(J-374) Identifying Activation Status of Lipopolysaccharide Stimulated Mouse Bone Marrow-Derived Dendritic Cells through 8 Color Flow Cytometry.

Introduction: Bone marrow (BM) derived dendritic cells (BMDCs) are noted due to their unique ability to produce an immune response by activating native T cells, making them a useful tool in immunology. Due to the difficulty of harvesting isolated dendritic cells (DCs) from tissues, protocols have been generated to allow the differentiation of DCs through primary culture. With the inclusion of GM-CSF and IL-4 growth factors within the culture media, a population of DCs and macrophages are able to be differentiated. Afterward, the DCs are available to be administered LPS treatment and outcomes can be observed through fluorescence assays such as flow cytometry (FC). Herein, we harvested BM cells from a mouse and obtained BMDCs over a 14-day culturing period. The cells were then treated with 200 ng/mL of lipopolysaccharide (LPS) and activation status was observed through 8-color FC. We hypothesized that the LPS-treated cell population would have increased antibody expression.

Materials and Methods: Mice femurs, tibias, and humerus are harvested from the mouse and the bone marrow is extracted using a centrifuge. The cells are then suspended in complete growth media with GM-CFS and IL4 growth factors to begin the differentiation process. On day 14, the cells are harvested and purified using EasySept selection cocktails and Votex Rapidsperes, which removes the macrophage population from the DCs. The purified DCs are then resuspended in media, with half the population being treated with 200 ng/mL LPS. After a 24-hour culturing period with the treatment, the cells are harvested and stained with APC-Cy7, CD45, CD11b, CD80, CD4, CD86, CD11c, CD8a, and MHC Class II antibodies and assayed through FC.

Results and Discussions: The cells were stained with live/dead stain to exclude dead cells in our analysis. We also included CD11c to ensure that the cells were indeed dendritic cells. We gated on Singlet Live CD11c+ cells for analysis of activation markers. The two stains observed were CD80 and CD40. CD80 is characterized by its presence in T-Cell activation and regulation of malignant cells and its expression quantified in FC is due to surface receptor activation of the DCs. CD40 is relevant as activated receptors distinguish an immune response and show different activation markers in DC populations. Untreated populations had lower signals of activation markers when observed using FC. When LPS-treated cells were observed, antibody expression went up significantly. CD80 markers, which relate to mature DCs, had higher levels of expression with LPS populations. When CD40 antibody results are quantified, we see the same trend.

Conclusion: LPS-treated cells experienced higher levels of antibody response when observed using FC. When observing CD80, we see that LPS-activated cells have higher levels of antibody expression. This trend is continued with the LPS-treated population stained with the CD40 antibody. This was in agreement with our hypothesis as LPS activates DCs through toll-like receptor engagement and increases the DC’s capacity to present pathogen-derived antigens. The LPS treatment primed the DCs for activation by interacting with the CD14 membrane receptor to increase cytokine generation, and thus, allowing for higher antibody response. BMDCs remain a powerful tool in immunology and their use is still pivotal in early-stage clinical trials.

Acknowledgments: This project was supported by the Washington University in St. Louis biomedical engineering department and the WashU Summer Undergraduate Engineering Fellowship program (WUSEF).

References: Fang R, Ismail N, Soong L, Popov VL, Whitworth T, Bouyer DH, Walker DH. Differential interaction of dendritic cells with Rickettsia conorii: impact on host susceptibility to murine spotted fever rickettsiosis. Infect Immun. 2007 Jun;75(6):3112-23. doi: 10.1128/IAI.00007-07. Epub 2007 Apr 2. PMID: 17403875; PMCID: PMC1932850.