IL-15 Augments in-Vitro Expansion and Functional Activity of Antigen-Specific Effector Memory T-Cells (TEM) While Co-Expression of IL-15 and IL-15 Rα on Antigen Presenting Cells Also Promotes Expansion of Central Memory T-Cells (TCM)
Authors: Hasan Aisha , Selvakumar Annamalai , Dupont Bo , Sadelain Michel , Riviere Isabelle , O’Reilly Richard J 
Published: October 2019

Abstract
Adoptive immunotherapy with in-vitro expanded antigen-specific T cells (TC) is often hampered by the extended culture times required to generate sufficient numbers of antigen- specific TC and their limited persistence in-vivo. IL-2 predominantly supports the generation of short-lived effector memory (TEM) and effector (TE) CD8+ TC without expanding the CD62L+ and CCR7+ central memory T cells (TCM), which may persist for long periods in vivo. We examined the potential of IL-15 to foster growth of CMVpp65- specific TC as well as expansion of both TEM and TCM CD8+ TC.
Accordingly, TC from 3 seropositive donors bearing HLA A0201 were sensitized in-vitro using NIH 3T3 based artificial antigen presenting cells transduced to express B7.1, ICAM-1, LFA-3, β2-M and HLA A0201 heavy chain as well as CMVpp65 (A2-AAPC) in presence of exogenous IL-2, IL-15 or IL-15 plus IL-2. To assess if trans-presentation of IL-15 by the receptor alpha on AAPCs leads to enhancement of IL-15 activity, we sequentially transduced A2-AAPCs with the IL-15 and IL-15 receptor alpha cDNA (A2- AAPC IL-15/IL-15Rα). TC were then cultured using A2-AAPC with either
IL-2 (20U/ml) IL-15 (10ng/ml) IL-2 plus IL-15 or with A2-AAPC IL-15/IL-15Rα or A2-AAPC IL-15Rα + IL-2 (20U/ml).
TC sensitized using either A2-AAPC IL-15/IL-15Rα or A2-AAPC + IL-15 demonstrated ~ 1500–2000 fold expansion of CMVpp65 A2-NLV tetramer (+) CD8+ TC, compared to a 300–600 fold expansion using A2-AAPC + IL-2 after 21–28 days in culture. Cultures containing IL-15 generated 25–70 ×106 A2-NLV tetramer (+) CD8+ TC in comparison to 10–18 ×106 in cultures with IL-2. At 7–10 days, ~20% of the tetramer (+) TC demonstrated a TCM phenotype (CD62L+ and CCR7+) in all cultures, with 75% TEM and 5% TE. By 21- 28 days, no TCM were detected among tetramer (+) TC in cultures containing exogenous IL-2, IL-15 or IL-2 plus IL-15. However, TC sensitized with A2-AAPC IL-15/IL-15Rα still contained 10–12% (~7×106) tetramer (+) CD8+ TCM which further increased through day 35. In functional assays, TC sensitized in the presence of IL-15 or AAPCs expressing IL-15 and IL-15Rα elicited superior CMV-specific responses with 7–20% CD8+ TC demonstrating CMVpp65 epitope specific interferon gamma production compared to 2–3% in cultures with IL-2. Cytotoxic activity against CMV pp65 peptide loaded autologous EBV transformed B-cell lines (E:T= 10:1) was higher for TC sensitized with IL-15 (80%) compared to cultures with IL-2 (60%). The cytotoxic activity against HLA mismatched targets or K562 was <5% in all cultures.
In conclusion, our data demonstrate higher yields and augmented function in antigen specific T cells cultured with IL-15. TC sensitized with A2-AAPC-CMVpp65 together with IL-15 +/− IL-2 only supported sustained expansion of TEM and TE. In contrast, TC sensitized with A2-AAPC IL-15/IL-15Rα also supported sustained expansion of TCM.