NY-ESO-1 Specific T-Cells Are Spontaneously Present in High-Risk Myeloma and Kill Primary Myeloma Cells.
Authors: van Rhee Frits , Szmania Susann M. , Moreno Amberly , Batchu Ramesh , Pomtree Mindy , Rosen Nancy , Cottler-Fox Michele , Barlogie Bart , Trico Guido J. 
Published: April 2020

Abstract
We have previously reported that the Cancer/Testis antigen NY-ESO-1 is frequently expressed in patients with high-risk myeloma or advanced myeloma characterized by cytogenetic abnormalities. We tested 3 HLA-A*0201 positive MM patients with abnormal cytogenetics for the spontaneous presence of NY-ESO-1 specific CD8+ T-cells in the peripheral blood. We were able to detect 0.1% and 0.6% NY-ESO-1 specific CD8+ T-cells respectively in 2 patients using HLA-A*0201/NY-ESO-1157-165 specific tetramers prior to any re-stimulation with peptide. The 3rd patient did not have NY-ESO-1 specific T-cells. This last patient had been very heavily pre-treated and in contrast to the other patients, influenza matrix peptide58–66 specific T-cells failed to expand indicating that this patient’s immune system may have been exhausted. CD3+/8+ T-cells were expanded in multiple experiments by re-stimulation with autologous monocyte derived dendritic cells (Mo-DCs) pulsed with NY-ESO-1157-C165V analogue peptide. Substitution of cysteine (C) for valine (V) at position 165 improves binding affinity for HLA-A*0201 and antigenicity by eliminating dimerization of the carboxy-terminus cysteine residue. The lines contained 45–79% tetramer positive T-cells after stimulation. The CTLs were highly specific and lysed NY-ESO-1 positive/ HLA-A*0201 positive primary myeloma cells (50%), the NY-ESO-1 positive/ HLA-*0201 positive myeloma cell line U266 (55%) and autologous PHA-blasts pulsed with NY-ESO-1157–165 wild-type peptide (75%), but not K562 or patient PHA-blasts pulsed with the HLA-A*0201 binding MAGE-3112–120 peptide at E:T ratios of 10:1. The CTLs were of Tc1 type (g-IFN positive and IL4 negative) and stained positive for granzymes suggesting killing via the perforin/granzyme pathway. Immunophenotyping showed that the CTLs expressed the IL2 receptor and the integrin VLA-4, which is associated with the ability to transgress endothelial barriers. Further, the CTLs predominantly belonged to the terminal effector compartment (CD45RO+/CCR7−). We were unable to detect or expand NY-ESO-1 specific T-cells in HLA-A*0201 positive normal donors or NY-ESO-1 negative MM patients suggesting that pre-existent T-cell immunity to NY-ESO-1 is a prerequisite for the expansion of NY-ESO-1 specific CTLs. It also ruled out the possibility that the use of autologous APC’s induced de novo NY-ESO-1 specific CTL. In conclusion, these data suggest that NY-ESO-1 positive myeloma tumors are able to elicit spontaneous T-cell immunity and that expanded CTLs from these patients can kill primary myeloma cells. These CTLs recognize on the cell surface of myeloma cells naturally processed and presented NY-ESO-1, which is displayed in the context of HLA-A*0201. These results suggest that patients with high-risk NY-ESO-1 positive myeloma have a pool of memory CTLs, which can be exploited for vaccine or other immuno-therapeutic approaches.