In Vitro Generation of Highly Purified Functional Invariant NKT Cells in Multiple Myeloma: A Strategy for Immunotherapy.
Authors: Song Weihua , van der Vliet Hans J.J. , Tai Yu-Tzu , Wang Ruojie , Prabhala Rao , Podar Klaus , Catley Laurence , Shammas Masood , Balk Steven P. , Anderson Kenneth C. , Exley Mark A. , Munshi Nikhil C. 
Published: April 2020

Abstract
Invariant NKT (iNKT) cells are important immunoregulatory cells that recognize glycolipid antigens with CD1d restriction and contribute to antitumor immune responses through the production of IFN-γ and IL-2. However, in progressive multiple myeloma (MM), the iNKT cell population is decreased along with its capacity to produce IFN-γ. Thus, a novel strategy for the immunotherapy of MM entails the enhancement of iNKT cell functions. In this study, we established iNKT cell lines from MM patients via enrichment with Vα24+ and subsequently with Vβ11+ cells, followed by several rounds of stimulation with α-GalCer-pulsed DCs. These techniques resulted in highly purified iNKT cell lines (&gt;97%). To evaluate potential in vivo interaction between iNKT cells and myeloma cells, we evaluated the CD1d expression on primary myeloma cells as well as MM cell lines. Gene expression profiling revealed compared to normal plasma cells, majority of primary MM cells (11 out of 15) expressed higher levels of CD1d; in contrast, all 6 MM cell lines tested had no expression. Flow cytometric analysis further confirmed the expression of CD1d on primary MM cells and lack of its expression on 12 different MM cell lines. A CD1d-transfected MM1S cell line (MM1S-CD1d) was therefore established for the functional study. To determine whether CD1d-expressing primary MM cells have the antigen presenting capacity, iNKT cell lines from healthy donors (n=2) and MM patients (n=2) were cocultured with 5 cases of CD1d positive primary MM cells with or without α-GalCer. Monitored by the CD25 expression, we demonstrated primary MM cells presented α-GalCer and also endogenous antigen(s) to activate iNKT cells. We have further evaluated the functional profile of expanded iNKT cell lines from MM patients (n=5). Upon stimulation with α-GalCer-pulsed MM.1S-CD1d cells, iNKT cells produced high levels of Th1-type cytokines (IFN-γ and IL-2) compared to low level Th2-type cytokine production (IL-4). Our results thus demonstrate that iNKT cell lines from MM patients were functionally restored by expansion with α-GalCer-pulsed DCs in vitro. To further augment iNKT cells function, we evaluated effects of lenalidomide on iNKT cell lines, an immunomodulatory drug which has been demonstrated to enhance T cell costimulation and NK cell activity. Lenalidomide did not directly stimulate iNKT cells in the presence or absence of α-GalCel. Importantly, upon CD1d-restricted activation by α-GalCer-loaded MM1S-CD1d cells, lenalidomide significantly enhanced the Th1-type immune responses of iNKT cell lines from both healthy donors and MM patients. Compared to those of controls, a significant increase of IFN- γ (healthy donor, p&lt; 0.001, n=7; MM patients, p&lt;0.05, n=3) and IL-2 (MM patients, p&lt;0.0015, n=3) occurred. Meanwhile, lenalidomide had no significant effect on the production of IL-4 by iNKT cell lines (healthy donor, p&gt;0.05, n=7; MM patients, p&gt;0.05, n=3). Taken together, our results provide preclinical feasibility and support a rationale to evaluate efficacy of adoptive transfer of iNKT cells in MM. Moreover, it provides a clinical basis for use of lenalidomide to enhance iNKT cell mediated immunotherapy in myeloma.