CD4+ T Cell Lines Reactive with Acute Myeloid Leukemia (AML) Cells Generated by Competitive Limiting Dilution Culture of AML Mononulear Cells (MNC) Eliminate Autologous AML by Apoptosis Induction.

Abstract

Traditionally, the focus of anti-tumor immunity has been on CD8+ CTL responses. It is now realized that CD4+ T cells play a relevant role in protective anti-tumor responses. HLA Class II molecules are expressed on AML blasts, predicting that AML cells may stimulate CD4+ T cells. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we developed a novel method of generating multiple autologous AML reactive T cell lines by competitive limiting dilution (LD) AML MNC culture. Most autologous AML reactive T cell lines generated from this culture were CD4+. These CD4+ T cell lines with high IFN-gamma secretion in response to autologous AML cells showed low to moderate specific lysis of autologous AML cells in 4-hour 51Cr release assays. However, co-culture assays demonstrated that these CD4+ AML reactive T cell lines exerted intense cytotoxicity toward autologous AML cells, depleting more than 95% of autologous AML line cells in 2 days, and more than 99% in 7 days, with no or weak toxicity to allogeneic AML cells and cell lines, HL60, NB4, U937 KG1a and ak LCL cell line. Anti-HLA class II, Dr, Dp, Dq mAb (67±16% inhibition) and anti-HLA-Dr mAb (69±11% inhibition) but not anti-HLA class I mAb significantly inhibited IFN-gamma secretion of six CD4+ T cell lines stimulated by autologous AML cells confirmed the HLA class II restriction of reactivity of these CD4+ T cell lines. Flow cytometry analysis showed that these CD4+ T cell lines induced significant Annexin-V expression on autologous AML cells. The induction of Annexin-V+ on AML cells by CD4+ T cell lines correlated significantly with IFN-gamma secretion in response to autologous AML cells (n=11; r=0.84). This result suggested that the most important mechanism of autologous AML cell elimination by CD4+ AML reactive T cells generated from LD-AML-MNC cultures was apoptosis induction related to IFN-gamma secretion.