Biological and clinical implications of invariant natural killer T cells in multiple myeloma: rising the potential for immunotherapy

Invariant natural killer T (iNKT) cells are a small subset of T cells that were first described in 1987. iNKT cells are characterized by the expression of a unique semi-invariant antigen-specific T cell receptor (TCR)-beta chain encoded by Valpha14.Jalpha18 in mice and Valpha24.Jalpha18 in humans. This allows them to recognize lipid antigens that are not detected by conventional T cells. The importance of iNKT cells in cancer was further highlighted after the discovery of the marine sponge-derived glycolipid alpha-Galactosylceramide (alpha-GalCer). alpha-GalCer is a potent agonist for the iNKT cell receptor and can be represented by the antigen-presenting cells (APC) such as dendritic cells (DCs) through their non-classical class I-like monomorphic major histocompatibility complex (MHC) molecule CD1d. After activation with alpha-GalCer, iNKT cells rapidly elicit their innate-like activity by a robust production of Th1 cytokines such as IFN-gamma, which in turn augments their anti-tumor response. Multiple myeloma (MM) is an incurable B cell malignancy caused by the accumulation of terminally differentiated plasma cells in the bone marrow (BM). The interactions between MM cells and the BM microenvironment are immunosuppressive for most immune cells including iNKT cells. Despite the promising results obtained from several clinical trials using soluble alpha-GalCer or alpha-GalCer-loaded DCs, iNKT cells become anergic in progressive MM with a deficiency in their IFN-gamma production. Thus, controlling the activity of iNKT cells is challenging and affects their implications in immunotherapy. In this thesis we focused on two aspects related to iNKT cells in the murine 5T33MM model: First, we evaluated iNKT cells and described their number and activity during the progression of MM in the 5T33MM model. We found that their number is variable in different tissues with the highest percentage detected in the liver. A dramatic drop in their number was seen in liver and spleen at the end stage of the disease. This was consistent to what we found in MM patients as we saw a significant decrease in circulating iNKT cells in relapsed patients compared to healthy donors, and this decrease was gradual during the stages of MM but ended with a sharp decrease in patients at relapse. We also demonstrated that the decline in iNKT cell number led to a deficiency in IFN-gamma secretion in response to alpha-GalCer loaded DCs. Furthermore, we showed that treatment of 5T33MM mice with alpha-GalCer loaded DCs on the same day of MM inoculation significantly prolonged the survival from 22 days to 29 days. In addition, we observed that alpha-GalCer injection induced T helper (Th) 1 responses (e.g. IFN-gamma secretion) in healthy and non-terminal sick mice. Secondly, based on the previous results which indicated the ability of iNKT cells in the 5T33MM model to respond to alpha-GalCer effectively and polarize the immune response toward Th1, we wanted to investigate whether alpha-GalCer-stimulated iNKT cells can be used to fight MM by reducing angiogenesis. We observed that alpha-GalCer treatment could significantly decrease both the tumor burden and the microvessel density (MVD) in the 5T33MM mice. Interestingly, the reduction of MVD in 4 mice out of 10 was not associated with the reduction in tumor load, suggesting a direct anti-angiogenic effect. We confirmed our observation by applying several in vivo and in vitro assays using conditioned media (CM) from alpha-GalCer-stimulated iNKT cells. We found that this CM reduced neovascularization, viability, proliferation, migration and network organization of endothelial cells (ECs) and to a lesser extent it in