Cancer Vaccines: From the State of the Art to the Most Promising Frontiers in the Treatment of Colorectal Cancer.

Colorectal cancer represents 10% of all new cancer cases each year and accounts for almost 10% of all cancer deaths. According to the WHO, by 2040 there will be a 60% increase in colorectal cancer cases. These data highlight the need to explore new therapeutic strategies. Classical interventions include surgical resection, chemotherapy and radiotherapy, which are invasive strategies that have many side effects on the patients and greatly affect their quality of life. A great advance in the treatment of this cancer type, as well as of all the others, could be the development of a vaccination strategy preventing the onset, the progression or the relapse of the pathology. In this review, we summarize the main vaccination strategies that are being studied for the treatment of colorectal cancer (CRC) and finally explore the possibility of using B-cells for the development of a new type of vaccine.

Purification of Murine and Human IL-10-Producing B Cells from Different Anatomical Compartments.

IL-10 is the best known and most studied anti-inflammatory cytokine and, in the last 20 years, it has acquired even greater fame as it has been associated with the regulatory phenotype of B cells. Indeed, although great efforts have been made to find a unique marker, to date IL-10 remains the main way to follow both murine and human regulatory B cells, hence the need of precise and reproducible methods to identify and purify IL-10-producing B cells for both functional and molecular downstream assays. In this chapter, we present our protocols to isolate these cells from the murine spleen and peritoneum and from human peripheral blood. Since the production of IL-10 by B cells is not only a weapon to counteract the adverse effect of pro-inflammatory cytokines but also a response to cellular activation, we focused on those B cells that are prone to IL-10 production and detectable following a short-term stimulation with phorbol-12-myristate-13-acetate, ionomycin, and lipopolysaccharide (murine system) or CpG (human system).

il-10 Gene Locus DNA Methylation in Regulatory B Cells.

Epigenetic studies are becoming increasingly common in the immunology field thanks to the support of cutting edge technology and to their potential of providing a large amount of data at the single cell level. Moreover, epigenetic modifications were shown to play a role in autoimmune/inflammatory disorders, paving the way for the possibility of using the results of epigenetic studies for therapeutic purposes. In recent years, epigenetic marks such as DNA methylation, histone modifications and nucleosome positioning were shown to regulate B cell fate and function during an immune response, but very little has been done in the context of one of the most recently discovered B cell subsets, that is regulatory B cells. Although no consensus has yet been found on the identity of these immunosuppressive B cells, the role of the IL-10 cytokine is consolidated, both in the murine and human setting. In this chapter we will focus on the analysis of the methylation profile of a gene of interest and we will specifically describe cloning and pyrosequencing bisulphite sequencing PCR (BSP). Given the specific context, we will provide tips and tricks for the analysis of the il-10 gene locus. Nonetheless, the methods presented are valid for the study of any gene of interest.