Revealing and harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells.

OBJECTIVE: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells. DESIGN: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products. RESULTS: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCRED)) and the CD39 encoding gene (ENTPD1), thus generating TCREDENTPD1KOHER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2+ patient-derived organoids in vitro and in vivo. CONCLUSION: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC.

Cytomegalovirus-specific T cells restricted for shared and donor human leukocyte antigens differentially impact on cytomegalovirus reactivation risk after allogeneic hematopoietic stem cell transplantation.

After allogeneic hematopoietic stem cell transplantation (HSCT), the emergence of circulating cytomegalovirus (CMV)- specific T cells correlates with protection from CMV reactivation, an important risk factor for non-relapse mortality. However, functional assays measuring CMV-specific cells are time-consuming and often inaccurate at early time-points. We report the results of a prospective single-center, non-interventional study that identified the enumeration of Dextramerpositive CMV-specific lymphocytes as a reliable and early predictor of viral reactivation. We longitudinally monitored 75 consecutive patients for 1 year after allogeneic HSCT (n=630 samples). The presence of ≥0.5 CMV-specific CD8+ cells/mL at day +45 was an independent protective factor from subsequent clinically relevant reactivation in univariate (P<0.01) and multivariate (P<0.05) analyses. Dextramer quantification correlated with functional assays measuring interferon-γ production, and allowed earlier identification of high-risk patients. In mismatched transplants, the comparative analysis of lymphocytes restricted by shared, donor- and host-specific HLA revealed the dominant role of thymic-independent CMV-specific reconstitution. Shared and donor-restricted CMV-specific T cells reconstituted with similar kinetics in recipients of CMV-seropositive donors, while donor-restricted T-cell reconstitution from CMV-seronegative grafts was impaired, indicating that in primary immunological responses the emergence of viral-specific T cells is largely sustained by antigen encounter on host infected cells rather than by cross-priming/presentation by non-infected donor-derived antigen-presenting cells. Multiparametric flow cytometry and high-dimensional analysis showed that shared-restricted CMV-specific lymphocytes display a more differentiated phenotype and increased persistence than donor-restricted counterparts. In this study, monitoring CMV-specific cells by Dextramer assay after allogeneic HSCT shed light on mechanisms of immune reconstitution and enabled risk stratification of patients, which could improve the clinical management of post-transplant CMV reactivations.

Flow cytometry data mining by cytoChain identifies determinants of exhaustion and stemness in TCR-engineered T cells.

The phenotype of infused cells is a major determinant of Adoptive T-cell therapy (ACT) efficacy. Yet, the difficulty in deciphering multiparametric cytometry data limited the fine characterization of cellular products. To allow the analysis of dynamic and complex flow cytometry samples, we developed cytoChain, a novel dataset mining tool and a new analytical workflow. CytoChain was challenged to compare state-of-the-art and innovative culture conditions to generate stem-like memory cells (TSCM ) suitable for ACT. Noticeably, the combination of IL-7/15 and superoxides scavenging sustained the emergence of a previously unidentified nonexhausted Fit-TSCM signature, overlooked by manual gating and endowed with superior expansion potential. CytoChain proficiently traced back this population in independent datasets, and in T-cell receptor engineered lymphocytes. CytoChain flexibility and function were then further validated on a published dataset from circulating T cells in COVID-19 patients. Collectively, our results support the use of cytoChain to identify novel, functionally critical immunophenotypes for ACT and patients immunomonitoring.

Lean adoption in hospitals: the role of contextual factors and introduction strategy.

BACKGROUND: In the scientific literature, many studies describe the application of lean methodology in the hospital setting. Most of the articles focus on the results rather than on the approach adopted to introduce the lean methodology. In the absence of a clear view of the context and the introduction strategy, the first steps of the implementation process can take on an empirical, trial and error profile. Such implementation is time-consuming and resource-intensive and affects the adoption of the model at the organizational level. This research aims to outline the role contextual factors and introduction strategy play in supporting the operators introducing lean methodology in a hospital setting. METHODOLOGY: The methodology is revealed in a case study of an important hospital in Southern Italy, where lean has been successfully introduced through a pilot project in the pathway of cancer patients. The originality of the research is seen in the detailed description of the contextual elements and the introduction strategy. RESULTS: The results show significant process improvements and highlight the spontaneous dissemination of the culture of change in the organization and the streamlined adoption at the micro level. CONCLUSION: The case study shows the importance of the lean introduction strategy and contextual factors for successful lean implementation. Furthermore, it shows how both factors influence each other, underlining the dynamism of the organizational system.

Exploiting Manipulated Small Extracellular Vesicles to Subvert Immunosuppression at the Tumor Microenvironment through Mannose Receptor/CD206 Targeting.

