Susanne Linderman with Dr. Eric Wilson, Microbiology and Molecular Biology

IgA is the most common antibody isotype involved in the immune system’s defense of mucosal membranes. In order to migrate and accumulate at these sites, IgA antibody secreting cells rely on chemokines. These chemokines, which are proteins differentially secreted in different tissues, bind to chemokine receptors on the cell’s surface. By discovering which receptors are important for immune cell migration to specific sites, we can gain a better understanding of how the immune system functions. This could eventually lead to better treatment of autoimmune and immunodeficiency diseases, as well as to the development of better vaccines. My goal was to demonstrate whether or not the chemokine receptor CCR9 affects homing, migration, and accumulation of IgA antibody secreting cells (ASCs) to the salivary glands of mice. While CCR10 has been shown to be a very influential receptor in immune cell migration to the sublingual gland, I hypothesized that CCR9 replaces CCR10 as the major receptor in cell migration to the submandibular gland. This would indicate that cell migration, even in very similar tissues like the salivary glands, is a highly regulated and tightly controlled process.

To determine the role of CCR9 in migration of IgA ASCs to the salivary glands, we began by measuring IgA mRNA levels in CCR9 knockout (KO) mice by real time quantitative PCR. While we expected IgA levels in the submandibular gland to decrease due to the lack of CCR9, IgA mRNA levels were actually statistically significantly higher in both the sublingual and submandibular glands (P<0.01) of CCR9 KO mice by a 1 tailed homoscedastic Student’s T test. While this was unexpected, there are several possible reasons for this result. For instance, since CCR9 has been shown to contribute to the migration of a high quantity of IgA ASCs to the small intestine, I hypothesize that IgA levels in the salivary glands increase in CCR9 KO mice because more cells are able to migrate to other chemokines expressed in the salivary glands since less cells migrate to the small intestine.

In addition to finding IgA mRNA level differences in CCR9 KO and wild type (WT) mice, we also found differences between different strains of mice. While previous research on Balb/C strain mice had shown a 12 fold difference in IgA levels between the sublingual and submandibular gland, C57 strain mice showed a significantly reduced difference between glands. The IgA mRNA levels in the C57 sublingual glands were significantly lower in C57 sublingual glands than in Balb/C sublingual glands (P<0.01). To determine if chemokine expression differences were responsible for the differences in IgA expression in these two strains, we also measured mRNA expression of the chemokines CCL28 and CCL25. The only significant difference was found in CCL25 expression in the submandibular gland (P<0.05). To verify these results we used ELISA assays to measure protein levels of IgA, CCL25, and CCL28 but CCL25 was difficult to measure, possibly due to its low concentration in the salivary glands.

Because we found significant strain differences, and because CCR9 KO and CCR10 KO mice were of different strains, we began backcrossing CCR9 KO and CCR9/CCR10 double KO mice onto a common Balb/C background. CCR9 KO mice were originally C57 strain mice. Heterozygous CCR9 KO mice were crossed with Balb/C WT mice 5 times and will be further backcrossed a minimum of 2 more times before heterozygous mice are crossed with each other to produce homozygous CCR9 KO mice. Since CCR10 KO mice were originally Balb/C strain mice, CCR9 +/- CCR10-/- mice were crossed with CCR10 KO mice. These double KO mice have been backcrossed for 6 generations and will be backcrossed a minimum of one more time before producing homozygous KO mice. CCR9/CCR10 KO mice were produced to determine if knocking out CCR10 could reduce the increased migration of IgA ASCs to the salivary glands found in CCR9 KO mice. Mice were genotyped by qPCR analysis using a primer that I designed for the neomycin resistance gene which is inserted in the KO genes. GAPDH expression was used as an endogenous control.

IgA ASCs migrating to the salivary glands may express the receptors CCR9 and CCR10 individually or concurrently. To determine which receptors were expressed by the cells migrating to each salivary gland, we performed several migrations assays in which cells collected from the spleen and the sublingual and submandibular salivary glands were allowed to migrate to CCL25 and CCL28 either individually or concurrently. To determine whether both CCR9 and CCR10 are expressed on IgA ASCs, cells were either migrated a second time to a second chemokine, or cells were “desensitized” by exposing the cells to a second chemokine before migration. In the latter case, it was expected that double positive cells expressing both receptors should not migrate because exposure to a high concentration of chemokine in the top well of the transwell migration chamber would inhibit migration to the chemokine in the bottom well. IgA ASCs which migrated were counted by either ELISPOT or flow cytometry.

Because the concentration of IgA ASCs in the salivary glands was so low, too few cells migrated to generate significant data. We therefore tried purifying the cells by magnetic column separation and by histopaque separation. However, while magnetic column separation increased the concentration of IgA ASCs in the sample, the number of cells migrating was still too low. Histopaque separation was also unsuccessful and I believe that a denser material should be used for separation in the future. Cells were also purified by cell sorting on a BD FacsVantage cell sorter. However, qPCR analysis of GAPDH, CCR9, and CCR10 on sorted cells was only partially successful. I believe that collection of more cells may be necessary, or that a better mRNA isolation protocol may be needed to accurately measure receptor expression on IgA ASCs.

Knowing how cells migrate differentially has promising applications and there are still many questions regarding how and why cells migrate to different tissues. This research, which has been outlined more fully in my honors thesis, has shown that strain differences can complicate this question, and that protocols need to be adjusted for tissues in which cell concentration is low, such as in the salivary gland. Based on migration assays of spleen IgA ASCs, I also believe that exposure of cells to CCL28 or CCL25 immediately before migration does not desensitize them as previously expected. This indicates that cells express only one of these receptors at a time, or that exposure of a cell to one chemokine does not necessarily inhibit migration to another chemokine. Although there were no significant differences between the migration of cells with or without desensitizing chemokines, I believe that a general trend indicates that exposure to two chemokines concurrently may change the migration pattern of cells and that migration to the salivary glands may therefore be even more complex than previously hypothesized.