Application Focus - Modeling Dendritic Cell and T-Cell Interactions in a 3D Environment
By George Kamphaus, Ph. D.

T-Cell and Dendritic Cell InteractionsThis blog describes a novel system for the study of T-cell activation by antigen-presenting dendritic cells (DC) and the use of a BTX ECM 830 electroporator to transfect quiescent (naïve or memory) T-cells. While there are many T-cell therapies that use genetically modified or transfected T-cells, in nearly all cases, the T-cells are activated before transfection. Such methods preclude the study of the physiologic mechanisms of activation by DC cells. To increase the understanding of the intercellular dynamics of quiescent CD8+ T-cell interaction with DC and CD-4+ helper cells, Abu Shah, et. al. have created a 3-D system that reconstitutes a tissue-like environment that allows for the integration of cytokines, DC and quiescent T-cells. Utilizing electroporation to transfect mRNA constructs of modified antigen-specific TCRs, the researchers were able to express functional TCR on the surface of the quiescent T-cells and show activation by interaction with DCs loaded with the corresponding peptide antigen.

First, a brief description of the electroporation protocol:
  • Harvest and wash T-cells three times with Opti-MEM (Thermo Fisher Scientific). Resuspend cells at 25 x 106 cells/ml.
  • Aliquot 100 to 200 µl (2.5 to 5 x 106 cells) into separate tubes and mix with the desired mRNA products.
  • For 106 CD8 T-cells, use 2 µg of each TCRα, TCRβ and CD3ζ RNA. For 106 CD4 T-cells, use 4 µg of TCRα, TCRβ RNA.
  • Add mixture to electroporation cuvette (Cuvette Plus, 2 mm gap, BTX).
  • Electroporation Parameters for the BTX ECM 830: 1 pulse (square wave) at 300 V, 2 ms pulse duration.
  • Collect cells from the cuvette and culture in 1 ml of pre-warmed media.
The in-situ system has a number of features that allow for many variables to be studied:
  • Labeling with different fluorescent markers allows different cell types to be monitored simultaneously by time-lapse video. In this study the motility of CD4+ and CD8+ T-cells and DC were all monitored together in a single system under identical conditions.
  • Different chemokines can be added to the matrix.
  • T-cells can be added before or after solidification of the collagen matrix to study varying kinetics of the DC / T-cell presentation.
  • The effect of TCR-antigen affinity can be studied by expression of various TCR in quiescent T-cells.
  • This can be used as a model system for immune-modulation therapies, such as PD-1 checkpoint inhibitors.
  • Because cells can be easily extracted after observation, cell surface markers and other characteristics can be studied by FACS or other flow cytometry methods.


This paper produced numerous important findings, including:
  • Using the BTX ECM 830 square wave electroporator, the efficiency of TCR expression varied among donors with an average value of 81 ± 7% (mean ± SD, n = 13), with more than 90% cell viability and 80–90% cell recovery, both of which were severely reduced using alternative electroporation approaches (Amaxa and Neon).
  • The induction of expression of an exogenous TCR in CD4 T-cells is considerably harder than in CD8 T-cells (Dai et al., 2009). Using natural sequences or the introduction of cysteine modifications used for 1G4 and 868 failed to induce expression of TCRs in naïve CD4 T-cells.
  • Cell motility of both CD4+ and CD8+ T-cells was faster than DC, either with or without homeostatic chemokines.
  • While both high affinity and low affinity peptide-TCR interactions were able to activate CD8+ T-cells, as evidenced by CD69 and CD25 expression, only T-cells with high-affinity TCR-antigen interactions showed arrested motility.
  • Two different anti-PD-1 antibodies enhanced the clustering of CD-8 T-cells around their targets, while not changing the motility dynamics.
  • Using the BTX ECM 830 square wave electroporator, the cells maintained the motile behavior and interaction dynamics of naïve CD8 T-cells expressing 1G4, at similar levels to those of untouched cells, on 2D stimulatory ‘spots.'

Click here to visit our Protocol Database, for electroporation protocols searchable by, system, cell/tissue type, application/transfectant, and citation.