We present a systematic study that deWnes molecular proWles of adjuvanticity and pyrogenicity induced by agonists of human Toll-like receptor molecules in vitro. Using P 3 CSK 4 , Lipid A and Poly I:C as model adjuvants we show that all three molecules enhance the expansion of IFN + /CD4 + T cells from their naïve precursors following priming with allogeneic DC in vitro. In contrast, co-culture of naive CD4 + T cells with allogeneic monocytes and TLR2/TLR4 agonists only resulted in enhanced T cell proliferation. Distinct APC molecular signatures in response to each TLR agonist underline the dual eVect observed on T cell responses. Using protein and gene expression assays, we show that TNF-and CXCL10 represent DC-restricted molecular signatures of TLR2/TLR4 and TLR3 activation, respectively, in sharp contrast to IL-6 produced by monocytes upon stimulation with P 3 CSK 4 and Lipid A. Furthermore, although all TLR agonists are able to up-regulate proIL-1 speciWc gene in both cell types, only monocyte activation with Lipid A results in detectable IL-1 release. These molecular proWles, provide a simple screen to select new immune enhancers of human Th1 responses suitable for clinical application.

Limiting dilution analysis (LDA) has been extensively employed as a quantitative method to estimate the precursor frequency of various T lymphocyte subsets according to their functional properties in vitro. We describe here an example of LDA experiment assessing antigen-specific T cell proliferation of microcultures in the presence or absence of adjuvant and illustrate how to estimate the frequencies of precursor T cells using an online tool that we made publicly available.