Kaitlyn Vance and Dr. Eric Wilson, Department of Microbiology and Molecular Biology

The aim of our research was to help elucidate the degree of specificity employed by antibody secreting cells (lymphocytes) through a study of the role of CCR10 in the differential homing of these cells to mucosal tissues. We used vitamin D3, known to upregulate expression of CCR10 on activated lymphocytes, as an adjuvant in our vaccines and tracked cell homing to mucosal tissues that express the CCR10 ligand CCL28 by detecting antigen specific antibody accumulation. Since mucosal tissues are ideal locations for vaccinations, the long-term aim was to develop a method for targeting antigen specific lymphocytes, and thus robust immune responses, to distinct mucosal tissues.

We began by immunizing mice with ovalbumin, using vitamin D3 as an adjuvant and following the methods established in the scientific literature. We measured immune responses using the ELISA assay and determining antibody concentrations in the milk, serum, and various mucosal tissues. Each set of mice took 8 weeks to obtain results, 4 for immunizing and 4 for breeding to obtain milk. As we got a few months into our research, we continued to find large variations between mice that were in the same treatment group, which made data analysis unpredictably difficult.

Dissatisfied with these results, we decided to try and simply replicate the work of Enioutina, et al¹, and paper that inspired our research originally. We spent a few months trying to replicate their work, measuring antibody levels in the serum and feces of immunized mice. Again, we found largely variable results between mice in the same group. So, we contacted R. Daynes, who put us in contact with Enioutina. After exchanging a few emails, she recommended that we try a more potent antigen, because ovalbumin was no always reliable. We redid the experiments using cholera toxin as an antigen, but our problem continued to make data analysis complicated.

After a long discussion with Dr. Wilson, we decided to change the direction of our research. Instead of using vitamin D3 as an adjuvant and tracking the production of antibodies, we decided to immunize mice with the more potent antigen, cholera toxin, and use different methods of mucosal immunization (intranasal, intraperitoneal, subcutaneous, sublingual, etc.). We then would look at the differences in migratory patterns for immune cells activated in these various manners. We decided to isolated cells from the spleens of these mice, and perform transwell migration to different chemokines. In this light, we could see what effect the method of immunization has on the overall chemotaxis of antibody secreting cells.

Beginning in August, we pursued this new but related research interest, in light of the complications we faced originally. I began to immunize mice with cholera toxin in the intraperitoneal (IP) method to determine a standard. We migrated these cells to the chemokine CCL28. At this time, we have only preliminary results for these experiments. Migration was not substantially efficient for cells specific for cholera toxin. It took a few months to determine ideal concentrations for the coating of EliSpot plates with cholera toxin as used in this assay. We have just begun to try oral immunizations. In addition, we discovered a potential difference in the antigen efficiency depending on whether the protein has been frozen or newly reconstituted at the time of immunization.

We finally have a reliable protocol for detecting cholera toxin specific immune cells by EliSpot. Interestingly, we found that many of these cells in the spleen are of isotype IgM, as opposed to the expected IgA isotype. We are currently investigating the significance of this finding, and just beginning to explore the differences in immunization methods.

Although we have encountered many set-backs this year, the ultimate goal of our research remains the same. By characterizing the differences in expression of chemokine receptors on antibody secreting cells activated by different immunization methods, we hope to determine the overall effect of mucosal immunization methods on the response of the immune system. With this knowledge, we will better understand the advantages of certain methods. This can in turn help cater mucosal immunizations for producing a robust response at the target tissue for different antigens.²

References

1. Enioutina, E. Y., D. Visic, Z. A. McGee, and R. A. Daynes. 1999. The induction of systemic and mucosal immune responses following the subcutaneous immunization of mature adult mice: characterization of the antibodies in mucosal secretions of animals immunized with antigen formulations containing a vitamin D3 adjuvant. Vaccine 17:3050-3064.
2. This research was performed by collaboration with Dr. Eric Wilson