Molecular characterization of human CD4+ IL-10-producing regulatory cells

Previous studies in the group led to the identification of CD4+FOXP3- cells with regulatory functions in human blood that coproduce IL-10 and IFN-gamma. These cells do not belong to the Treg cell lineage since they are Foxp3- but they show some similarities with Th1 cells since they express CCR5, T-bet and produce high levels of IFN-gamma. Thus, they share relevant characteristics with both T regulatory type I cells (Tr1) and Th1 cells and we called them Th1-10 cells. In this study we presented a molecular characterization of Th1-10 cells that includes a gene expression and a microRNA profiling and performed functional studies to assess Th1-10 cells regulatory properties. We demonstrated that Th1-10 cells have a high regulatory potential being able to block the proliferation of activated CD4 naïve T cells to a similar extent as conventional Treg cells, and that this suppression capacity is at least partially mediated by secreted IL10. We showed also that Th1-10 cells are closely related to Th1 effector memory cells and express genes involved in cytotoxicity. In particular, they express the transcription factor EOMES and the cytotoxic effector molecules GZMA and GZMK, and they release cytotoxic granules upon stimulation. Moreover, we found that Eomes regulates cytotoxic functions in CD4+ T cells. We demonstrated that miR-92a, selectively downregulated in Th1-10 cells, directly targets the 3’UTR of EOMES.and this finding identifies miR-92a as a possible mediator of Th1-10 cytotoxicity. Th1-10 cells retain some proliferative capacity when sorted ex vivo and activated in vitro via their TCR, and this effect is markedly enhanced by IL-15, which also had a pro-survival effect on Th-10 cells. Thus, in contrast to conventional cytotoxic T cells, Th1-10 cells have cytotoxic and regulatory functions and are not terminally differentiated, since they retain proliferative capacity.

Use of E. coli OMVs as delivery system to elicit neutralizing antibodies against Chlamydia infectivity: the HtrA protein example

The obligate intracellular pathogen Chlamydia trachomatis is a gram negative bacterium which infects epithelial cells of the reproductive tract. C. trachomatis is the leading cause of bacterial sexually transmitted disease worldwide and a vaccine against this pathogen is highly needed. Many evidences suggest that both antigen specific-Th1 cells and antibodies may be important to provide protection against Chlamydia infection. In a previous study we have identified eight new Chlamydia antigens inducing CD4-Th1 and/or antibody responses that, when combined properly, can protect mice from Chlamydia infection. However, all selected recombinant antigens, upon immunization in mice, elicited antibodies not able to neutralize Chlamydia infectivity in vitro. With the aim to improve the quality of the immune response by inducing effective neutralizing antibodies, we used a novel delivery system based on the unique capacity of E. coli Outer Membrane Vesicles (OMV) to present membrane proteins in their natural composition and conformation. We have expressed Chlamydia antigens, previously identified as vaccine candidates, in the OMV system. Among all OMV preparations, the one expressing HtrA Chlamydia antigen (OMV-HtrA), showed to be the best in terms of yield and quantity of expressed protein, was used to produce mice immune sera to be tested in neutralization assay in vitro. We observed that OMV-HtrA elicited specific antibodies able to neutralize efficiently Chlamydia infection in vitro, indicating that the presentation of the antigens in their natural conformation is crucial to induce an effective immune response. This is one of the first examples in which antibodies directed against a new Chlamydia antigen, other than MOMP (the only so far known antigen inducing neutralizing antibodies), are able to block the Chlamydia infectivity in vitro. Finally, by performing an epitope mapping study, we investigated the specificity of the antibody response induced by the recombinant HtrA and by OMV-HtrA. In particular, we identified some linear epitopes exclusively recognized by antibodies raised with the OMV-HtrA system, detecting in this manner the antigen regions likely responsible of the neutralizing effect.