IL-15 Induced Polyclonal CTL Generated From Expanded CBT Cells Against Leukemia Cell Lines Constitutes IFN-γ Producing Cells and TCRγδ Cells
Authors: Antony Jeyaraj , Chen Xiaohua , Szabolcs Paul 
Published: October 2019

Abstract
Abstract 1910

Background:
We have previously demonstrated that ex vivo expanded CBT cells expanded with CD3/CD28 co-stimulatory beads + IL-2 and IL-7 were receptive to subsequent in vitro priming against killed lymphoid and myeloid leukemia cells in the presence of IL-7, IL-12, and IL-15, (Davis et al. Cancer Research 2010;70(13):5249).

Hypothesis and Objectives:
Here, we wanted to test 1) the minimum prerequisite cytokine requirement, hypothesizing that higher specificity may be obtained if less exogenous cytokine is employed. 2) Determine the mechanism of CTL's kill by employing blocking antibodies against HLA-ABC, HLA-DR and TCRγδ recognition sites. 3) Characterize the critical cellular phenotype of the CTL to identify whether the cytotoxicity is specific to CD4, CD8, TCRαβ, or TCRγδcell populations or the observed cytotoxicity is the contribution from multiple cellular phenotypes?

Methods:
CTL was generated from already expanded >98% pure T cells either with a combination of cytokines (IL-7, -12 & -15) versus IL-15, versus IL-7 alone and were compared from the same cultures by matched pair Student T-test. After 3 weeks of CTL cultures, the cytotoxicity (specific lysis) was tested against fresh IM9 leukemia cells (loaded with BATDA) at an Effector: Target ratio of 40:1, 20:1 and 10:1. The blocking with HLA class I, class II and TCRγδ antibodies were done before performing the CTL assay. ELISPOT assay tested the specificity (non-specific target recognition) and magnitude of activity as measured by IFN-γ spot forming cells (SFC).

Results:
From the data shown in Figure 1, it is evident that IL-15 alone is sufficient to support both priming and expansion of IM-9 specific CTL. After 3 weeks of CTL cultures, the observed specific lysis of fresh IM9 leukemia with IL-15 alone versus in combination was 73% and 75% respectively at an Effector: Target ratio of 40:1. The generated CTL exhibit no cytotoxicity against non-specific targets against a myeloid leukemia cell line (U937). While cytotoxicity by Europium release assay 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) (Figure 2A). Though the relative (fold) expansion of CTL observed in the group with all cytokines is significantly higher, the numbers are comparable between the combination of cytokines and IL-15 alone towards the later weeks of priming and expansion, data not shown.
By blocking experiments, it is evident that, even though no single MoAb completely abolished cytotoxicity, the most significant diminution was identified when the effectors were blocked with TCRγδ (Figure 2B). Multicolor FACS analysis indicates that after exposing the CTLs to specific targets, ∼7–8% of the cells are secreting IFN- γ and ∼ 20–25% of the generated CTLs are TCRγδ positive. Immunoscope analysis of TCRγδ spectratype identifies oligoclonal restriction of CTL cultures with lytic activity compared to identical cytokine expanded T cells lacking leukemia–specific CTL activity (Figure 3).

Conclusion:
A polyclonal leukemia specific CTL can be generated with IL-15 alone from previously expanded and partially mature CB T cells. While the cytotoxicity is preserved compared to those primed/expanded by multiple cytokines the specificity is higher in IL-15 alone. Notably, the CTL activity is not confined to a single cellular subset and possibly the most significant activity resides in the TCRγδ expressing T cell population.

Disclosures:
No relevant conflicts of interest to declare.