IL-15 Alone Supports in Vitro Generation of Polyclonal CTL From CD3/CD28 Co-Stimulated Expanded CB T Cells Against Leukemia Cells

Background: Ex vivo expanded CBT cells (with CD3/CD28 co-stimulatory beads + IL-2 and IL-7) are receptive to subsequent in vitro priming against killed lymphoid and myeloid leukemia cells in the presence of IL-7, IL-12, and IL-15. This had been reported from our lab (Davis et al. Cancer Research 2010, 70(13): 5249).

Objectives: We tested 1) the minimum cytokine and priming requirements, hypothesizing that higher specificity may be obtained if fewer exogenous cytokine is employed.  2) Determine the critical recognition element of cytotoxicity. 3) Characterize the cellular phenotype associated with the CTL activity.

Methods: CTL was generated from already expanded (<3% of a typical cord blood graft) >98% pure T cells either with a combination of cytokines IL-7, -12 & -15 (n=4) versus IL-15 (n=5), versus IL-7 (n=3) alone. After 2nd and 3rd weeks of CTL cultures, the cytotoxicity was tested against fresh IM9 leukemia cells using europium ligand  (EuTDA) release assay. The blocking with HLA class I & II and TCRγd antibodies were done before performing the CTL assay (n=3). ELISPOT assay was done to test the specificity as measured by IFN-γ spot forming cells (SFC). Eight colors FACS analysis was done using FACS canto II, to analyze the CTL's phenotype.

Results: After 3 weeks of priming, the mean specific lysis of CTL generated using IL-15 alone versus combination of cytokines on fresh IM9 cells was 79% and 80% respectively at an E/T ratio of 40:1 (Figure-1). CTL generated using IL-15 alone has a specific lysis of 90% at the end of second priming (n=4). The CTL's exhibit no cytotoxicity against non-specific targets (myeloid leukemia cell line U937).  The expanded CBT cells primed with IL-15 alone, but without being exposed to IM9 cells did not acquire specific cytotoxicity (n=4). While cytotoxicity was comparable, CTL from IL-15 alone cultures displayed higher specificity in ELISPOT assays with much less non-specific target recognition as measured by IFN-γ spot forming cells (SFC) (n=2).

Eventhough, no single blockingMoAb completely abolished cytotoxicity, the most significant diminution was identified when the effectors were blocked with TCRγd.  Multicolor FACS analysis indicates that after exposing the CTLs to specific targets, ~6-7% of the cells are secreting IFN-γ in response and 6-20% are TCRγd positive. Immunoscope analysis of TCRγd spectratype identifies oligoclonal restriction of CTL cultures with lytic activity compared to identical cytokine expanded T cells lacking leukemia–specific CTL activity.

Conclusion:  Polyclonal leukemia specific CTL can be generated with IL-15 alone from previously CD3/CD28 expanded CB T cells.  While the cytotoxicity is comparable to those primed/expanded by combinations of cytokines, the CTL specificity is higher with IL-15 alone. Notably, the CTL activity is not confined to a single cellular subset and resides in the TCRγd+ T cell.