Chronic Lymphocytic Leukemia (CLL) Cells Are Susceptible to γδ-T Cell Mediated Killing, Provided CLL-Derived γδ-T Cell Dysfunction Can be Reversed
Authors: de Weerdt Iris , Terpstra Sanne , Hofland Tom , Lameris Roeland , de Bruin Renee C.G. , Levin Mark-David , Remmerswaal Ester B.M. , ten Berge Ineke J.M. , Tonino Sanne H. , de Gruijl Tanja D , van der Vliet Hans , Kater Arnon P. 
Published: October 2019

Abstract
Background:
Although T cell immunotherapy is considered a promising therapeutic approach in B cell malignancies, autologous T cell based therapy proved to be far less effective in CLL than in more aggressive B cell malignancies. This has been attributed to an acquired state of T cell dysfunction. Disturbances in conventional (αβ-)T cells include expansion of CD4+ and CD8+ T cells, increased expression of exhaustion markers and impaired cytotoxicity and cytokine production. Vγ9Vδ2-T cells are a conserved subset of cytotoxic T lymphocytes with potent antitumor activity, due to recognition of phosphoantigen-induced changes in CD277 in tumor cells. Aminobisphosphonate (ABP) treatment leads to intracellular accumulation of phosphoantigens and increased Vγ9Vδ2 antitumor responses. Vγ9Vδ2-T cells have been shown to effectively kill malignant B cell lines in vitro. Moreover, in clinical trials Vγ9Vδ2-T cells have been shown to recognize and kill B cell lymphomas. Whether Vγ9Vδ2-T cells could be exploited for CLL immunotherapy has not yet been explored. The aim of this study is to investigate the phenotype and function of Vγ9Vδ2-T cells in CLL patients, in order to determine whether Vγ9Vδ2-T cells can effectively kill CLL cells.
Results:
Frequencies of Vγ9Vδ2-T cells do not differ between untreated CLL patients (n=46) and age-matched healthy controls (HC) (n=20) as assessed by flow cytometry. Vγ9Vδ2-T cell subpopulations are skewed towards effector type (CD27- CD45RA-) in CLL patients, while numbers of naïve (CD27+ CD45RA+) Vγ9Vδ2-T cells are decreased. Expression of exhaustion markers PD-1 and BTLA is comparable between CLL and HC, as is expression of CD16, mediating antibody-dependent cellular cytotoxicity. Next, we compared the functionality of Vγ9Vδ2-T cells from CLL patients and HC. We first examined cytokine production and CD107a expression, a marker of degranulation. Production of TNFα and IFNγ upon PMA/ionomycin stimulation was significantly diminished in CLL Vγ9Vδ2-T cells as compared to HC Vγ9Vδ2-T cells. Similarly, CD107a expression was significantly reduced. Overnight coculture with primary CLL cells or the Vγ9Vδ2-T cell sensitive Daudi lymphoma cell line also induced expression of TNFα, IFNγ and CD107a. However, upon co-culture, HC Vγ9Vδ2-T cells expressed significantly more TNFα, IFNγ and CD107 than CLL Vγ9Vδ2-T cells. Subsequently, we compared cytotoxicity of Vγ9Vδ2-T cells towards Daudi cells. HC-derived Vγ9Vδ2-T cells killed Daudi cells 3-4 times more effectively at 1:5 and 1:2.5 effector:target ratios. Although ABP pretreatment of Daudi cells increased both CLL-derived and HC-derived Vγ9Vδ2-mediated killing, differences between CLL and HC could not be overcome. We then looked at Vγ9Vδ2-T cell cytotoxicity towards CLL cells. Vγ9Vδ2-T cells from HCs effectively recognized and killed primary CLL cells, irrespective of ABP pretreatment. CLL-derived Vγ9Vδ2-T cells killed allogeneic CLL cells significantly less efficiently.
Finally, we investigated whether the Vγ9Vδ2-T cell dysfunction in CLL patients was reversible upon ex vivo activation without the presence of leukemic B cells. Purified Vγ9Vδ2-T cells were cocultured with mature monocytic-derived dendritic cells in the presence of ABP for 8 days. Following these culture conditions, no difference was observed in production of TNFα, IFNγ and IL-4 upon PMA/ionomycin stimulation between HC- and CLL-derived activated Vγ9Vδ2-T cells. Likewise, there was no difference in CD107a expression. The activated Vγ9Vδ2-T cells of HCs and CLL patients were equally effective at killing Daudi cells.
Conclusion:
Vγ9Vδ2-T cells are capable of recognizing and killing CLL cells. Yet, CLL-derived Vγ9Vδ2-T cells are functionally impaired in terms of cytokine production and cytotoxic capacity in comparison to age-matched HCs. Functional impairments of Vγ9Vδ2-T cells are reversible upon ex vivo activation. If dysfunction can be overcome effectively, the antileukemic properties of autologous Vγ9Vδ2-T cells can be efficiently employed.

Disclosures
No relevant conflicts of interest to declare.