Recovery of rare cells and single cells analysis: different opportunities and challenging applications

Single-cell biology is a new discipline which aims to address and solve the problem of cellular heterogeneity. Single-cell omic, which allows the molecular investigation of different cell types in a high throughput manner, is driving the Precision and Personalized Medicine approaches. Single B cell isolation strategies, the starting points of the single-cell omics, have become essential research procedures for efficiently sampling the natural repertoire of immunized animals and humans (Tiller et al, 2008). The goal of the first study is to demonstrate the feasibility of a novel approach to rapidly generate recombinant mAbs recovering rare antigen-specific plasma cells from complex samples derived from immunized mice. TLS Foundation, where this work has been carried out, has recently invested in Precision Medicine activities and, in particular, in the development of an alternative method to hybridoma technology to isolate rare antigen-specific B cells from blood of immunized or infected individuals. In Study 1 we have set-up a FACS sorter free method for a fast identification and isolation of antigen-specific plasma cells producing IgG with unique and desirable features and, moreover, we have obtained high yield TAP-PCR products. This could simplify the actual hybridoma technology procedure for the identification and molecular cloning of antigen-specific antibodies from single-B cells. Single-cell biology is also used to address and solve problems related to tumour heterogeneity. Cancer is one of the research areas that has greatly benefited from single-cell analysis. The complexity of immune responses to cancer has hampered the development of novel therapeutical approaches with the exception of monoclonal antibody based-therapies that target specific immunomodulators. In the last 10 years PD-1 blockade monoclonal therapy has indeed revolutionized cancer treatments but a substantial population of patients is still unresponsive. To rescue unresponsive patients, the mechanism of unresponsiveness and phenotype must be elucidated. The second part of this work (Study 2) deals with the possibility to reveal one of the mechanism responsible for T cells exhaustion in patients that are non-responsive to monoclonal anti-PD1 therapy. We demonstrated that there is a correlation between the overexpression of TFX (Transcriptional Factor X, the real name of the factor has been hidden for confidentiality) and the onset of the characteristic features of exhaustion. Indeed the KO of this gene restores in T cells their functionality through a more active metabolism. Further in-depth study of the modulation of this transcription factor may elucidate the pathways and the genes responsible for the exhaustion phenotype in T cells.