negative cases we found over-expression of DNA-methyl transferase family members, consistent to hypo-expression of the hsa-miR-29 family. This finding suggests an alternative way for the activation of lymphomagenesis in these cases, based on global changes in methylation landscape, aberrant DNA hypermethylation, lack of epigenetic control on transcription of targeted genes, and increase of genomic instability. In addition, we observed an over-expression of another MYC family gene member, MYCN that may therefore represent a cooperating mechanism of MYC in driving the malignant transformation in those cases lacking an identifiable MYC translocation but expressing the gene at the mRNA and protein levels. CONCLUSIONS: Collectively, our results showed that MYC translocation-positive and MYC translocation-negative Burkitt lymphoma cases are slightly different in terms of microRNA and gene expression. MYC translocation-negative Burkitt lymphoma, similarly to other aggressive B-cell non Hodgkin's lymphomas, may represent a model to understand the intricate molecular pathway responsible for MYC dysregulation in cancer.

The Epstein Barr-encoded BART-6-3p microRNA affects regulation of cell growth and immuno response in Burkitt lymphoma.

BACKGROUND: Burkitt lymphoma is an aggressive B-cell lymphoma presenting in three clinical forms: endemic, sporadic and immunodeficiency-associated. More than 90% of endemic Burkitt lymphoma carry latent Epstein-Barr virus, whereas only 20% of sporadic Burkitt lymphoma are associated with Epstein-Barr infection. Although the Epstein-Barr virus is highly related with the endemic form, how and whether the virus participates in its pathogenesis remains to be fully elucidated. In particular, the virus may impair cellular gene expression by its own encoded microRNAs. METHODS: Using microRNA profiling we compared Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for both cellular and viral microRNAs. The array results were validated by qRT-PCR, and potential targets of viral microRNAs were then searched by bioinformatic predictions, and classified in functional categories, according to the Gene Ontology. Our findings were validated by in vitro functional studies and by immunohistochemistry on a larger series of cases. RESULTS: We showed that a few cellular microRNAs are differentially expressed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases, and identified a subset of viral microRNAs expressed in Epstein-Barr-positive Burkitt lymphomas. Of these, we characterized the effects of viral BART6-3p on regulation of cellular genes. In particular, we analyzed the IL-6 receptor genes (IL-6Rα and IL-6ST), PTEN and WT1 expression for their possible relevance to Burkitt lymphoma. By means of immunohistochemistry, we observed a down-regulation of the IL-6 receptor and PTEN specifically in Epstein-Barr-positive Burkitt lymphoma cases, which may result in the impairment of key cellular pathways and may contribute to malignant transformation. On the contrary, no differences were observed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for WT1 expression. CONCLUSIONS: Our preliminary results point at an active role for the Epstein-Barr virus in Burkitt lymphomagenesis and suggest new possible mechanisms used by the virus in determining dysregulation of the host cell physiology.

HIV-1 Tat induces DNMT over-expression through microRNA dysregulation in HIV-related non Hodgkin lymphomas.

BACKGROUND: A close association between HIV infection and the development of cancer exists. Although the advent of highly active antiretroviral therapy has changed the epidemiology of AIDS-associated malignancies, a better understanding on how HIV can induce malignant transformation will help the development of novel therapeutic agents. METHODS: HIV has been reported to induce the expression of DNMT1 in vitro, but still no information is available about the mechanisms regulating DNMT expression in HIV-related B-cell lymphomas. In this paper, we investigated the expression of DNMT family members (DNMT1, DNMT3a/b) in primary cases of aggressive B-cell lymphomas of HIV-positive subjects. RESULTS: Our results confirmed the activation of DNMT1 by HIV in vivo, and reported for the first time a marked up-regulation of DNMT3a and DNMT3b in HIV-positive aggressive B-cell lymphomas. DNMT up-regulation in HIV-positive tumors correlated with down-regulation of specific microRNAs, as the miR29 family, the miR148-152 cluster, known to regulate their expression. Literature reports the activation of DNMTs by the human polyomavirus BKV large T-antigen and adenovirus E1a, through the pRb/E2F pathway. We have previously demonstrated that the HIV Tat protein is able to bind to the pocket proteins and to inactivate their oncosuppressive properties, resulting in uncontrolled cell proliferation. Therefore, we focused on the role of Tat, due to its capability to be released from infected cells and to dysregulate uninfected ones, using an in vitro model in which Tat was ectopically expressed in B-cells. CONCLUSIONS: Our findings demonstrated that the ectopic expression of Tat was per se sufficient to determine DNMT up-regulation, based on microRNA down-regulation, and that this results in aberrant hypermethylation of target genes and microRNAs. These results point at a direct role for Tat in participating in uninfected B-cell lymphomagenesis, through dysregulation of the epigenetical control of gene expression.

Gene expression analysis uncovers similarity and differences among Burkitt lymphoma subtypes.

Burkitt lymphoma (BL) is classified into 3 clinical subsets: endemic, sporadic, and immunodeficiency-associated BL. So far, possible differences in their gene expression profiles (GEPs) have not been investigated. We studied GEPs of BL subtypes, other B-cell lymphomas, and B lymphocytes; first, we found that BL is a unique molecular entity, distinct from other B-cell malignancies. Indeed, by unsupervised analysis all BLs clearly clustered apart of other lymphomas. Second, we found that BL subtypes presented slight differences in GEPs. Particularly, they differed for genes involved in cell cycle control, B-cell receptor signaling, and tumor necrosis factor/nuclear factor κB pathways. Notably, by reverse engineering, we found that endemic and sporadic BLs diverged for genes dependent on RBL2 activity. Furthermore, we found that all BLs were intimately related to germinal center cells, differing from them for molecules involved in cell proliferation, immune response, and signal transduction. Finally, to validate GEP, we applied immunohistochemistry to a large panel of cases and showed that RBL2 can cooperate with MYC in inducing a neoplastic phenotype in vitro and in vivo. In conclusion, our study provided substantial insights on the pathobiology of BLs, by offering novel evidences that may be relevant for its classification and possibly future treatment.