The Importance of Be(ginn)Ing Naïve: Implications for Cancer Immune-Gene Therapy

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 (T_N) 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 T_N proved superior engraftment and longer persistence than transduced central memory (T_CM) cells, and xenoreactivity comparable to that of unmanipulated lymphocytes. Engineered T_N, but not T_CM, maintained engraftment and xenoreactivity in serial transplantation experiments, indicating unique self-renewal abilities. Given the great potentials of this novel T_N-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 T_N and T_CM lymphocytes. Because of this and of the self-renewal abilities displayed in vivo, we termed them as precursor to T_CM (T_preCM). We next sought to identify the natural counterpart of this T_preCM population in healthy donors, exploiting some of the markers present in the T_preCM signature, such as CD95 which is expressed by all memory T subsets but not by T_N, 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 CD45RO^dim and CD95⁺ lymphocytes, which represent bona fide T_preCM. To functionally characterize natural T_preCM, 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.