Toll-like receptor 4 gene polymorphism influences dendritic cell in vitro function and clinical outcomes in vaccinated melanoma patients

Abstract

Toll-like receptor 4 (TLR4) is expressed on dendritic cells (DCs), sensing environmental danger molecules that induce their activation and maturation. Recently, we reported a method for the production of therapeutic DCs against melanoma, called tumor antigen-presenting cells (TAPCells), using a heat-shocked allogeneic melanoma cell lysate (TRIMEL) as an activation factor and antigen provider. Since TRIMEL contains endogenous TLR4 ligands, we evaluated the role of TLR4 in TAPCells differentiation by antibody neutralization and the association of a Tlr4 polymorphism (896A/G) (Asp299Gly), determined by PCR-RFLP, with the in vitro activation capacity and the clinical outcome of TAPCells-vaccinated patients. Antibody blocking of monocyte TLR4 inhibited surface expression, determined by flow cytometry, of the major histocompatibility complex class I, CCR7, CD80, CD83 and CD86 on TAPCells, reduced interleukin (IL)-6 and tumor necrosis factor -α gene expression evaluated by qRT-PCR, and also inhibited the TAPCells-mediated interferon-γ (IFN-γ) secretion of melanoma-specific CD8(+) T cells determined by ELISpot (p < 0.01). Moreover, CD8(+) T-cell activation capacity was significantly reduced in TAPCells bearing the TLR4 Asp299Gly receptor (p < 0.05). Finally, TAPCells-vaccinated stage-IV melanoma patients bearing the Tlr4 896G allele showed a shortened post-therapy median survival rate compared with those carrying the Tlr4 896A allele (p < 0.05; log-rank test). Our results indicate that TLR4 is a key receptor for the tumor lysate-mediated in vitro generation of clinically efficient antigen-presenting cells. Further analysis of patients included in different vaccine protocols is necessary for definitively establishing a role for TLR4 polymorphism in clinical responses.

TAPCells, the Chilean dendritic cell vaccine against melanoma and prostate cancer

“…Importantly, 60% of evaluated metastatic patients (62 of 102) developed a DTH reaction against TRIMEL, indicating the development of immunological memory against the tumor that correlates with prolonged survival of patients. The in vitro analysis of TRIMEL revealed that it contains DAMPs such as nuclear protein HMGB-1 induced by heat shock, which are capable of eliciting, through Toll-like receptors the maturation of DCs, improving TAA cross-presentation (Aguilera et al, 2011;Tittarelli et al, 2012). Tissue biopsies from DTH sites revealed the presence of CD45RO + CD8 + and CD4 + TL able to release pro-infl ammatory cytokines after in vitro stimulation.…”

“…This heat shock consists of exposing the cells to temperatures of 42 °C for a period of 1 hour and then 2 hours at physiological temperature (37 °C). Our data indicate that this conditioning of tumor cells from which the lysate is produced triggers danger signals that are essential to activate DCs and enhance antitumor signals to TL (Aguilera et al, 2011;Tittarelli et al, 2012;Reyes et al, 2013).…”

“…During the past 10 years, interaction between basic immunologists, clinicians and oncologists has facilitated a major international contribution to basic immunology and the development of cancer immunotherapy by our research group Escobar et al, 2005;López et al, 2006;Salazar-Onfray et al, 2008;López et al, 2009;Aguilera et al, 2011;Tittarelli et al, 2012;Durán et al, 2012;Reyes et al, 2013).…”

Trial Watch

“…Thus, TLRs appear to play a prominent role not only in the orchestration of innate immune responses against infectious pathogens, but also in anticancer immunity, be it spontaneous or elicited by (chemo)therapeutic interventions 23 , 27 , 57 , 58 . In accord with this notion, functionally relevant polymorphisms in the genes encoding several TLRs (i.e., TLR1, TLR2, TLR3, TLR4, TLR6, TLR9 and TLR10) have been shown to influence the natural development of a wide array of neoplasms, including tumors that are not associated with a microbial etiology, 59 - 77 as well as to affect the response of cancer patients to chemotherapy and immunotherapy, at least in some settings 54 , 78 - 80 . Moreover, the expression of several TLRs including TLR2, TLR4, TLR7 and TLR9 by malignant cells appear to evolve not only along with oncogenesis and tumor progression, but also in response to microenvironmental cues, 81 - 90 suggesting that, at least in some types of cancer, TLRs may influence disease progression in a direct fashion rather than as a consequence of immunological effects.…”

Trial Watch

“…Accordingly, SNPs that reduce the activity of various TLRs have been shown to negatively impact the response of cancer patients to therapy in multiple scenarios 83 , 86 - 88 . At least in part, this reflects the key role that some TLRs play in the perception of cell death as immunogenic 79 , 80 .…”