Immunosuppression at tumor microenvironment (TME) is one of the major obstacles to be overcome for an effective therapeutic intervention against solid tumors. Tumor-associated macrophages (TAMs) comprise a sub-population that plays multiple pro-tumoral roles in tumor development including general immunosuppression, which can be identified in terms of high expression of mannose receptor (MR or CD206). Immunosuppressive TAMs, like other macrophage sub-populations, display functional plasticity that allows them to be re-programmed to inflammatory macrophages. In order to mitigate immunosuppression at the TME, several efforts are ongoing to effectively re-educate pro-tumoral TAMs. Extracellular vesicles (EVs), released by both normal and tumor cells types, are emerging as key mediators of the cell to cell communication and have been shown to have a role in the modulation of immune responses in the TME. Recent studies demonstrated the enrichment of high mannose glycans on the surface of small EVs (sEVs), a subtype of EVs of endosomal origin of 30-150 nm in diameter. This characteristic renders sEVs an ideal tool for the delivery of therapeutic molecules into MR/CD206-expressing TAMs. In this review, we report the most recent literature data highlighting the critical role of TAMs in tumor development, as well as the experimental evidences that has emerged from the biochemical characterization of sEV membranes. In addition, we propose an original way to target immunosuppressive TAMs at the TME by endogenously engineered sEVs for a new therapeutic approach against solid tumors.

Trans-dissemination of exosomes from HIV-1-infected cells fosters both HIV-1 trans-infection in resting CD4+ T lymphocytes and reactivation of the HIV-1 reservoir.

Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4+ T lymphocytes by means of mechanisms defined as trans-infection and trans-dissemination, respectively. We previously reported that exosomes from HIV-1-infected cells activate both uninfected quiescent CD4+ T lymphocytes, which become permissive to HIV-1, and latently infected cells, with release of HIV-1 particles. However, nothing is known about the effects of trans-dissemination of exosomes produced by HIV-1-infected cells on uninfected or latently HIV-1-infected CD4+ T lymphocytes. Here, we report that trans-dissemination of exosomes from HIV-1-infected cells induces cell activation in resting CD4+ T lymphocytes, which appears stronger with mature than immature DCs. Using purified preparations of both HIV-1 and exosomes, we observed that mDC-mediated trans-dissemination of exosomes from HIV-1-infected cells to resting CD4+ T lymphocytes induces efficient trans-infection and HIV-1 expression in target cells. Most relevant, when both mDCs and CD4+ T lymphocytes were isolated from combination anti-retroviral therapy (ART)-treated HIV-1-infected patients, trans-dissemination of exosomes from HIV-1-infected cells led to HIV-1 reactivation from the viral reservoir. In sum, our data suggest a role of exosome trans-dissemination in both HIV-1 spread in the infected host and reactivation of the HIV-1 reservoir.

Latent HIV-1 is activated by exosomes from cells infected with either replication-competent or defective HIV-1.

BACKGROUND: Completion of HIV life cycle in CD4(+) T lymphocytes needs cell activation. We recently reported that treatment of resting CD4(+) T lymphocytes with exosomes produced by HIV-1 infected cells induces cell activation and susceptibility to HIV replication. Here, we present data regarding the effects of these exosomes on cells latently infected with HIV-1. RESULTS: HIV-1 latently infecting U937-derived U1 cells was activated upon challenge with exosomes purified from the supernatant of U937 cells chronically infected with HIV-1. This effect was no more detectable when exosomes from cells infected with HIV-1 strains either nef-deleted or expressing a functionally defective Nef were used, indicating that Nef is the viral determinant of exosome-induced HIV-1 activation. Treatment with either TAPI-2, i.e., a specific inhibitor of the pro-TNFα-processing ADAM17 enzyme, or anti-TNFα Abs abolished HIV-1 activation. Hence, similar to what previously demonstrated for the exosome-mediated activation of uninfected CD4(+) T lymphocytes, the Nef-ADAM17-TNFα axis is part of the mechanism of latent HIV-1 activation. It is noteworthy that these observations have been reproduced using: (1) primary CD4(+) T lymphocytes latently infected with HIV-1; (2) exosomes from both primary CD4(+) T lymphocytes and macrophages acutely infected with HIV-1; (3) co-cultures of HIV-1 acutely infected CD4(+) T lymphocytes and autologous lymphocytes latently infected with HIV-1, and (4) exosomes from cells expressing a defective HIV-1. CONCLUSIONS: Our results strongly suggest that latent HIV-1 can be activated by TNFα released by cells upon ingestion of exosomes released by infected cells, and that this effect depends on the activity of exosome-associated ADAM17. These pieces of evidence shed new light on the mechanism of HIV reactivation in latent reservoirs, and might also be relevant to design new therapeutic interventions focused on HIV eradication.

Exosomes from human immunodeficiency virus type 1 (HIV-1)-infected cells license quiescent CD4+ T lymphocytes to replicate HIV-1 through a Nef- and ADAM17-dependent mechanism.

