Aisha Nasreen Hasan

Soluble and membrane-bound interleukin (IL)-15 Rα/IL-15 complexes mediate proliferation of high-avidity central memory CD8(+) T cells for adoptive immunotherapy of cancer and infections.

The lack of persistence of infused T cells is a principal limitation of adoptive immunotherapy in man. Interleukin (IL)‐15 can sustain memory T cell expansion when presented in complex with IL‐15Rα (15Rα/15). We developed a novel in‐vitro system for generation of stable 15Rα/15 complexes. Immunologically quantifiable amounts of IL‐15 were obtained when both IL‐15Rα and IL‐15 genes were co‐transduced in NIH 3T3 fibroblast‐based artificial antigen‐presenting cells expressing human leucocyte antigen (HLA) A:0201, β2 microglobulin, CD80, CD58 and CD54 [A2‐artificial antigen presenting cell (AAPC)] and a murine pro‐B cell line (Baf‐3) (A2‐AAPC15Rα/15and Baf‐315Rα/15). Transduction of cells with IL‐15 alone resulted in only transient expression of IL‐15, with minimal amounts of immunologically detectable IL‐15. In comparison, cells transduced with IL‐15Rα alone (A2‐AAPCRα) demonstrated stable expression of IL‐15Rα; however, when loaded with soluble IL‐15 (sIL‐15), these cells sequestered 15Rα/15 intracellularly and also demonstrated minimal amounts of IL‐15. Human T cells stimulated in vitro against a viral antigen (CMVpp65) in the presence of 15Rα/15 generated superior yields of high‐avidity CMVpp65 epitope‐specific T cells [cytomegalovirus‐cytotoxic T lymphocytes (CMV‐CTLs)] responding to ≤ 10− 13 M peptide concentrations, and lysing targets cells at lower effector : target ratios (1 : 10 and 1 : 100), where sIL‐15, sIL‐2 or sIL‐7 CMV‐CTLs demonstrated minimal or no activity. Both soluble and surface presented 15Rα/15, but not sIL‐15, sustained in‐vitro expansion of CD62L+ and CCR7+ central memory phenotype CMV‐CTLs (TCM). 15Rα/15 complexes represent a potent adjuvant for augmenting the efficacy of adoptive immunotherapy. Such cell‐bound or soluble 15Rα/15 complexes could be developed for use in combination immunotherapy approaches.

Abstract B050: Functional characterization of virus specific stem cell like memory T cells, their maturation in vitro, and potential contribution to secondary T cell responses

Antigen-specific T cells are crucial components for antitumor or antivirus immunity. Adoptive transfer of antigen specific T-cells can induce remission of disease; however, the quality of the T cells that are selected for expansion and adoptive transfer has been identified as a critical factor that determines the persistence of transferred cells. Memory stem (TSCM) T-cells demonstrate evidence of stem cell like properties, such as differentiation potential as well as self-renewal. Thus far, there are no data characterizing antigen-specific TSCM cells with respect to their differentiation pattern, TCR clonotype, or function in comparison to different memory T-cell subsets. In this study, we demonstrate successful generation of CMV-specific T cells derived from naive (TN) and memory T-cell subsets (TSCM, TCM and TEM) from HLA*A0201 CMV seropositive donors. T cells were sensitized using artificial antigen presenting cells transduced to express HLA-A*0201 and CMVpp65 in the presence of the cytokines IL-7 and IL-15. We then characterized CMVpp65-specific T cells isolated by their binding to tetramers of HLA-A*0201 bearing the immunodominant CMVpp65495-503 peptide, NLVPMVATV (NLV). Upon in vitro expansion in the presence of CMV antigen, both Tet+ TN and TSCM-derived cells differentiated into TCM and TEM. However, during prolonged stimulation with CMV antigen, Tet+ TSCM-derived cells sustained the population of the less differentiated Tcm memory phenotype, as well as a higher proliferative capacity leading to superior expansion of Tet+ CMV-specific T-cells compared to CMV-specific TCM and TEM memory T cell subsets. In functional assays, both TN and TSCM-derived Tet+ cells were able to secrete high level of IFN-γ and TNF-α, as well as CD107a in an antigen-dependent manner. Over 30 days of antigen-specific stimulation, T cell receptor (TCR) gene analysis of the Tet+ T-cells demonstrated a graduated clonal overlap from TSCM to TCM to TEM among the CMV specific T-cells. Furthermore, certain TCR clonotypes were shared within CMV specific T-cells derived from all memory T-cell subsets, indicating that T-cells recognizing the same epitope were derived from the same mother clones. In conclusion, these data provide evidence that antigen-specific TSCM cells constitute an earlier differentiation stage within memory T-cells compared to TCM and TEM cells, with a superior expansion capacity. TSCM cells may therefore serve not only as a durable reservoir, but also an initiator of the memory immune response upon antigen challenge resulting from reactivation of a latent infection. These findings have implications for the design of adoptive immunotherapy strategies employing virus specific CTLs to maximize generation of T-cell population, optimized for in vivo expansion, function and persistence. Citation Format: Tzu-yun Kuo, Aisha N. Hasan, Richard J. O9Reilly. Functional characterization of virus specific stem cell like memory T cells, their maturation in vitro, and potential contribution to secondary T cell responses [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B050.