Survival fate of neonatal pDC following CpG activation.

<p>(A) IFN-α responses of neonatal pDCs to CpGA were plotted against the survival rate. Cell survival is shown by annexin V and propidium iodide staining (PI). (B) Neonatal pDCs were stimulated with CpGA or IAV in the absence or presence of IL-3 (10 ng/ml) or GMCSF (10 ng/ml) for 24 hrs. IFN-α was detected from supernatants.</p

IL-12p40 producing pDCs represent a small subset of CD2+pDC.

<p>(A) Neonatal and Adult MNCs were stimulated with IAV-HI CpGA then CD2⁺CD5⁺, CD2⁺CD5⁻ and CD2⁻CD5⁻ pDCs subsets were gated to analyze their capacity to produce IL-12 p40 detected by intracellular cytokine staining. CpGA stimulated mDC (BDCA1⁺CD20⁻CD14⁻), monocytes (CD14⁺) and B cells (CD20⁺) from adult PBMC were also stained for IL-12p40 intracellularly. Numbers indicate the percentage of cytokine-producing cells among totally gated cells. (B) Expression of co-stimulatory markers CD40, CD86 and HLADR on neonatal BDCA4⁺CD123⁺pDCs defined as CD2⁺CD5⁺, CD2⁺CD5⁻ and CD2⁻CD5⁻ subsets under steady state. Data are representative of 3 experiments.</p

Innate responses of cord blood pDCs to viruses.

<p>Adult or neonatal whole blood (WB) (A and C), mononuclear cells (MNC) (A and C) or purified pDCs (A–C) were stimulated with CpGA (50 µg/ml for WB, 5 µg/ml for others in A), live IAV (1000 HAU/ml for WB, 10 HAU/ml for others), heat-inactivated IAV (HI-IAV in C) live HIV (10⁵ infected T cells in A) and live HSV-1 (10⁶ pfu/ml in A) for 24 hrs. Plasma or supernatants were collected and tested for IFN-α (A and C) or TNF-α, CCL3 and CCL4 (B). NA: non applicable. The number of donors is indicated for each group (n) as well as <i>P</i> values for adults and neonates comparison. In B, n = 8 for adult and n = 7 for neonates.</p

Heterogeneity of neonatal pDCs.

<p>(A) The percentages CD123^hiBDCA2⁺ pDCs within CD45+ MNC were calculated from neonatal cord blood and adults. pDCs could be subdivided by CD2 and CD5, and the accumulated data for CD2⁺, CD2^- pDCs subsets and CD2⁺CD5⁺, CD2⁺CD5⁻ pDCs subsets are shown in (B). (C) The innate IFN-α production of sorted CD2⁺ and CD2⁻ pDCs subsets to CpGA, live IAV and HIV (the results are incated as ng/ml). (D) Neonatal and Adult MNCs were stimulated as indicated and CD2⁺CD5⁺, CD2⁺CD5⁻ and CD2⁻CD5⁻ pDCs subsets were gated to analyze their capacity to produce IFN-α detected by intracellular cytokine staining. Numbers indicate the percentage of cytokine-producing cells among gated cells. Data are representative of 5 experiments. The number of donors is indicated for each group (n) as well as <i>P</i> values for adults and neonates comparison.</p

Cord blood pDCs are the main contributor for IFN-α response.

<p>(A) Neonatal cord blood was left untreated or exposed to IAV, for 6 hrs and stained intracellularly for IFN-α. pDCs are identified by CD123 and BDCA4 and monocytes by CD14 expression. (B) Total CBMC (1), or CBMC depleted of pDCs (2) or of monocytes (3) were stimulated with CpGA, live IAV and HI-IAV as in Fig. Plasma or supernatants were collected and tested for IFN-α (A). Depletion efficiency is shown for pDCs identified by CD123 and CD45RA and monocytes by CD14 and CD11b expression. One experiment representative of three is shown.</p

The Quest for Anticancer Vaccines: Deciphering the Fine-Epitope Specificity of Cancer-Related Monoclonal Antibodies by Combining Microarray Screening and Saturation Transfer Difference NMR

The identification of MUC1 tumor-associated Tn antigen (αGalpNAc1-<i>O</i>-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays and saturation transfer difference NMR, we have characterized the fine-epitope mapping of a MUC1 chemical library (naked and Tn-glycosylated) toward two families of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference for glycopeptides containing the Tn-Ser antigen rather than the Tn-Thr analogue, stressing the role of the underlying amino acid (serine or threonine) in the binding process. The reported strategy can be employed, in general, to unveil the key minimal structural features that modulate antigen–antibody recognition, with particular relevance for the development of Tn-MUC1-based anticancer vaccines