HDAC Inhibitors as Epigenetic Regulators

The SIINFEKL-loaded splenocytes were labeled with 1?M CFSE (CFSEhigh target population), while the control splenocytes, loaded with an irrelevant peptide, were labeled with 0.1?M CFSE (CFSElow cell population). Therefore, vaccination with an exogenous antigen formulated with Santacruzamate A SMIP2.1 is a successful strategy for the induction of a cytotoxic T cell response along with antibody production. manipulation of DCs.5,6 These approaches suffer from difficulties in manufacturing, as well as the high costs. A promising strategy is the use of adjuvants, molecules that are added to vaccine formualtions in order to modulate the immune response and ultimately increase protection. Although many experimental adjuvants have been evaluated in animal models, until 10 y ago only squalene-based oil in water emulsions and aluminum-based salt adjuvants had been licensed for inclusion in human vaccines.1 These adjuvants are effective at eliciting humoral responses, but fail to stimulate CD8+ T cell immunity. Alternative vaccine adjuvants aimed at eliciting both antibody and cellular responses are based on the activation of receptors of the innate immune system, such as TLRs. Engagement of TLRs with either natural or synthetic agonists, results in a robust activation of innate immune cells and leads to the production of proinflammatory cytokines.7,8 Many pre-clinical studies support the safety of TLRs agonists in vaccine formulations as well as their ability to increase adaptive immune responses.9,10 TLR agonists have also been shown to enhance therapeutic vaccination against cancer and chronic viral infections.8,11,14 Indeed, vaccines containing the adjuvant AS04, made by the alum-absorbed TLR4 agonist monophosphoryl lipid A (MPL), have been approved for human use in 2005.1,15 Here we explored the ability of SMIP2.1, a novel synthetic lipopeptide-based TLR2 agonist, to induce cross-presentation by both mouse and human APCs. Using and experiments we showed that SMIP2.1 can activate the innate immune system via a TLR2-dependent mechanism, induce the maturation of APCs, and elicit a strong antibody response against influenza and tetanus toxoid antigens. In mice, TLR2 agonists can induce an antigen-specific CD8+ T cell response, especially when linked to the antigen.16-18 Here, we show that SMIP2.1 is also a good inducer of a CTL response when mixed with the antigen as Santacruzamate A aqueous suspension using either mice or human cells. Mice that received OVA-specific OT-I TCR transgenic cells by adoptive transfer showed increased CD8+ T cell proliferation, cytokine production, and cytotoxic activity upon inclusion of SMIP2.1 Santacruzamate A in the Santacruzamate A OVA vaccine formulation. We investigated which APCs populations could be the target for SMIP2.1-induced cross-presentation and showed that both CD8+ and CD8? DCs could cross-present. While it is already known that DCs can cross-present exogenous antigens, the role of B cells in this process is less clear.19-21 Using transnuclear B cells that express a BCR specific for OVA, we demonstrated for the first time that B cells can cross-present OVA upon TLR2 stimulation. Likewise, upon stimulation with SMIP2.1, human PBMCs were able to cross-present the CMV pp65 protein to human CMV (HCMV)-primed CD8+ T cells. This Pik3r1 study shows that SMIP2.1 could assist in the generation of antigen specific CTL along with the robust activation of CD4+ T cells, and thus could be exploited in the design of effective adjuvants for antitumor and antiviral vaccines. Results Identification of a new TLR2 agonist A series of high-throughput screens on a chemical library of 1 1.8 million compounds were performed. Briefly, the TLR2 expressing human B cell line RI-I and monocytic cell line THP-1 were screened in arrayed, 1536 well format in single point (10?M in DMSO) using TNF as a readout (data not shown). Compounds able to stimulate these leukocyte cell lines were counter-screened using mouse lymphocytes as well as HEK293 clones stably transfected with the luciferase gene under control of transcription factor NF-kB and different human TLRs (data not shown). This strategy resulted in the identification of a group of triacetylated lipopeptides active only on both human and mouse TLR2 which differed in the amino acid component and in the length of the acyl chain. This class of lipopeptide bears a triacylated cysteine glycerol core, similar to the known TLR2 agonist Pam3CSK4, but differs in the serine and lysine amino acid residues.22 A representative of this class of lipopeptides is shown in Figure 1A as SMIP2.1. The dipeptide portion of SMIP2.1.