Analysis of CCR7 mediated T cell transfectant migration using a microfluidic gradient generator

Abstract

a b s t r a c t T lymphocyte migration is crucial for adaptive immunity. Manipulation of signaling molecules controlling cell migration combined with in vitro cell migration analysis provides a powerful research approach. Microfluidic devices, which can precisely configure chemoattractant gradients and allow quantitative single cell analysis, have been increasingly applied to cell migration and chemotaxis studies. However, there are a very limited number of published studies involving microfluidic migration analysis of genetically manipulated immune cells. In this study, we describe a simple microfluidic method for quantitative analysis of T cells expressing transfected chemokine receptors and other cell migration signaling probes. Using this method, we demonstrated chemotaxis of Jurkat transfectants expressing wild type or C terminus mutated CCR7 within a gradient of chemokine CCL19, and characterized the difference in transfectant migration mediated by wild type and mutant CCR7. The EGFP tagged CCR7 allows identification of CCR7 expressing transfectants in cell migration analysis and microscopy assessment of CCR7 dynamics. Collectively, our study demonstrated the effective use of the microfluidic method for studying CCR7 mediated T cell transfectant migration. We envision this developed method will provide a useful platform to functionally test various signaling mechanisms at the cell migration level.

Chemotactic Responses of Jurkat Cells in Microfluidic Flow-Free Gradient Chambers

“…This suggests that CXCL12 saturate chemotactic responses of Jurkat cells at concentrations above 75 nM in a manner similar to the saturation of T cells responses to CCL19 or CCL21 concentrations above 100 nM [59,60]. Also, using a different microfluidic system, Jurkat cells were shown to have significantly reduced chemotactic response to a CCL19 gradient when the CCL19 concentration was reduced from 100 nM to 10 nM while preserving their average migration speed [61] similar to our chemotaxis results. Overall, these observations and our results suggest that both the concentration gradient and the average concentration of the chemokines directs the chemotactic behavior of different immune cells and that both Jurkat cells and T cells share saturable motility responses to chemotactic cues albeit to different chemokines.…”

Live-cell microscopy reveals that human T cells primarily respond chemokinetically within a CCL19 gradient that induces chemotaxis in dendritic cells

“…The data presented in this study showed that human polyclonal T cells, unlike mDCs, respond to a CCL19 gradient largely chemokinetic, showing a random walk with directional bias and showing similarities to the response to a uniform CCL19 concentration. Previously published data showed that the human Jurkat T cell line, transfected with CCR7 to respond to CCL19, responded chemotacticly to a 100 nM CCL19 gradient under experimental flow conditions (Wu et al 2015). However, the authors of this study used a fibronectin-coated microfluidic device, and it has been reported that T lymphocytes can orientate their migration based on the direction of fluid flow during integrin-mediated migration (Valignat et al 2013).…”

Live-Cell Microscopy Reveals That Human T Cells Primarily Respond Chemokinetically Within a CCL19 Gradient That Induces Chemotaxis in Dendritic Cells

“…The data presented in this study showed that human polyclonal T cells, unlike mDCs, respond to a CCL19 gradient largely chemokinetically, showing a random walk with directional bias, and showing similarities to the response to a uniform CCL19 concentration. Previously published data showed that the human Jurkat T cell line, transfected with CCR7 to respond to CCL19, responded chemotacticly to a 100 nM CCL19 gradient under experimental flow conditions (19). However, the authors of this study used a fibronectin-coated microfluidic device, and it has been reported that T lymphocytes can orientate their migration based on the direction of fluid flow during integrinmediated migration (20).…”