IL-15 Augments in-Vitro Expansion and Functional Activity of Antigen-Specific Effector Memory T-Cells (T_EM) While Co-Expression of IL-15 and IL-15 Rα on Antigen Presenting Cells Also Promotes Expansion of Central Memory T-Cells (T_CM)

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 (T_EM) and effector (T_E) CD8⁺ TC without expanding the CD62L⁺ and CCR7⁺ central memory T cells (T_CM), 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 T_EM and T_CM 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 T_CM phenotype (CD62L⁺ and CCR7⁺) in all cultures, with 75% T_EM and 5% T_E. By 21- 28 days, no T_CM 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⁺ T_CM 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 T_EM and T_E. In contrast, TC sensitized with A2-AAPC IL-15/IL-15Rα also supported sustained expansion of T_CM.