The lymph node is a highly organized and dynamic structure that is critical for facilitating the intercellular interactions that constitute adaptive immunity. Most ex vivo studies of the lymph node begin by reducing it to a cell suspension, thus losing the spatial organization, or fixing it, thus losing the ability to make repeated measurements. Live murine lymph node tissue slices offer the potential to retain spatial complexity and dynamic accessibility, but their viability, level of immune activation, and retention of antigen-specific functions have not been validated. Here we systematically characterized live murine lymph node slices as a platform to study immunity. Live lymph node slices maintained the expected spatial organization and cell populations while reflecting the 3D spatial complexity of the organ. Slices collected under optimized conditions were comparable to cell suspensions in terms of both 24-h viability and inflammation. Slices responded to T cell receptor cross-linking with increased surface marker expression and cytokine secretion, in some cases more strongly than matched lymphocyte cultures. Furthermore, slices processed protein antigens, and slices from vaccinated animals responded to ex vivo challenge with antigen-specific cytokine secretion. In summary, lymph node slices provide a versatile platform to investigate immune functions in spatially organized tissue, enabling well-defined stimulation, time-course analysis, and parallel read-outs.

A major hub of adaptive immunity is the lymph node, which has a highly organized and dynamic structure. Most techniques currently used to study the lymph node ex vivo begin by reducing the tissue to a cell suspension, thus losing the spatial organization, or fixing the tissue, thus losing the ability to make measurements over time or after stimulation. Live tissue slices offer the potential to retain spatial complexity and provide the dynamic accessibility of traditional in vitro measurements, but this approach has not been rigorously validated for murine lymph node tissue. Here we describe a systematic characterization of live murine lymph node slices as a platform to study immunity. Live lymph node slices maintained the expected spatial organization and were comprised of the expected cell populations. Slices collected under optimized conditions were comparable to cell suspensions both in terms of 24-hr viability and inflammation. Slices processed protein antigens and responded to T cell receptor cross-linking with expected surface marker expression and cytokine secretion. Interestingly, IFN-γ but not IL-2 secretion from slices was higher than from cell suspensions after both CD3 and R848 stimulation. Furthermore, slices from vaccinated animals responded to ex vivo antigen challenge with antigen-specific cytokine secretion. In summary, lymph node slices provide an experimental platform that maintains spatial organization and temporal dynamics, while allowing for controlled stimulus-response experiments with multiple parallel read-outs. We anticipate that tissue slices may serve as a versatile tool to investigate immune functions within the lymph node.

Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the use of live ex vivo LN slices to study the dynamics of adaptive immunity. However, when working with reactive lymph nodes from vaccinated animals, the tissues frequently became dislodged from the supportive agarose matrix during slicing, leading to damage that prevented downstream analysis.Because reactive lymph nodes expand into the surrounding adipose tissue, we hypothesized that dislodging was a result of excess lipids on the collagen capsule of the LN, and that a brief wash with a mild detergent would improve LN interaction with the agarose without damaging tissue viability or function. Therefore, we tested the use of digitonin on improving slicing of vaccinated LNs. Prior to embedding, LNs were quickly dipped into a digitonin solution and washed in saline.Lipid droplets were visibly removed by this procedure. A digitonin wash step prior to slicing significantly reduced the loss of LN during slicing from 13 -75 % to 0 -25 %, without substantial impact on viability. Capture of fluorescent microparticles, uptake and processing of protein antigen, and cytokine secretion in response to a vaccine adjuvant, R848, were all unaffected by the detergent wash. This novel approach will enable ex vivo analysis of the generation of adaptive immune response in LNs in response to vaccinations and other immunotherapies.