Precision-cut lung slices (PCLS) are a novel tool to study cells of the lower airways. As PCLS retain the integrity and architecture of the lung, they constitute a robust model for studying the cells of the lower respiratory tract. Use of PCLS for imaging has been previously documented; however, other applications and techniques can also be applied to PCLS to increase their use and therefore decrease the number of animals needed for each experiment. We present a detailed protocol for generating PCLS from the murine lung. We show that cultured PCLS remain viable up to at least 8 days of culture, that RNA can be isolated from the tissue, and that flow cytometry can be carried out on the cells obtained from the PCLS. Furthermore, we demonstrate that cytokines and chemokines can be detected in the culture supernatants of PCLS exposed to viruses. Overall, these protocols expand the use of PCLS, especially for infection studies. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Precision-cut lung slices (PCLS) Basic Protocol 2: PCLS culture and viability Basic Protocol 3: RNA isolation from PCLS, cDNA conversion, and RT-qPCR Basic Protocol 4: Staining of cells from PCLS for flow cytometry Basic Protocol 5: In vivo RSV administration and ex vivo PCLS RSV exposure.
© 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.

Foxp3+ T regulatory (Treg) cells promote immunological tumor tolerance, but how their immune-suppressive function is regulated in the tumor microenvironment (TME) remains unknown. Here, we used intravital microscopy to characterize the cellular interactions that provide tumor-infiltrating Treg cells with critical activation signals. We found that the polyclonal Treg cell repertoire is pre-enriched to recognize antigens presented by tumor-associated conventional dendritic cells (cDCs). Unstable cDC contacts sufficed to sustain Treg cell function, whereas T helper cells were activated during stable interactions. Contact instability resulted from CTLA-4-dependent downregulation of co-stimulatory B7-family proteins on cDCs, mediated by Treg cells themselves. CTLA-4-blockade triggered CD28-dependent Treg cell hyper-proliferation in the TME, and concomitant Treg cell inactivation was required to achieve tumor rejection. Therefore, Treg cells self-regulate through a CTLA-4- and CD28-dependent feedback loop that adjusts their population size to the amount of local co-stimulation. Its disruption through CTLA-4-blockade may off-set therapeutic benefits in cancer patients.
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