The Importance of Be(ginn)Ing Naïve: Implications for Cancer Immune-Gene Therapy
Authors: Cieri Nicoletta , Camisa Barbara , Cocchiarella Fabienne , Provasi Elena , Magnani Zulma , Bondanza Attilio , Mondino Anna , Mavilio Fulvio , Bicciato Silvio , Recchia Alessandra , Bonini Chiara 
Published: October 2019

Abstract
Abstract 2052
T cell engineering against tumor antigens aims at ameliorating current immunotherapeutic strategies. To date, however, the suboptimal persistence of the transferred cells represents a serious limitation of this approach. The most appropriate T cell subset to be infused should ensure optimal in vivo persistence and yet appropriate anti-tumor activity. Here we report that culturing highly purified naïve T (TN) cells with beads conjugated to anti-CD3 and anti-CD28 antibodies, allows the retrieval of a novel post-mitotic CD45RA+ CD62L+ CCR7+ T cell population, which requires IL-7 and IL-15 for expansion and maintenance. This population is highly proliferative and sensitive to RV and LV transduction, expresses low levels of γIFN and cytotoxic molecules and is best defined as IL-7Rα+ CXCR4+ c-kit+ CCR5− HLA-DR− PD-1−. When infused in immunodeficient mice, genetically manipulated and in vitro expanded TN proved superior engraftment and longer persistence than transduced central memory (TCM) cells, and xenoreactivity comparable to that of unmanipulated lymphocytes. Engineered TN, but not TCM, maintained engraftment and xenoreactivity in serial transplantation experiments, indicating unique self-renewal abilities. Given the great potentials of this novel TN-derived cell population for immune-gene therapy, we further characterized it by molecular profiling. The gene expression signature is typical of antigen-experienced lymphocytes and classifies these cells between naturally occurring TN and TCM lymphocytes. Because of this and of the self-renewal abilities displayed in vivo, we termed them as precursor to TCM (TpreCM). We next sought to identify the natural counterpart of this TpreCM population in healthy donors, exploiting some of the markers present in the TpreCM signature, such as CD95 which is expressed by all memory T subsets but not by TN, and we selected the pp65 protein of cytomegalovirus (CMV) as a model antigen. CMV persistent infection induces a T-cell response that is maintained throughout life, indicating that a self-renewing memory T cells are generated. We studied the phenotype of CMV pp65-specific CD8+ T cells in seropositive donors and identified antigen-specific CD45RA+CD62L+. Among CD45RA+CD62L+ cells CMV-specific cells were enriched for CD45ROdim and CD95+ lymphocytes, which represent bona fide TpreCM. To functionally characterize natural TpreCM, we sorted CD45RA+CD62L+ cells according to their CD95 expression and challenged them with increasing doses anti-CD3 antibody, with or without a costimulatory signal. We found that among CD45RA+CD62L+ cells, only CD95+ lymphocytes were responsive to TCR-triggering alone, while CD95− cells required costimulation to proliferate. In conclusion, we identified a novel memory T cell subset, and identified conditions able to gene-modify and expand these memory lymphocytes while preserving their functional characteristics. Exploitation of these concepts might improve cancer adoptive immunotherapy.

Disclosures:
Bonini: MolMed: Consultancy.