Heterodimer-Specific Stimulation Of Toll-Like Receptor 2 Induces Divergent Downstream Effects In Primary Samples Of Precursor B Cell Acute Lymphoblastic Leukemia
Authors: Rolf Nina , Kariminia Amina , Ivison Sabine M , Reid Gregor SD , Schultz Kirk R. 
Published: October 2019

Abstract

Introduction
Reports of spontaneous acute lymphoblastic leukemia (ALL) remissions following severe bacterial infections or administration of Coley's bacteria extract intriguingly suggest that bacterial lipopeptides may trigger effective immune-mediated eradication of ALL. Coley's extract is now known to have delivered a “perfect storm of Toll-like receptor (TLR) agonists” providing potent immune-stimulation. Considerable preclinical and clinical data verified strong anticancer effects for purified TLR ligands. To date, however, TLR2 ligands remain the least studied despite several unique characteristics of TLR2: i) recognition of most bacterial and other pathogens through hetero-dimerization with TLR1 or 6 (all surface-localized, easily ligand-accessible, consistently expressed on most immune cells); ii) mediation of direct cytotoxicity against malignant cells; and iii) distinction of healthy from damaged self by recognizing endogenous ligands from damaged cells. Building on work previously presented at ASH, the aim of this study was to investigate the effects of TLR2 stimulation on primary ALL and to delineate whether heterodimer-specific TLR2 stimulation resulted in divergent functional downstream effects that could contribute to the generation of anti-leukemia immune activity.

Methods
Pam3CSK4 (Pam3) and Pam2CSK4 (Pam2) are two well-characterized heterodimer-specific synthetic TLR2 ligands not known to cause overwhelming sepsis in vivo. Recently, their exact binding properties were identified by crystallography, offering a reliable in-vitro model to study the potentially divergent functional downstream effects of TLR2/1 (Pam3) vs TLR2/6 (Pam2) stimulation. We investigated whether both TLR2 ligands a) modulate CD40 expression and b) induce death in 4 primary ALL samples and 4 ALL cell-lines. Furthermore, using the best responding cell-line, we investigated which downstream signaling pathways were activated by either ligand.

Results
TLR1, 2 and 6 expression was confirmed on all primary ALL and ALL cell lines. As shown in Table 1, all 4 primary ALL samples were highly responsive to stimulation with TLR2 ligands as both Pam2 and Pam3 strongly upregulated CD40 expression. However, only Pam3 induced death of leukemic blasts. A similar response pattern was observed with cell lines, albeit stronger and more consistent in primary ALL. Antibody blockade of TLR2 abrogated Pam2-mediated CD40 expression. Blocking of both TLR2 and TLR1 was required to reduce Pam3-mediated CD40 upregulation. Pam3-induced cell death was identified as caspase-mediated apoptosis without ROS activation. Both Pam2 and Pam3 strongly induced NFkB and PI3K pathways, though with distinct signaling kinetics that may underlie their divergent effects on ALL blasts.

Conclusions
Our results reveal that synthetic TLR2 ligands are potent stimulators of primary ALL blasts and we demonstrate that heterodimer-specific stimulation resulted in distinct functional downstream responses not previously reported. Close association of danger signals with dying cells critically shapes the immune response: although both TLR2 ligands stimulate ALL cells, the ability of Pam3 to augment CD40 expression while simultaneously inducing cell death achieves the conditions recognized as necessary for generating potent immunological responses. Our study, therefore, indicates that the TLR2/1 ligand Pam3 possesses significant potential for generating anti-ALL immune activity through its direct effects on leukemic blasts.

Disclosures:
No relevant conflicts of interest to declare.