Human blood mDC subsets exhibit distinct TLR repertoire and responsiveness

Abstract

Human blood DCs encompass pDCs and two subsets of mDCs: CD1c(+) mDCs and CD141(+) mDCs. The rare CD141(+) DC population is thought to be the equivalent of mouse CD8α(+) cDCs that play a significant role in antigen cross-presentation. Here, we analyzed by Q-PCR TLR1-10 expression in blood DC subsets. Whereas CD1c(+) DCs express all TLR except TLR9, CD141(+) DCs present a more restricted pattern with high expression of TLR3 and -10, expression of TLR1,-2, -6, and -8, and lack of TLR4, -5, -7, and -9. The in vitro analysis of isolated mDC subset reponsiveness to an extensive panel of TLR ligands confirmed these results, with CD141(+) DCs responding only to TLR1/2, -3, and -7/8. The cytokine/chemokine production profile of isolated CD141(+) DCs was also more restricted, as they produced mainly proinflammatory cytokines but no IL-12 and to a lower level, in comparison with CD1c(+) DCs, except for CXCL10, CCL5, and IFN-β. In contrast, with the use of a whole blood assay, we found that CD141(+) DCs produce IL-12 in response to TLR1/2, -3, and more surprisingly, -9. Finally, both mDC subsets are potent inducers of Th1 response, particularly after TLR3 triggering. Taken together, these data confirmed functional differences between blood mDC subsets. The major response of CD141(+) mDCs to TLR3 ligand and their cytokine production pattern suggest a role for these cells in antiviral immunity.

CMRF-56+blood dendritic cells loaded with mRNA induce effective antigen-specific cytotoxic T-lymphocyte responses

“…24 T2 cells were pulsed with 10 mg/mL WT1 126-134 , Kallikrein 4 11¡19 (KLK4; FLGYLILGV)) or MART1 [26][27][28][29][30][31][32][33][34][35] peptide in X-Vivo 15 for 2 h and stained with 2.5 mM calcein-AM. Antigen specific cytotoxicity was measured after 3 h incubation at 37 C using a 50:1 effector:target ratio on an Enspire plate reader (Perkin Elmer) with an excitation at 485 nm and fluorescent emission at 520 nm.…”

“…24 [25][26][27][28][29][30][31][32][33][34][35] (ELAGIGILTV) peptides (Mimotopes) or expanded using peptide loaded, mitomycin C (Sigma-Aldrich, M-4287-2MG) treated autologous PBMC as described. 24 WT1-specific T cells generated following T-cell expansion were monitored using HLA-A Ã 0201:WT1 126-134 dextramers (Immudex).…”

“…However, the properties of Mo-DC were shown to differ substantially from those of primary "in vivo" derived BDC, [23][24][25] and it is now well accepted that there are significant transcriptional and functional differences between Mo-DC and BDC. 12,14,17,22,[25][26][27][28][29][30] A major concern regarding the use of Mo-DC for therapeutic vaccination has been their well-documented failure to migrate from sites of injection to draining lymph nodes (LN). 31,32 This suggests that the efficacy seen in some studies may relate to an adjuvant or antigen-transfer effect to host DC present at the site of injection.…”

The role of human CD1c+ DCs at activating innate and adaptive immune responses

“…CD8 + CD103 + DCs in mice that express TLRs 3, 4 and 9 and produce IL-12p70 upon stimulation with poly:IC (PIC -a TLR 3 agonist), LPS (a TLR 4 agonist) and CpG (a TLR 9 agonist) respectively (Hochrein et al, 2001). However, stimulation of CD141 + DCs with a wide range of TLR agonists including PIC and R848 or employment of secondary T cell-mediated signals including CD40 ligand (CD40L) fail to enhance IL-12p70 production (Hemont et al, 2013, Jongbloed et al, 2010, Nizzoli et al, 2013, Poulin et al, 2010. Taken together, current studies to 26 | P a g e date suggest that this DC subset is not the major producer of IL-12p70.…”

“…Like their murine counterparts, CD141 + DCs selectively express C-type lectin CLEC9A (Caminschi et al, 2008, Huysamen et al, 2008, Sancho et al, 2009), nectin-like protein 2 (NELC2) (Galibert et al, 2005) and chemokine receptor XCR1 (Bachem et al, 2010, Crozat et al, 2010, express high levels of toll-like receptor (TLR) 3 which recognises double-stranded (ds) RNA (Hemont et al, 2013, Jongbloed et al, 2010 and express TLR 8 which recognises single-stranded (ss) RNA (Jongbloed et al, 2010). Furthermore, CD141 + DCs are the major producers of IFN-λ in response to dsRNA, a feature uniquely shared with its mouse CD8 + /CD103 + counterpart (Lauterbach et al, 2010).…”