Cell-cell interaction with APC, not IL-23, is required for naive CD4 cells to acquire pathogenicity during Th17 lineage commitment

Subpopulations of pathogenic or nonpathogenic Th17 cells were reported to develop when presensitized CD4 cells were activated with their target Ag during polarization by either IL-23 or IL-6 and TGF-β, respectively. In this study, we generated two Th17 subpopulations by using a system in which naive CD4 cells from TCR transgenic mice specific to hen egg lysozyme (HEL) are polarized with IL-6/TGF-β and, concurrently, are activated either with HEL presented by APCs, or with anti-CD3/CD28 Abs. Only the former cells were pathogenic, inducing inflammation in eyes expressing HEL. Naive CD4 cells activated by the anti-CD3/CD28 Abs acquired pathogenicity, however, when cocultured with HEL/APC. Importantly, the naive CD4 cells did not acquire pathogenicity when cocultured with APCs stimulated with LPS or when separated from the HEL-presenting cells by a semipermeable membrane. Unlike with presensitized Th17, soluble IL-23 does not participate in pathogenicity acquisition by naive CD4 cells; no pathogenicity was induced by adding IL-23 to cultures activated with anti-CD3/CD28 Abs. Furthermore, Abs against IL-23 or IL-23R did not inhibit acquisition of pathogenicity in cultures of naive CD4 cells activated by HEL/APC. Our data thus show that, unlike presensitized CD4 cells, naive CD4 cells polarized toward Th17 phenotype acquire pathogenicity only by direct interaction with APCs presenting the Ag, with no apparent involvement of soluble IL-23. We suggest that the Th17 lymphocytes derived from naive CD4 cells participate in pathogenic and other immune processes, along with the IL-23-dependent Th17 cells.     

Activation of invariant NKT cells ameliorates experimental ocular autoimmunity by a mechanism involving innate IFN-gamma production and dampening of the adaptive Th1 and Th17 responses

Invariant NKT cells (iNKT cells) have been reported to play a role not only in innate immunity but also to regulate several models of autoimmunity. Furthermore, iNKT cells are necessary for the generation of the prototypic eye-related immune regulatory phenomenon, anterior chamber associated immune deviation (ACAID). In this study, we explore the role of iNKT cells in regulation of autoimmunity to retina, using a model of experimental autoimmune uveitis (EAU) in mice immunized with a uveitogenic regimen of the retinal Ag, interphotoreceptor retinoid-binding protein. Natural strain-specific variation in iNKT number or induced genetic deficiencies in iNKT did not alter baseline susceptibility to EAU. However, iNKT function seemed to correlate with susceptibility and its pharmacological enhancement in vivo by treatment with iNKT TCR ligands at the time of uveitogenic immunization reproducibly ameliorated disease scores. Use of different iNKT TCR ligands revealed dependence on the elicited cytokine profile. Surprisingly, superior protection against EAU was achieved with alpha-C-GalCer, which induces a strong IFN-gamma but only a weak IL-4 production by iNKT cells, in contrast to the ligands alpha-GalCer (both IFN-gamma and IL-4) and OCH (primarily IL-4). The protective effect of alpha-C-GalCer was associated with a reduction of adaptive Ag-specific IFN-gamma and IL-17 production and was negated by systemic neutralization of IFN-gamma. These data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving innate production of IFN-gamma and a consequent dampening of the Th1 as well as the Th17 effector responses.     

Trans protein splicing of cyanobacterial split inteins in endogenous and exogenous combinations

Inteins are autocatalytic protein domains that post-translationally excise from protein precursors and ligate their flanking regions with a peptide bond, in a process called protein splicing. Intein-containing DNA polymerases of cyanobacteria and nanoarchaea are naturally split into two separate genes at their intein domain. Such naturally occurring split inteins rapidly self-associate and reconstitute protein-splicing activity in trans. Here, we analyze the in vitro protein-splicing activity of three naturally split inteins from diverse cyanobacteria: Oscillatoria limnetica, Thermosynechococcus vulcanus, and Nostoc sp. PCC7120. N- and C-terminal halves of these split inteins were mixed in nine combinations, resulting in three endogenous (wild-type) and six exogenous combinations. Protein splicing was detected in all split-intein combinations, despite a 30-50% sequence variation between the homologous proteins. Splicing activity proceeded under a variety of conditions, including the presence of denaturants and reductants and high temperature, ionic strength, and viscosity. Still, in a high concentration of salt (2 M) or urea (6 M), specific combinations spliced significantly better than others. Additionally, copper ions were found to inhibit trans splicing in a reversible double-lock reaction. Our comparative analysis of naturally split inteins in endogenous and exogenous combinations demonstrates the modularity of trans protein-splicing elements and their robust activity. It suggests tight interactions between split-intein halves and conditions for modifying the specificity of intein parts. These results promote the biotechnological use of split inteins for controlled assembly of protein fragments either in vivo or in vitro and under moderate or extreme conditions.     

Divided we stand: splitting synthetic cells for their proliferation

With the recent dawn of synthetic biology, the old idea of man-made artificial life has gained renewed interest. In the context of a bottom-up approach, this entails the de novo construction of synthetic cells that can autonomously sustain themselves and proliferate. Reproduction of a synthetic cell involves the synthesis of its inner content, replication of its information module, and growth and division of its shell. Theoretical and experimental analysis of natural cells shows that, whereas the core synthesis machinery of the information module is highly conserved, a wide range of solutions have been realized in order to accomplish division. It is therefore to be expected that there are multiple ways to engineer division of synthetic cells. Here we survey the field and review potential routes that can be explored to accomplish the division of bottom-up designed synthetic cells. We cover a range of complexities from simple abiotic mechanisms involving splitting of lipid-membrane-encapsulated vesicles due to physical or chemical principles, to potential division mechanisms of synthetic cells that are based on prokaryotic division machineries.