Dendritic cells trigger an acquired immune response against pathogens and tumor cells
Dendritic cells are antigen-presenting immune cells that actively search the tissue for pathogens. For example, once they have detected and ingested viruses or bacteria, they become highly mobile and express the chemokine receptor CCR7 in their cell membrane, which specifically recognizes the two chemokines CCL19 and CCL21. The two attractants trigger a directed migratory movement, called chemotaxis, in the dendritic cells and lead them through the tissue to the lymphatic vessels and finally to the nearest lymph node. Once there, the dendritic cells launch an acquired immune response by presenting fragments (antigens) of the previously ingested pathogen to other immune cells, the T cells, on their surface.
Coordinated cell migration plays a central role in specific immune defense against pathogens or cancer. Different signals leading to immune cell activation and migration determine the efficacy of the triggered immune response. We investigate the molecular mechanisms by which different immune cells recognize messenger molecules in order to be able to migrate purposefully.
To analyze the migratory properties of CCR7 expressing cells in space and time in depth microscopically in real time, we use a 3D collagen matrix migration assay. With this method, we analyze the migration of cells in the direction of increasing CCL19 or CCL21 concentrations in a physiological three-dimensional network of collagen fibers. Thanks to the availability of self-produced fluorescent chemokines, we observed that in this system CCL19 and CCL21 build different shapes of chemokine gradients (Figure 1), which correlated with the chemotactic efficiency of human dendritic cells. Immediately after addition of red fluorescent CCL19-S6Dy549P1 to the reservoir adjacent to the collagen matrix containing embedded dendritic cells, the fluorescent signal was very high, decreased rapidly over the first hundred micrometers of the 3D collagen matrix, and extended throughout the matrix during the measurement period. In contrast, CCL21-S6Dy549P1 was only able to penetrate the matrix over a short distance during the first 300 minutes and the fluorescent signal decreased steeply with increasing distance from the chemokine reservoir.
Figure 1: Different shapes of CCL19 and CCL21 chemokine gradients. (A) Microscopy images of a section of the 3D collagen matrix chambers for observation of migrating human dendritic cells. Bright field (BF) microscopy image and fluorescence images without addition of chemokine (cell medium only) and with addition of fluorescent (S6Dy549P1 coupled) chemokine, respectively, at the time of application (t=0 min; chemokine source at the top of the image) and 180 min later after distribution of the molecules in the gel matrix. The distance across the gel matrix is 1 mm. (B-C) Distribution of S6Dy549P1 coupled CCL19 and CCL21 molecules, respectively, in the collagen matrix chambers. The mean fluorescence intensity (MFI) of chemokines diffusing into the matrix was determined microscopically in the 3D matrix for 5 hours and colored using a "one-phase decay model": blue (t = 0min) to red (t = 300min). [Figure modified from Artinger, M. et al. (2022) Front Immunol, open-access article (CC BY)].
An important goal of our research is to discover new molecules and signaling pathways to specifically alter chemotaxis and the activation of immune cells. For example, we have discovered that the inflammatory mediator prostaglandin E2 plays a key role in the activation of dendritic cells and exerts a decisive influence on their chemotaxis and the success of the specific immune response.
In the course of the last years, we have developed for the first time a human dendritic cell line migrating against CCL19 (Video 1) and CCL21 as well as a model for the production of immortalized bone marrow-derived dendritic cell progenitors (BMDCs). These cells can be easily genetically manipulated, in contrast to primary cell cultures, which are isolated and grown directly from an organ or tissue. Thus, we are now able to observe spatio-temporal signal transduction pathways not only in established cancer cell lines but also in migrating dendritic cells thanks to a series of fluorescent biosensors or signaling proteins developed in our laboratory.
Video 1: Detection of the accumulation of the green fluorescent PIP3 biosensor PH-Akt-GFP at the cell front of migrating CAL-1 cells. Gene-manipulated CAL-1 cells stably expressing PH-Akt-GFP were differentiated with GM-CSF for three days and subsequently matured with the synthetic toll-like receptor TLR7 ligand resiquimod (R848) for 18 hours. The migration of cells in a CCL19 gradient in the 3D collagen matrix migration assay was imaged by confocal microscopy. The chemokine source is located at the right side of the video. Time-lapse confocal microscopy video (duration: 16min.)
Migration of cancer cells and formation of metastases
Certain types of cancer cells express CCR7, which is otherwise naturally present only on immune cells. Like immune cells, they recognize the chemokines CCL19 and CCL21 and thus also migrate to the lymph nodes and other organs of the lymphatic system, where they can form metastases.
We are developing modern and highly precise methods to study cellular events that occur after CCL19 or CCL21 binding to CCR7. Using these methods, we aim to determine which signaling molecules lead to the expression of CCR7, how a gradient is formed along which cells move to the site of highest chemokine concentration, and how cell migration is regulated. In particular, we are looking for similarities and differences in the migratory behavior of cancer and immune cells, which should help to develop new immunological therapeutic approaches to prevent CCR7-mediated metastasis of tumor cells.
