Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - PERSYST (Generation and maintenance of long-lived memory T cells in humans)

Teaser

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...

Summary

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. In this project we seek to identify the molecular signals governing the generation and persistence of long-lived memory T cells in humans with enhanced functional capacity. This is important because modulating these signals during vaccination strategies or upon adoptive T cell transfer (a protocol were T cells capable to recognize tumors or viruses are infused in a patient) may favour immune responses against the target. To achieve our aims, we will study 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 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. The stem cell-like 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 these cells (transcriptomics, proteomics, etc.), we will identify the molecular mechanisms that are responsible for their maintenance in the long-term. Such molecular signals will then be exploited in the laboratory to favour the generation of stem cell-like memory T cells from undifferentiated precursors. From these studies, we expect to identify additional molecular regulators of memory T cell differentiation and potent immunity.

Work performed

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 are now using is to study antigen-specific T cells in the context of bone marrow transplantation (BMT).
As part of Work Package 1 (WP1), we are currently defining the phenotype, functionality and T cell receptor diversity of T cells that are adoptively transferred with the graft. We are using cytomegalovirus (CMV)-specific T cells as a model, as we recently demonstrated that CMV-specific T cells that are infused with the graft are capable to persist in the patient and clonally expand following viral reactivation (occurring in 50% of transplanted patients). In parallel, we are studying Epstein-Barr virus (EBV)-specific responses as a comparison. Similar to CMV, EBV chronically infects healthy individuals but rarely reactivates following this type of BMT. We developed 30-parameter flow cytometry panels to investigate multiple features of T cells that are indicative of their immune response to these viruses, and are currently analyzing samples from patients at different time points post-BMT. In this way, we aim to identify correlates of persistence and immune response in the long-term, and expect to modulate these signals in uncommitted T cell precursors to generate stem-cell like T cells (WP3).
30-parameter flow cytometry data are complex because they measure up to 30 parameters on millions of single T cells in a multidimensional space. We recently developed new pipelines of computational analysis to dissect the complexity of single cell data. In this way, we could identify new subtypes of T cells that are currently being studied with molecular assays. The same technologies are currently being used to define the features of another type of T cells that are adoptively transferred with the graft, i.e. T cells capable to respond to tumor antigens (WP2). In addition, we are using single cell RNA sequencing, a technology capable to define the whole transcriptome at the level of single cells. This approach will define the genes that are involved in the differentiation and long-term maintenance of the infused cells.
In WP3, we took advantage of preliminary data to identify genes specifically involved in stem cell-like memory T cell differentiation in humans. In particular, we are focusing our attention on transcription factors, that is molecules capable to regulate the expression of hundreds of genes and thus defining the identity of cell populations. We identified candidate molecules that are specifically overexpressed by a specialized population of memory T cells with long term persistence capacity, i.e. the T memory stem cells (TSCM). Due to the paucity of these cells in humans, we initially optimized an in vitro system to generate large number of these cells from more abundant unprimed precursors (Zanon et al, Eur J Immunol, 2017 Sep;47(9):1468-1476). This approach takes advantage of specific stimulation via the T cell receptor and cytokine cocktails. Via the use of modified viruses, we were able to upregulate and downregulate specific genes and study the functionality of cells thereof. Bioinformatic analysis of gene expression profiles identified additional metabolic pathways that are differentially activated in TSCM vs more differentiated cells.Current experiments are aiming to modulate such pathways in order to influence memory T cell differentiation.

Final results

Our work is still in progress and the most recent results still have to be published. Our future work is expected to identify the pivotal mechanisms at the basis of long-term memory formation in humans. Our results may be exploited to favour vaccination strategies and adoptive T cell transfer approaches, which rely on long-term memory. A paper on the signals regulating the development of stem cell-like memory T cells from uncommited precursors has been submitted. 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 recently optimized high-dimensional, 30-parameter flow cytometry and are currently the only facility in Italy and one of the few in Europe to host such a technology. This development puts us at the forefront of single cell analysis of the immune sytem. 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.