Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-Mediated Calcium Signaling Is Active in Memory CD4+ T Cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP), identified as one of the most potent calcium-mobilizing second messengers, has been studied in different eukaryotic cell types, including lymphocytes. Although aspects of NAADP-mediated calcium release in lymphocytes are still under debate, the organelles pertaining to NAADP-mediated calcium release are often characterized as acidic and related to lysosomes. Although NAADP-mediated calcium release in different subsets of T cells, including naïve, effector and natural regulatory T cells, has been studied, it has not been widely studied in memory CD4+ T cells, which show a different calcium flux profile. Using a pharmacological approach, the effect of Ned-19, an NAADP pathway antagonist, on the involvement of NAADP in TCR activation in murine memory CD4+ T cells and their downstream effector functions, such as proliferation and cytokine production, was studied. According to this study, Ned-19 inhibited TCR-mediated calcium flux and its downstream effector functions in primary memory CD4+ T cells. The study also revealed that both extracellular and intracellular calcium stores, including endoplasmic reticulum and lysosome-like acidic calcium stores, contribute to the TCR-mediated calcium flux in memory CD4+ T cells. NAADP-AM, a cell permeable analogue of NAADP, was shown to release calcium in memory CD4+ T cells and calcium flux was inhibited by Ned-19.

Synthesis and Evaluation of a Self-Adjuvanting Pseudomonal Vaccine Based on Major Outer Membrane Porin OprF Epitopes Formulated with Low-Toxicity QS-21-Containing Liposomes.

Pseudomonas aeruginosa (PA) is a Gram-negative pathogen that the World Health Organization has ranked as a priority 1 (critical) threat. One potential prophylactic approach to preventing or reducing the incidence of PA would be development of a long sought-after vaccine. Both antibody and CD4+ T-cell responses have been noted as playing key roles in protection against infection. In these studies, we have designed a prototype vaccine consisting of several known linear B-cell epitopes derived from an outer membrane porin F (OprF). The resulting thiol-containing protein was conjugated to a version of the lipopeptide-based Toll-like receptor agonist Pam3CysSK4Mal (10) containing a maleimide moiety and formulated into dipalmitoylphosphatidylcholine (DPPC)/cholesterol (Chol) liposomes. Mice immunized with the resulting vaccine generated antibodies that bound PA14 (serotype O10) in vitro and induced opsonization in the presence of rabbit complement and murine macrophage RAW264.7 cells. The liposome was optimized to contain 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), Chol, Pam3CysSK4-OprF (12) and the Quillaja saponaria-derived saponin adjuvant QS-21. The resulting vaccine formulation produced significantly higher antibody titers, increased the IgG2a antibody isotype, and increased the number of IgG-producing B-cells as well as splenic primed T-cells. In summary, the liposomal vaccine platform was found highly useful for the generation of a robust and balanced TH1/TH2 response.