Alfred G. and Meta A. Ennis Professor University of Pennsylvania, United States
Introduction:: The body’s immune system acts as the defense system against foreign particles and pathogens. Of all the leukocytes involved in the immune system, T cells play a crucial role in adaptive immunity. For T cells to enact their functions, it is crucial for them to be able to move locations where they are needed. An interesting phenomenon where T cells, which are adhered to ICAM-1 molecules, migrate upstream has been widely reported. While prior research in this phenomenon has elucidated which integrin-ligand pair is responsible for the upstream migration, there is very little understanding about the actual mechanism of upstream migration by T cells. By utilizing Traction Force Microscopy (TFM), this research aims to understand and shed light upon how T cells migrate upstream by generating a traction force map. With the result from this research, it would be possible to understand exactly how T cells exert forces to migrate upstream and in turn, better manipulate the motion of T cells. This would provide a whole new avenue to engineer and utilize T cells, which are already being widely used for treatment purposes, such as the CAR T therapy.
Materials and Methods:: Making of the Polyacrylamide gel
Polyacrylamide gel with a known elastic modulus with fluorescent beads embedded in gel is made, with reference to the work by Sarah Hyunji Kim et al. (Sarah Hyunji Kim et al, 2021, Mol Biol Cell). Activated slide supporting the polyacrylamide gel is also made in reference to work by Sarah Kim. The polyacrylamide gel is functionalized with protein A/G (ThermoFisher, 21186) and ICAM-1 Fc chimera from R&D systems (R&D systems, 720-IC).
Cells and Media
KG1a cells used for validation experiments were cultured in IMDM media enriched with 20% v/v FBS. Primary CD4+ T cells used in the experiments were obtained through the Human Immunology Core at the University of Pennsylvania and maintained in RPMI1640 media enriched with 10% v/v FBS.
Cell tracking, Data Analysis and Traction Force Map Generation
The motility of cells was imaged using Nikon TE-300 microscope over 30 minutes. Utilizing Fiji and ImageJ (https://imagej.nih.gov/ij/, NIH, Bethesda, MD), cells were analyzed for their upstream migration behaviors and aspect ratios. Libtrc software developed by Yu-Li Wang and Micah Dembo is utilized to calculate the traction force map with the inputs of the location of the fluorescent beads under stress and at their relaxed state.
Results, Conclusions, and Discussions:: Through the validation experiments using KG1a cells, it has been shown that the functionalization of gel is successful, and the gel can support upstream migration from the cells. By recreating and recapturing the upstream motion with primary T cells, creating traction force maps of T cells in upstream migration would come naturally. The traction force map would help elucidate clearly how T cells migrate upstream. This result would have a lot of physiological significance, especially with how much T cell research is being performed to engineer and manipulate T cells for therapeutic purposes. Furthermore, with the availability of a technique that can study upstream migration in real time, it would open new doors to performing a systematic analysis on the impacts of different integrins and proteins on the ability for a leukocyte to undergo upstream migration. Such research would certainly open new avenues for the understanding and engineering of other leukocytes for the benefit of healthcare and therapeutics.
Acknowledgements (Optional): : This work was supported by National Institutes of Health [GM143357].
References (Optional): : Kim, S. H. J., & Hammer, D. A. (2021). Integrin cross-talk modulates stiffness-independent motility of CD4+ T lymphocytes. Molecular Biology of the Cell, 32(18), 1749–1757. https://doi.org/10.1091/MBC.E21-03-0131
