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Generation and maintenance of long-lived memory T cells in humans

Periodic Reporting for period 4 - PERSYST (Generation and maintenance of long-lived memory T cells in humans)

Reporting period: 2020-03-01 to 2021-05-31

A specialized population of immune cells called T cells is important for protection from invading microbes. Vaccination strategies elicit the development of T cells that are capable to remember the encounter with the pathogen upon a second rechallenge, and thus are called memory T cells. Such memory endows them to mediate a faster and more effective response, hence confering protection. To better understand how memory is formed and mantained, we studied a condition where T cells from a donor are infused into a patient, i.e. in the context of haploidentical bone marrow transplantation for hematological malignancies. We demonstrated that memory T cells adoptively-transferred with the graft are capable to persist in the long-term in a fashion similar to stem cells, that is to self-renew and to be multipotent. These memory T cells actively respond to viral reactivation, thus leading to hypothesize that they are important to limit viral dissemination and thus confer protection. By studying the molecular profiles of memory cells (transcriptomics, proteomics, etc.), we have identified novel subsets and molecular signals related to the generation and persistence of these cells in humans. We used molecular approaches to interfere with the terminal differentiation of T cells and thus induce stem-like memory T cells with enhanced potency against tumors upon an immunotherapeutic approached call adoptive cell transfer. We have further identified a distinct subset of stem-like memory T cells that is precursor of dysfunctional cells and has relevance in chronic pathological conditions such as viral infections and tumors. We are currently working on inhibiting those molecular signals at the basis of dysfunction, so to promote T cells with enhaced functionality in the long term. Our research will have important implications for the generation of durable immunity in the context of vaccination strategies and anti-tumor immunity.
Our work uses cellular and molecular technologies to analyze the complex heterogeneity of T cell responses at the level of single cells. Despite organized in cell populations and performing similar functions, every cell in the immune system is different. Therefore single cell analysis is a very powerful tool to identify rare functions that otherwise would be missed by bulk analysis (i.e. of the average population). In this regard, we recently optimized a unique technology, 28-color, 30-parameter flow cytometry and used it to study antigen-specific T cells in the context of haploidentical stem cell transplantation with post-transplant cyclophosphamide (HSCT with pt-cy).
We defined the phenotype, functionality and T cell receptor diversity of T cells that are adoptively transferred with the graft. We used cytomegalovirus (CMV)-specific T cells as a model, as we have demonstrated in the past that CMV-specific T cells infused with the graft are capable to persist in the patient and clonally expand following viral reactivation. We developed 30-parameter flow cytometry panels to investigate multiple features of T cells and found that CMV-specific T cells are primed early post HSCT, initially display a proliferating/activated phenotype, that is replaced by a terminal effector phenotype. One year after transplant, CMV-specific T-cell profiles were similar to those of the CMV-seropositive donor, suggesting reestablishment of physiological homeostasis. Uncontrolled viral replication associated with lower abundance of distinct CMV-specific CD4+ T-cell immunophenotypes, hinting at a possible role of these cells in CMV control following haplo-HSCT with pt-cy. Analysis of the blood of patients receiving infusions of immune cells from the donor revealed that CMV-specific T cells are capable to persist, thereby indicating they can provide anti-viral immunity in the long term.

We combined single cell technologies, such as sequencing and 30-parameter flow cytometry, to study the complexity of memory T cells in humans. We recently developed new pipelines of computational analysis to dissect the complexity of single cell data, and were able to identify previously unrecognized populations of cells that have long-lived characteristics but at the same time share features with those dysfunctional cells found in solid tumors. Intriguingly, these cells are the preferential targets of immunotherapy approaches directed to reinvigorate the function of the immune system in solid tumors (i.e. those with checkpoint blockade). We have elucidated the molecular characteristics of these dysfunctional cells, and are currently exploiting this information to improve the function of T cells in response to checkpoint blockade immunotherapy or in response to an additional immunotherapeutic approach that is very effective for the treatment of hematological malignancies, i.e. infusion of anti-tumor T cells generated in the lab. In particular, we have shown that growing T cells in the lab in the presence of inexpensive antioxidants improves the anti-tumor immune response considerably at the preclinical level. We have also identified novel transcription factors (molecules capable to regulate the expression of hundreds of genes) that block the function of these potent T cells. We anticipate the genetic engineering of T cells aiming to disrupt these inhibitory networks will improve the anti-tumor immune response in the long term.
Our results may be exploited to favour vaccination strategies and adoptive T cell transfer approaches, which rely on long-term memory. We have published the novel finding that inhibiting reactive oxygen species metabolism with antioxidants induces long-lived memory T cell development and improves anti-tumor immunity upon adoptive transfer. Induction of such cells to be used T cell infusions for cancer immunotherapy is a major goal. We thus expect our contribution to change the way how T cells are generated for such purpose. In addition, we were among the first to optimize high-dimensional, 30-parameter flow cytometry, putting us at the forefront of single cell analysis of the immune system. We expect our work to influence future approaches of single cell analysis by flow cytometry and subsequent data analysis. Flow cytometry is also widely used in diagnostic for cancer, immunodeficiencies and immune-related diseases. We expect such a complex technology to become more popular in the near future and our work to become a reference not only for research centers but also for hospitals. Our recent discovery on long-lived memory T cells with dysfunctional characteristics have important implications for vaccine development, cancer immunotherapy and adoptive cell transfer. In fact, a large effort worldwide is currently devoted to induce functional long-lived memory cells that are instead resistant to dysfunction, and thus can mediate more potent and longer-lived responses in cancer and chronic viral infections. In this regard, our publication revealed novel molecular mechanisms that we are currently exploiting in follow-up projects.
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T cell differentiation