Pediatric pre-B acute lymphoblastic leukemia is the most common childhood cancer. Although current therapies achieve a high rate of remission, relapse of pre-B ALL remains a significant clinical challenge and new forms of therapy are needed. The graft-vs-leukemia (GVL) effect after bone marrow transplantation has shown that the immune system is capable of producing an effective anti-tumor response, which suggests that immune-mediated therapies may provide a complementary treatment strategy. Toll-like receptors (TLR), found on many immune cells, including B cells, have been shown to be important molecules in both innate and adaptive immune responses. Ligation of TLRs with their respective ligands results in an increase in the immunogenicity of antigen presenting cells (APC), through upregulation of MHC antigens and costimulatory molecules and production of cytokines and chemokines. We have previously reported that stimulation of pre-B ALL cells with the TLR9 ligand, CpG DNA, enhances the induction of Th1 immune responses by allogeneic T cells. In this study we examined the expression profile of TLRs 1-8 in precursor-B ALL cells and the effects of TLR ligation on the immune stimulatory capacity of precursor-B ALL cell lines. Eight precursor-B ALL cell lines were used in this study (Nalm6, REH, Sup-B15, KOPN-57bi, 380, 697, OP-1 and RS4:11). Standard RT-PCR analysis was used to examine the expression of TLRs 1-8 by the cell lines. The cell lines were stimulated with ligands for TLR2 (peptidoglycan), TLR3 (poly I:C) and TLR4 (LPS) and evaluated for changes in costimulatory molecule expression and allogeneic T cell stimulation. Non-quantatative PCR detected each TLR, with the exception of TLR8, in the majority of the cell lines. TLR8 was only detected, at low level, in RS4:11 cells. Despite the broad expression profile of the TLRs, significant differences in the effect of TLR ligation were observed between cell lines. In general, only modest increases in CD40 and CD86 expression were observed on responsive cell lines, with the majority of the lines showing no significant changes in response to TLR2, 3 or 4 ligation, despite receptor detection by PCR. Changes in allogeneic T cell proliferation in response to TLR stimulated ALL cell lines were observed, with the largest increases occurring with peptidoglycan and LPS. As was the case with costimulatory molecule expression, no T cell proliferative response change was common to all cell lines. However, analysis of cytokine production by T cells revealed a consistent increase in IFN-gamma and reduction in IL-5 levels in response to peptidoglycan stimulated ALL cells. The results reported in this study indicate that precursor-B ALL cell lines express several TLR molecules and that TLR ligation alters the immunogenicity of the majority of these lines. Interestingly, ligation of TLR2 with peptidoglycan induced a profound shift towards Th1 cytokine production. These observatios suggest that TLR ligation, most notably TLR2 ligation, may provide a strategy to influence anti-ALL immune responses and enhance immune mediated control of this disease.