UNLABELLED: Resting CD4+ T lymphocytes resist human immunodeficiency virus (HIV) infection. Here, we provide evidence that exosomes from HIV-1-infected cells render resting human primary CD4+ T lymphocytes permissive to HIV-1 replication. These results were obtained with transwell cocultures of HIV-1-infected cells with quiescent CD4+ T lymphocytes in the presence of inhibitors of exosome release and were confirmed using exosomes purified from supernatants of HIV-1-infected primary CD4+ T lymphocytes. We found that the expression of HIV-1 Nef in exosome-producing cells is both necessary and sufficient for cell activation as well as HIV-1 replication in target CD4+ T lymphocytes. We also identified a Nef domain important for the effects we observed, i.e., the 62EEEE65 acidic cluster domain. In addition, we observed that ADAM17, i.e., a disintegrin and metalloprotease converting pro-tumor necrosis factor alpha (TNF-α) in its mature form, associates with exosomes from HIV-1-infected cells, and plays a key role in the HIV-1 replication in quiescent CD4+ T lymphocytes. Treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in resting CD4+ T lymphocytes. TNF-α is the downstream effector of ADAM17 since the treatment of resting lymphocytes with anti-TNF-α antibodies blocked the HIV-1 replication. The data presented here are consistent with a model where Nef induces intercellular communication through exosomes to activate bystander quiescent CD4+ T lymphocytes, thus stimulating viral spread. IMPORTANCE: Overall, our findings support the idea that HIV evolved to usurp the exosome-based intercellular communication network to favor its spread in infected hosts.

Cell activation and HIV-1 replication in unstimulated CD4+ T lymphocytes ingesting exosomes from cells expressing defective HIV-1.

BACKGROUND: A relevant burden of defective HIV-1 genomes populates PBMCs from HIV-1 infected patients, especially during HAART treatment. These viral genomes, although unable to codify for infectious viral particles, can express viral proteins which may affect functions of host cells as well as bystander ones. Cells expressing defective HIV-1 have a lifespan longer than that of cells producing infectious particles. Hence, their interaction with other cell types, including resting lymphocytes, is expected to occur frequently in tissues where HIV actively replicates. We investigated the effects of the expression of a prototype of functionally defective HIV-1 on bystander, unstimulated CD4+ T lymphocytes. RESULTS: We observed that unstimulated human primary CD4+ T lymphocytes were activated and became permissive for HIV-1 replication when co-cultivated with cells expressing a functionally defective HIV-1 (F12/Hut-78 cells). This effect depended on the presence in F12/Hut-78 supernatants of nanovesicles we identified as exosomes. By inspecting the underlying mechanism, we found that ADAM17, i.e., a disintegrin and metalloprotease converting pro-TNF-α in its mature form, associated with exosomes from F12/Hut-78 cells, and played a key role in the HIV-1 replication in unstimulated CD4+ T lymphocytes. In fact, the treatment with an inhibitor of ADAM17 abolished both activation and HIV-1 replication in unstimulated CD4+ T lymphocytes. TNF-α appeared to be the downstream effector of ADAM17 since the treatment of unstimulated lymphocytes with antibodies against TNF-α or its receptors blocked the HIV-1 replication. Finally, we found that the expression of NefF12 in exosome-producing cells was sufficient to induce the susceptibility to HIV-1 infection in unstimulated CD4+ T lymphocytes. CONCLUSIONS: Exosomes from cells expressing a functionally defective mutant can induce cell activation and HIV-1 susceptibility in unstimulated CD4+ T lymphocytes. This evidence highlights the relevance for AIDS pathogenesis of the expression of viral products from defective HIV-1 genomes.

Extracellular vesicle-based anti-HOXB7 CD8+ T cell-specific vaccination strengthens antitumor effects induced by vaccination against Her2/neu.

We previously developed an innovative strategy to induce CD8+ T lymphocyte-immunity through in vivo engineering of extracellular vesicles (EVs). This approach relies on intramuscular injection of DNA expressing antigens of interest fused at a biologically-inactive HIV-1 Nef protein mutant (Nefmut). Nefmut is very efficiently incorporated into EVs, thus conveying large amounts of fusion proteins into EVs released by transfected cells. This platform proved successful against highly immunogenic tumor-specific antigens. Here, we tested whether antigen-specific CD8+ T cell immune responses induced by engineered EVs can counteract the growth of tumors expressing two "self" tumor-associated antigens (TAAs): HOXB7 and Her2/neu. FVB/N mice were injected with DNA vectors expressing Nefmut fused to HOXB7 or Her2/neu, singly and in combination, before subcutaneous implantation of breast carcinoma cells co-expressing HOXB7 and Her2/neu. All mice immunized with the combination vaccine remained tumor-free, whereas groups vaccinated with single Nefmut-fused antigens were only partly protected, with stronger antitumor effects in Her2/neu-immunized mice. Double-vaccinated mice also controlled tumor growth upon a later tumor cell re-challenge. Importantly, co-vaccination also contained tumors in a therapeutic immunization setting. These results showed the efficacy of EV-based vaccination against two TAAs, and represent the first demonstration that HOXB7 may be targeted in multi-antigen immunotherapy strategies.