CD137 Expression Identifies Leukemia Specific CTL after in Vitro Priming of Cord Blood T Cells Previously Expanded By CD3/CD28 Co-Stimulation

Introduction: We previously reported that cord blood T-cells (CBT), obtained from <3% of cord blood graft can be expanded, and primed against (lymphoid and myeloid leukemia cell lines in the presence of IL12, IL7 and IL15 (Cancer Research, 2010; 70(13): 5249).  Recently, we demonstrated that IL15 alone can support the priming and expansion of CTL.

Hypothesis: CD137 (4-1BB) is a member of the TNFR- family, expressed on activated T cells, which augment the survival and proliferation. Here we tested the hypothesis that de-novo CD137 expression may identify leukemia-specific CTL.

Methods: IM9-specific CTL was generated from pre-expanded CBT cells using weekly stimulations with irradiated targets and IL15. After 3 weeks, cytotoxicity was tested, re-stimulated to sort CD137 expressing cells (BD Biosciences) and were re-tested for cytotoxicity and TCRβ CDR3 spectratyping was done to identify their clonality. RT² profiler^TM PCR array human Th1 & Th2 responses (SABiosciences) were employed to identify the differential gene expression.

Results: After 3 weeks of priming/expansion, 2-3 % of the CTL were CD137+.  However, after overnight re-stimulation, CD137 expression increased to a range of 12-39%  (Figure-1). The cytotoxicity of bulk cells segregated into the CD137+ cells after FACS sort (Figure-2). Amongst the 23 TCR Vβ families tested, distinct oligoclonal expansion of Vβ7, Vβ5.1 and Vβ21 were observed in the CD137+ population (Figure-3). While post-sort RT qPCR analysis demonstrated significantly increased message for CCR4, EBI3, TNFRSF9 and TNFSF4 genes, CD40LG, IL2, IL6, IL7R, SFTPD and SPP1 expressions were significantly decreased in CD137+ cells with high frequencies.

Conclusion:  Cytotoxicity against leukemia cell line from expanded cord blood cells dominates in the CD137 positive subset, therefore CD137 positive selection can be used to isolate and enrich leukemia specific CTLs. Notably, CTL expanded from different sources have a predictable TCRVβ repertoire shaped by the leukemic target. These findings will have great influence on attaining clinically applicable leukemia-specific CTL protocols.