title: Imaging the immunological synapse between dendritic cells and T cells.
abstract: Immunological synapse formation between antigen-specific T cells and antigen presenting cells (APC) involves reorganization of the cellular cytoskeleton (polymerization of filamentous actin) and recruitment of adhesion molecules (e.g. LFA-1, ICAM-1). This engagement is critical for the generation of specific immune responses. Until recently, quantitative, high-throughput measurements of these interactions have not been possible. Instead, previous assessment was reliant on qualitative microscopy of live cells, where typically the APC is adhered to a surface and the suspended T cell is required to migrate to facilitate synapse formation. While this methodology can demonstrate the capacity for synapse formation, it cannot accommodate quantification of large numbers of interacting cell pairs, nor does it allow for statistically robust comparison between test conditions. We have developed a method for assessing immunological synapse formation between purified ex vivo dendritic cells (DCs) and responder antigen-specific CD4(+) T cells using imaging flow cytometry, allowing us to quantify LFA-1 and f-actin rearrangement at the interface between DC/T cell pairs. This novel application of imaging flow cytometry represents a major advance in dendritic cell function and immunological synapse research as it facilitates quantitative, high throughput analysis of the interaction between live, ex vivo DC and T cells.
authors.affiliation: QIMR-Berghofer Medical Research Institute, Brisbane, QLD, Australia; Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia. Electronic address: kate.markey@qimr.edu.au.
authors.forename: Kate A
authors.initials: KA
authors.lastname: Markey
authors.affiliation: QIMR-Berghofer Medical Research Institute, Brisbane, QLD, Australia.
authors.forename: Kate H
authors.initials: KH
authors.lastname: Gartlan
authors.affiliation: QIMR-Berghofer Medical Research Institute, Brisbane, QLD, Australia.
authors.forename: Rachel D
authors.initials: RD
authors.lastname: Kuns
authors.affiliation: QIMR-Berghofer Medical Research Institute, Brisbane, QLD, Australia.
authors.forename: Kelli P A
authors.initials: KP
authors.lastname: MacDonald
authors.affiliation: QIMR-Berghofer Medical Research Institute, Brisbane, QLD, Australia; Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia.
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Hill
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D000938:Antigen-Presenting Cells / Q000166:cytology / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002450:Cell Communication / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D019044:Image Cytometry / Q000379:methods; D054992:Immunological Synapses / Q000378:metabolism / Q000502:physiology*; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008213:Lymphocyte Activation / Q000502:physiology; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL
chemical_list: D000199:Actins; D015815:Cell Adhesion Molecules; D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1
keywords: Amnis ImageStream; Imaging flow cytometry; LFA-1; Phalloidin; Transgenic T cell; dendritic cell