Matthew Greer and Dr. Greg Burton, Department of Chemistry and Biochemistry
I received an ORCA grant for a project entitled “Reactivation of latent HIV in CD4+ T cells through FDC costimulation.” The original hypothesis of this research was that the factor FDCs (follicular dendritic cells) produce to reactivate latent HIV in T cells is a protein signaling molecule that can be identified through analyzing the proteins FDCs produce. While this project is still proceeding, my resources during the last year since I received an ORCA grant have been dedicated to ironing out a few preliminary wrinkles to this project. Specifically, I have troubleshot and optimized the method our lab group uses to isolate follicular dendritic cells.
FDCs are found in the follicles of lymph nodes such as the tonsils and spleen and participate in a wide range of immune activity. Of particular interest to our group is FDC activity in the presence of an HIV infection. Not only do these cells trap and harbor HIV particles, but they also produce signaling molecules to direct HIV pathogenesis in other cells, especially in CD4+ T cells. While FDCs present a promising avenue for research in both viral activity and immunological system function, such research is not without serious challenges. FDCs contain octopus-like dendrites that form a network in the core of lymph nodes. In order to separate FDCs from all of the other cells, lymph nodes1 must be physically and enzymatically digested, separated on a gradient, labeled with antibodies, and finally sorted on a flow cytometer—an instrument that reports florescent activity associated with the antibodies and separates cells with those attached antibodies from all other cells.
When I started my ORCA project, the members of our lab who had experience sorting FDCs finished their degrees and we struggled to reproduce their procedures. In the process of recreating their experiments, we found many necessary controls to ensure optimal cell isolation were forgotten or neglected. I researched additional ways we could control for optimal sorting purity by labeling FDCs with two antibodies instead of one and keeping controls of cells that do not have any antibodies attached or just the primary2 or secondary3 antibody attached. I also hypothesized that much of the difficulty we experienced in the final steps in the process was due to a lack of care or thought in the initial steps of cutting up tonsils and running the digested tonsils over a separation gradient.
Even though our initial results were discouraging and inconsistent, we found a way to optimize our FDC sorting procedure. Many of the tonsils were not completely cut up in the initial steps which inhibited enzymatic digestions in the next steps and clogged up filters and gradients in latter steps. To remedy this, we instructed all in the lab to take better care in cutting up tonsils so that the resulting tissue could be passaged through a medium sized pipet tip. I also experimented with different gradients to separate out the cells. Because FDCs have a different size and weight than most other cells in tonsils, the digested tonsil can be run over a sugar gradient that will separate out cells based on their size. Multiple sugars and protocols exist for these gradients, but we found a procedure that had been nearly abandoned in our lab that involves creating a continuous gradient in an ultracentrifuge gave the best separation of FDCs, although this particular procedure was more difficult to perform than others.
To ensure the primary antibody we were using was binding to the FDCs and not anything else, which is often the case in many antibody binding reactions, we began using a different antibody that binds to a separate spot on FDCs than the first antibody we were using does. I tested the identity and purity of this second antibody by performing an immunoblot of this antibody, a procedure used to determine binding specificity and presence of a protein in a sample. By using these two antibodies, we were able to then obtain a dual report on flow cytometer with two separate secondary antibodies with two distinct florophores, or florescent molecules. This served as a double positive control to select only those cells that contained both distinct markers recognized by two distinct primary antibodies.
A final control we instituted was an assay to determine the activity of sorted FDCs post flow cytometry sorting. FDCs produce a soluble factor that causes T cells to proliferate and divide much faster than they normally would when cultured by themselves. Therefore, we can culture FDCs with T cells and measure thymidine incorporation for a quantitative measure of the success of our FDC isolation. Thymidine is a molecule that cells use to synthesize new DNA and so if radioactive thymidine is inserted into T cell culture, a simple measure of radioactivity in cells twenty-four hours post culture can give a measure of how successful our FDC isolation was. We can now measure at least a two-fold increase in thymidine incorporation in T cells when our isolated FDCs are present, indicative of a successful isolation procedure.
My original project is now beginning to gain more steam now that our FDC isolation problems have been solved. However, lest I begin to think research will be smooth and easy, the mass spectrometer I proposed to use for this experiment has encountered more than its fair share of technical difficulties and will be out of commission for the next month. Together these experiences have taught me many lessons in patience and diligence as well as the rewards of research. Although I did not reach my final goal in the allotted time for the ORCA grant, those funds gave me priceless insight into the proper way to troubleshoot and overcome unforeseen obstacles in a research setting. This grant also gave me the opportunity to share my gained insight into FDC sorting with my coworkers in weekly lab meetings and will undoubtedly open the doors for future scientific publications on the activity of FDCs in the presence of HIV.