Latent-Membrane 2a Specific CTL's but Not Latent-Mebrane 1 Specific CTL's Lysis Efficiently Hodgkin Lymphoma Cells.

Abstract

Relapse is the leading cause of treatment failure after allogeneic SCT of Hodgkin Disease (HD). As Ebstein-Barr infection (EBV) is associated with 60% of all HD cases, adoptive immunotherapy with donor derived EBV-specific T-cells lines has resulted in disease control of allogeneic SCT. Potential targets for the adoptively transferred T-cells are the type II latency protein LMP-1 and LMP-2a, which are both homogenously expressed by HD cells. In healthy individuals, both LMP-1 and LMP-2a elicits subdominant CD8+ T-cell responses with frequencies of less than 1:10000. LMP-1 and LMP-2a specific T-cells from 1x108 PBMC derived from HLA A*0201+healthy donors were stimulated with the HLA A*0201 LMP1-epitopes YLLEMLWRL and YLGQNWWTL and the HLA A*0201 LMP-2a epitope CLGGLLTM. Activated T-cells were selected by the cytokine secretion assay and expanded for 10 days. In 85% of donors 1.7 x106 (range 0.7 –4.5 x106; n=13) LMP-1 or LMP-2a specific CD8+ T-cell could be generated with an average purity of 83% as determined by tetramer staining. LMP1- and LMP2a-specific CD8+ T-cells were then expanded 3000 x in 14 d by the rapid expansion protocol and evaluated functionally for cytokine production and specific lysis. Both LMP-1 and LMP-2a specific CD8+ T-cells retained specific cytokine production if stimulated with peptide pulsed targets, efficiently lysed peptid pulsed targets. Surprisingly, if LMP-1 was presented endogenously by EBV positive targets or by targets cells transduced with LMP-1, no cytokine production or specific lysis was detected despite protein expression of LMP-1 in all targets. In contrast, IFN-γ production could be readily detected in LMP-2a-specific CD8+ T-cells after stimulation with target cells processing endogenously the LMP-2a antigen as well as specific lysis of EBV positive target cells. Furthermore, LMP2a specific CD8+ demostrated also specific lyse of Hodgkin-cells expressing the LMP2a (30:1 E/T ratio; 29,3%) where as LMP-1-specific CD8+ T-cells could not lyse HD-cells. In summary, LMP-1 and LMP-2a specific T-cells, although present at undectable levels in healthy donors, can be readily selected and expanded to up to 6x109 antigen-specific T-cells in less than 4 weeks starting from 1x108 PBMC. Based on this data, adoptive immunotherapy of relapsed EBV positive HD after allogeneic SCT should be preferentially performed with LMP-2a specific CD8+ T-cells rather than with LMP-1 specific CD8+ T-cells.