ResponseEfficient APC System for Activating Th1 IL-10-Deficient Dendritic Cells as a Highly Molecular Basis for the Potency of

Identiﬁcation and targeting of novel immunobiological factors that regulate the induction of Th1 cells are crucial for designing effective vaccines against certain intracellular pathogens, including Chlamydia. IL-10-deﬁcient dendritic cells (DC) are potent APCs and effective cellular vaccines that activate a high frequency of speciﬁc Th1 cells. To elucidate the molecular basis for the potency of the IL-10-deﬁcient APC system, we tested the hypothesis that Chlamydia Ag-primed IL-10 knockout (IL-10KO) DC are quantitatively and qualitatively distinct in their metabolic characteristics relating to T cell activation. Using a combination of RT-PCR, two-dimensional gel electrophoresis, and MALDI-TOF-based proteomics analyses, the transcriptional and translational activities of Chlamydia -pulsed DC from wild-type and IL-10KO mice were assessed. IL-10 deﬁciency caused early maturation and activation of pulsed DC (i.e., high CD11c, CD40, CD80, CD83, CD86, IL-1, IL-12, and the T cell-attracting chemokine CCL27/ CTACK) and consequently an enhanced ability to process and present Ags for a rapid and robust T cell activation. Supporting comparative proteomics revealed further that IL-10 deﬁcient DC possess speciﬁc immunobiological properties, e.g., the T cell-attracting chemokine CCL27/CTACK, calcium-dependent protein kinase, and the IL-1/IL-12 inducer, NKR-P1A (CD161), which differentiated them immunologically from wild-type DC that express molecules relating to anti-inﬂammatory, differentiative, and metabolic processes, e.g., the anti-IL-12 molecule peroxisome proliferator-activated receptor- (cid:1) and thymidine kinase. Collectively, these results provide a molecular basis for the high Th1-activating capacity of IL-10KO APC and may provide unique immunomodulation targets when designing vaccines against pathogens controlled by T cell immunity. The Journal of Immunology, 2005, 174: 4860–4869. used the features of IL-10-deﬁcient to molecular and biological basis the high T cell activating capacity of efﬁcient APC system. The results from of pathways that can be ratio-nally during delivery to an optimal Peptides in the selected spots were extracted using three 20-min incubations in 10 mM am- monium bicarbonate, 60% acetonitrile solution. The different extractions were pooled, and the volume was reduced to (cid:5) 10 (cid:3) l in a Savant Speed Vac. A volume of 2 (cid:3) l of this mixture was mixed with 8 (cid:3) l of (cid:2) -cyano-4-hydrocinnamic acid (10 mg/ml in 30% methanol, 50% acetonitrile, 0.1% TFA, water), and 2 (cid:3) l were spotted on a MALDI target. Mass spectra for the different spots were acquired on a Bruker Reﬂex IV MALDI-TOF MS equipped with pulsed ion extraction and a nitrogen laser. The peaks corresponding to speciﬁcally identiﬁed peptides were picked using XMass software (Bruker Reﬂex IV). All the mass spectra were internally cali-brated with trypsin autolysis peaks. The resulting peptide mass ﬁngerprints were searched against a local copy of the nonredundant database NCBInr (http://prospector.ucsf.edu) search program adjusted for murine proteins. The parameters used in the search were as follows: peptide mass, isoelectric point (pI), tolerance 50 ppm, 1 missed cleavage, carboxymethylated cysteine, and N-terminal hydrogen. immunoassay analysis of supernatants from Chlamydia pulsed DC, revealing that IL-10KO DC do not secret signiﬁcant IL-10. The early expression of CD11c, IL-1 (cid:4) , IL-12, CD40, and CD80 in Chlamydia -pulsed IL-10KO was statistically signiﬁcant compared with the expression by Chlamydia- pulsed WT DC ( p (cid:7) 0.001, 0.001, 0.006, 0.001, and 0.014, respectively). cell during Ag presentation and T cell activation. These results revealed that unlike WT DC, Chlamydia -pulsed IL-10KO DCs rapidly acquired markers (CD11c, CD40, and CD83) that are associated with matured DCs, suggesting that early maturation of DCs is an important factor in the enhanced T cell activation. In fact, early maturation of DCs leads to the rapid acquisition of the capacity to express crucial costimulatory molecules such as IL-1 (cid:4) , CD40, and IL-12 that are important for the differentiative activation of T cells along the Th1 pathway. Our results are further corroborated by the fact that under normal physiological conditions of the mammalian host, DC exist as relatively immature mononuclear cells scattered in various mucosal and nonmucosal tissues in the body. On en-counter of an antigenic signal through the TLR-mediated signals recognition of pathogen-associated molecular CpG, LPS, dsRNA), DC a differentiative maturation the immature (highly endocytic cells) mature activated cells up-regulated costimulatory molecules enhanced Ag presentation and T cell activation The maturational process their capacity to process MHC-peptide complexes, their migration toward second-ary lymphoid tissues such as lymph nodes, acquisition of costimulatory molecules, and efﬁcient Ag presentation for T cell activation. whereas Ag handling by fully mature DC T cells and prime immune responses, immature DC induce or response. In T cells differ-entiate into speciﬁc the TCR-epitope