Lauren Zagieboylo and Dr. Eric Wilson, Microbiology and Molecular Biology
Main Text
B-1 B cells are an understudied type of white blood cell that provide T cell-independent antibody production, allowing them to play an important role in the early immune response to many bacterial and viral pathogens. They are especially important for defense in mucosal sites, such as the intestinal tract, where many infections occur.
At a time when antibiotic resistance has become a prevalent threat in medicine, many scientist are looking for new ways to defend the body. Several of the hopeful new alternatives we are looking for may come from processes that are actually very old. Nature and the body have developed intricate systems that—while imperfect—have effectively defended life against pathogens for millions of years. One of these defenses comes from B-1 B cells. It is our hope that understanding the mechanisms involved in lymphocyte migration will help us find ways to better regulate immune responses and create more efficient mucosal vaccines because it will allow us to utilize working systems and processes already in place in the body. This research helps us discover how to take those inherent systems and alter them for our benefit.
It has been shown that the chemokines CCL25 and CCL28 regulate the migration of the more common B-2 B cells to the intestines (Heishima et al. 2004); I hypothesized that CCL25 is also key in the migration of B-1 B cells to the small intestine and that CCL28 directs the migration of B-1 B cells to the large intestine.
I encountered numerous obstacles and, I suppose, many of the common frustrations of research in my attempts to uncover which chemokines were involved in B-1 B cell migration. It all began promisingly. I had learned how to use the confocal microscope to visualize cells using fluorescent antibodies and believed I had found evidence of B-1 B cells in the intestines of mice. I compared the tissues from a normal mouse to those of a mouse with a receptor called Lymphotoxin-β receptor (LtBr) missing. The absence of this receptor was supposed to prevent the B cell-T cell interactions necessary for B cells to produce IgA antibodies. Because B-1 B cells can produce IgA independent of T cell interaction, any IgA I detected should be from the cells I wanted. Whereas the normal mice had an even spread of IgA cells, I found only clusters of these cells in the mutant mice.
The microscopy results led me to believe I could detect levels of B-1 B cells by measuring IgA antibody levels from the mice. My lab had bred some mice that were missing CCR9 and CCR10, receptors for the chemokines CCL25 and CCL28 respectively. If the lack of the CCR9 chemokine receptor negatively impacted the ability of B-1 B cells to accumulate in the intestinal tissue, for example, I should see it as a decrease in the IgA concentrations of CCR9 LtBr knockout mice. I used ELISAs to compare relative IgA levels in CCR9 LtBr and CCR10 LtBr knockout mice to wild type mice. The results were not only confusing, but also inconsistent. No matter what I tried. And I tried an endless number of approaches and tweaks.
Undeterred, I performed other tests such as migration assays to see whether cells from the intestines of mice would migrate towards either CCL28 or CCL25 (suggesting that that chemokine is necessary for B-1 B cells to migrate to the intestines). I also performed RT-qPCR’s, which quantitatively measure the expression of a particular protein, to see whether there were higher levels of either CCR9 or CCR10 in the LtBr knockout mice (also to show whether CCL28 or CCL25 was important to migration). I synthesized primers to detect the presence of a particular antibody only expressed by B-1 B cells. I also obtained and cultivated a particular strain of bacteria that was supposed to stimulate growth of B-1 B cells in the hopes that it would help me detect these cells more easily. Sadly, it seemed no matter what I tried, nothing worked.
After over a year of working on this project, I came across an article entitled “Dynamic interactions between bacteria and immune cells leading to intestinal Iga synthesis” by Tsuji et al. that suggested my focus had been misplaced. I had been trying to detect IgA-secreting B-1 B cells in the intestines, but this paper stated that both B-2 and B-1 B cells no longer migrate to the intestines in the LtBr knockout mice I had used for all my experiments.
This knowledge was not only depressing, but it also left me once again without a reliable means of detecting and testing B-1 B cells. At present, without any other feasible alternative solutions to my dilemma, I have moved on to other, hopefully more promising, projects. Despite the setbacks and sad outcome, I learned a lot about the research process and developed my own determination, creativity, and comprehension of scientific articles as I tried to find ways to make my project work. I also feel that this experience marked an excellent stepping stone for future, more successful, projects.
References
- Hieshima K, Kawaski Y, Hanamoto H, Nakayama T, Nagakubo D, Kanamaru A, Yoshie O. CC Chemokine Ligands 25 and 28 Play Essential Roles in Intestinal Extravasation of IgA Antibody-Secreting Cells. J Immunol. 2004 Sep 15;173(6):3668-75.
- Tsuji M, Suzuki K, Kinoshita K, Fagarasan S. Dynamic interactions between bacteria and immune cells leading to intestinal IgA synthesis. Seminars in Immunology 2008 Feb;20(1):59-66.