The role of intestinal lymph DCs in intiating immune responses

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DCs may be thought of as the scouts of the immune system. Their function is to pass through the tissues of the body and, when appropriate, initiate adaptive immune responses to attack invading pathogens. In order to perform this function, they are constantly migrating from tissues, via the lymph, to lymph nodes where they are able to interact with circulating lymphocytes. In rats, we are able to collect the DCs migrating from the intestine with minimal in vitro manipulation. This is achieved by first removing the mesenteric lymph nodes (MLN) and allowing the afferent and efferent lymphatics to heal. In this way, DCs that would normally be trapped in the lymph nodes are able to continue through the lymph and can be harvested by placing a cannula into one of the major lymphatic vessels, the thoracic duct. Thoracic duct cannulation is a long-established technique that has proven extremely useful in defining the functions of DCs in vivo, enabling us to show that DCs are critical both for initiating immune responses to antigens introduced into the intestine, and in preventing damaging responses against harmless self antigens. We have found, to our surprise, that a compound (a TLR7/8 ligand, the small-molecule R-848) that increases the number of migrating DCs in lymph, and activates DCs in lymph nodes, is not able to stimulate an immune response against a protein (ovalbumin) when the two are mixed and fed to animals. Conversely, feeding E. coli heat labile enterotoxin (Etx) mixed with ovalbumin stimulates a strong anti-ovalbumin immune response without increasing the number of migrating DCs or causing detectable changes in the activation state of lymph node DCs. Subsequent experiments have suggested that increasing the number of DCs carrying ovalbumin in the lymph is likely to be one of a number of factors critical to the outcome of any immune stimulation. We plan to manipulate purified DCs to define the DC characteristics most important for initiating immune responses. Having identified the most important characteristics of immunogenic DCs in vivo, we will be able to develop methods for using cultured DCs to screen potential immunogenic compounds. Two other recent observations have provided the basis for new avenues of research into intestinal lymph. The first is the discovery that lymph DCs fall not into two, as previously understood, but three distinct subsets. The previously-identified CD172+ DCs can be separated into two groups based on their expression of both CD11b/c and CD32. The CD172+ CD11b/chigh DCs are the most numerous in intestinal lymph, while CD172+ CD11b/clow DCs are able to secrete IL-12, an immunostimulatory cytokine. Because we are able to collect migratory DCs before they reach the MLN, the experiments described in this application will enable us to define the different functional properties of these 3 subsets migratory DCs. We have also observed that thoracic duct lymph contains many membrane-bound vesicles between 50-200nm in diameter. These vesicles resemble exosomes, vesicles that are generated by a number of cell types in vitro, including DCs. Exosomes, when mixed with antigen-presenting cells in vitro are able to stimulate or suppress immune responses, and are being tested in clinical trials for cancer immunotherapy. However, exosomes have not previously been identified in lymph and their physiological importance is unclear. We will therefore purify and characterize the exosomes from intestinal lymph. We will also investigate whether they are involved in the control of immune responses, either in he steady state or after the administration of oral adjuvants. We believe that these experiments will advance understanding of the role of intestinal DCs in oral vaccination, and help with the development of a new generation of oral adjuvants.