In Vivo Imaging of T Cell Immunological Synapses and Kinapses in Lymph Nodes

Abstract

T cells can become activated in lymph nodes following a diverse set of interactions with antigen-presenting cells. These cellular contacts range from short and dynamic to stable and long-lasting interactions, termed kinapses and synapses, respectively. Here, we describe a methodology to generate naïve T cells expressing a fluorescent probe of interest through the generation of bone marrow chimeras and to image T cell dynamics using intravital two-photon microscopy. In these settings, the formation of kinapses and synapses can be triggered by the administration of low and high affinity peptides, respectively. Finally, 3D cell tracking can help classify distinct T cell behaviors. These approaches should offer new possibilities for dissecting the process of T cell activation in vivo.

Termination of T cell priming relies on a phase of unresponsiveness promoting disengagement from APCs and T cell division

“…At different times after peptide injection (3, 24, 30, 48, and 72) lymph nodes were harvested and digested in RPMI with 1 mg/ml collagenase and 0.1 mg/ml DNase (Sigma) for 20 min at 37°C. Alternatively, mice were anesthetized and the popliteal lymph nodes prepared for intravital imaging as previously described ( Moreau and Bousso, 2017 ). In some experiments, a second cohort of OT-I CD8 + T cells labeled with 2.5 µM SNARF (Invitrogen) was adoptively transferred into mice by i.v.…”

Multi-Compartment Lymph-Node-on-a-Chip Enables Measurement of Immune Cell Motility in Response to Drugs

“…Moreover, the ability of B and T cells to move among strategic locations in the LN is critical to achieve a fully humoral response [ 31 ]. Two-photon laser microscopy has allowed to characterize B and T cell movement within the LN and has shown that lymphocytes move by a random walk [ 32 , 33 ]. In order to address whether the LN-on-a-chip supports cellular motility we analyzed in real time the movement of T and B immune cells by time-lapse microscopy.…”

“…ton laser microscopy has allowed to characterize B and T cell movement w and has shown that lymphocytes move by a random walk [32,33]. In orde whether the LN-on-a-chip supports cellular motility we analyzed in real tim ment of T and B immune cells by time-lapse microscopy.…”

“…Lymphocytes move for searching and presenting antigens, for interacting with other cells and for proliferating. Inside the lymph node, naïve T cells have been described to move by a random walk behaving as sentinels looking for foreign antigens [ 32 , 33 ]. We selected Jurkat T and Raji B cells because they are, respectively, classical T and B cellular models in immunology, extensively used during the last 20 years to characterize the biology and function of T and B lymphocytes [ 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ].…”