The Effect of Inducıble Costımulatory Lıgand (Icos-Lg) Expressıng Myeloıd Leukemıa Cells on Helper T Lymphocyte Actıvatıon and Exhaustıon

Abstract

T cell exhaustion is characterized by progressive loss of cytokine production, decreased proliferation and resistance to reactivation especially upon continuous antigen stimulation. Despite being potent stimulators of helper T (Th) lymphocytes, AML cells employ intriguing mechanisms to limit anti-tumor responses. Thus, this study proposes an in vitro co-stimulation dependent Th cell exhaustion model that may represent a novel immune escape mechanism in AML. Firstly, ICOS-LG expression kinetics were evaluated on AML cell lines of different maturation status (HL-60, THP-1, U937, Kasumi-1, KG-1) in comparison to CD14+ monocytes by flow cytometry under steady state and pro-inflammatory conditions. Then, CD4+ T cells were co-cultured with AML cells or monocytes at different ratios and stimulated with various anti-CD3 concentrations. Media and aCD3 was regularly refreshed in extended culture periods. CD25, CD127, FoxP3, CD69, PD-L1, PD-1, CTLA-4, ICOS, TIM-3, LAG3, CD38, CD154, CCR7, HLA-DR expression, together with proliferation and viability were evaluated by flow cytometry. Assays were also performed under ICOS-LG, PD-L1/PD-L2 and CD80/CD86 blockade. IL-2, IL-10, IL-4, TGF-, IFN-γ and TNF-α levels were measured by ELISA. Exhausted (TIM-3mo/hi) and non-exhausted Th cells (TIM-3-/low) were enriched and re-stimulated for functional exhaustion assays. In the co-cultures, Th cells possessed high levels of LAG3, TIM-3, PD-1, ICOS and CTLA-4. Th cells' proliferation and activation with AML cells or monocytes were similar; however, exhaustion was significantly higher in AML co-cultures. These exhausted cells produced lower IL-2, IFN-γ and TNF-α. Exhaustion was independent of Th cells' initial proliferative activity. Co-stimulatory signals derived from AML cells were more critical than aCD3 stimulation in Th cell exhaustion. Exhaustion could be reversed by exogenous IL-2. Our findings indicate a novel immune escape mechanism employed by AML cells that can induce a co-stimulation dependent Th cell exhaustion.