The role of intestinal lymph DCs in intiating immune responses

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.

The ligation of pattern recognition receptors has profound direct and indirect effects on cells of the immune system, but does not always lead to the initiation of immune responses. The only cells known to initiate immune responses are dendritic cells (DCs). DCs are unable to support CD4+ T cell differentiation when activated indirectly by inflammatory mediators (2). Our data demonstrate that direct sensing of pathogen-associated signals can also be insufficient to stimulate an immune response. The TLR7/8 ligand R-848 exerts dramatic in vivo effects on DCs yet fails to act as an adjuvant. Conversely, the E. coli derived heat-labile enterotoxin (Etx) acts as a strong oral adjuvant. We aim to compare the effects on DCs of pathogen-mediated signals that stimulate immunity (e.g E. coli heat labile enterotoxin) and non-immunising signals (e.g. R-848, a synthetic ligand for TLR7/8). We will examine the migratory properties, activation status, antigen uptake, antigen presenting capacity, cytokine secretion, and molecular phenotype of the subsets of lymph DCs, which we are able to collect with minimal in vitro manipulation as they migrate from the intestine to the MLN via lymph. Having identified the effects on DCs that are required for the initiation mucosal immune responses, we will develop in vitro methods for screening potential mucosal adjuvants. We will culture bone marrow-derived DCs (BMDCs) using Fms-Like Tyrosine Kinase 3 Ligand (FLT3L). This produces BMDCs that closely resemble in vivo DCs. We will use these BMDCs to identify the in vitro effects that correlate with the in vivo adjuvant effects uncovered by our first sets of experiments. Having identified correlates for adjuvant activity, these BMDCs will be used to screen potential novel adjuvants, enabling us to both streamline the process of identifying molecules with adjuvant activity, and use fewer animals in the process. We have previously described two distinct populations DCs in intestinal lymph, identified by their expression of CD172 and CD4. This distinction has proven extremely useful, enabling the definition of a potentially tolerogenic DC subset which constitutively transports antigens derived from apoptotic enterocytes to the MLN (3), and an immunogenic subset that is better able to stimulate activation of naive T cells (4). We have recently discovered that the CD172+ DCs can be split based on their expression of CD11b/c. CD172+ CD11b/chigh DCs are the most numerous DCs in intestinal lymph and express the highest levels of the FcgII receptor (CD32). On the other hand, the CD172+ CD172low DCs are the only DCs that secrete IL-12p40 and IL-6 after in vivo ligation of TLR8. We aim to characterize the immunostimulatory or tolerogenic properties of these lymph DCs after treating animals with oral adjuvants. We will therefore deepen our understanding of the roles of migrating DCs in maintaining the balance between self-tolerance and immune activation. We have recently identified exosomes in lymph. We aim to discover whether these exosomes have an immunological role. We believe this is likely for a number of reasons. Exosomes derived from DCs in vitro have been shown to promote the exchange of functional peptide-MHC complexes between DCs (5). Mast cell-derived exosomes are able to induce functional maturation of DCs (6). Secreted vesicles similar to exosomes have been proposed to mediate the tolerogenic effects of transferred DCs that do not themselves reach draining lymph nodes (7). Having characterized the protein content of vesicles by Western blotting, we will use adoptive transfer strategies to discover whether exosomes purified from steady state lymph are involved in maintaining oral tolerance, and if exosomes from orally immunized animals are able to stimulate immune responses. These experiments will yield basic scientific information about DC biology, and lead to the development of useful tools for the generation of novel oral adjuvants.