Inflammasomes are multiprotein innate immune complexes formed in response to infections, tissue damage, or cellular stress that promote the maturation and release of IL-1β/IL-18 and are implicated in lytic cell death. The NLRP3 inflammasome is canonically activated by an initial priming event followed by an activation stimulus, leading to rapid cell death that occurs through caspase-1 (CASP1) and gasdermin D (GSDMD) activation, called pyroptosis. CASP1- and GSDMD-deficient cells are protected from the rapid LPS plus ATP-induced pyroptosis. However, innate immune responses physiologically occur over time, extending beyond minutes to hours and days. Therefore, in this study, we assessed lytic cell death beyond the early timepoints. While cells lacking the innate immune sensor NLRP3 were protected from cell death induced by the canonical NLRP3 trigger, LPS priming and ATP stimulation (LPS plus ATP), for extended time, CASP1- and GSDMD-deficient cells started to lyse in a time-dependent manner after 2 h. Nevertheless, robust IL-1β and IL-18 release was still dependent on CASP1 activation. These data suggested that NLRP3 engages an additional innate immune, lytic cell death pathway. Indeed, LPS plus ATP induced the activation of caspases and RIPKs associated with PANoptosis in WT cells, and cells deficient in PANoptosis machinery were protected from cell death for extended times. A PANoptosome complex containing NLRP3, ASC, CASP8, and RIPK3 was observed by microscopy in WT, as well as CASP1- or GSDMD-deficient, cells by 30 min post-stimulation. Overall, these findings highlight the central role of NLRP3 as a PANoptosome sensor. Given the physiological role of innate immune cell death, PANoptosis, in health and disease, our study emphasizes the importance of a comprehensive understanding of PANoptosomes, and their components, as therapeutic targets.

For decades, the Vibratome served as a standard laboratory resource for sectioning fresh and fixed tissues. In skilled hands, high quality and consistent fresh unfixed tissue sections can be produced using a Vibratome but the sectioning procedure is extremely time consuming. In this study, we conducted a systematic comparison between the Vibratome and a new approach to section fresh unfixed tissues using a Compresstome. We used a Vibratome and a Compresstome to cut fresh unfixed lymphoid and genital non-human primate tissues then used in situ tetramer staining to label virus-specific CD8 T cells and immunofluorescent counter-staining to label B and T cells. We compared the Vibratome and Compresstome in five different sectioning parameters: speed of cutting, chilling capability, specimen stabilization, size of section, and section/staining quality. Overall, the Compresstome and Vibratome both produced high quality sections from unfixed spleen, lymph node, vagina, cervix, and uterus, and subsequent immunofluorescent staining was equivalent. The Compresstome however, offered distinct advantages; producing sections approximately 5 times faster than the Vibratome, cutting tissue sections more easily, and allowing production of larger sections. A Compresstome can be used to generate fresh unfixed primate lymph node, spleen, vagina, cervix and uterus sections, and is superior to a Vibratome in cutting these fresh tissues.

The development of in situ major histocompatibility complex (MHC) tetramer (IST) staining to detect antigen (Ag)-specific T cells in tissues has radically revolutionized our knowledge of the local cellular immune response to viral and bacterial infections, cancers, and autoimmunity. IST combined with immunohistochemistry (IHC) enables determination of the location, abundance, and phenotype of T cells, as well as the characterization of Ag-specific T cells in a 3-dimensional space with respect to neighboring cells and specific tissue locations. In this review, we discuss the history of the development of IST combined with IHC. We describe various methods used for IST staining, including direct and indirect IST and IST performed on fresh, lightly fixed, frozen, and fresh then frozen tissue. We also describe current applications for IST in viral and bacterial infections, cancer, and autoimmunity. IST combined with IHC provides a valuable tool for studying and tracking the Ag-specific T cell immune response in tissues.