DOI: 10.6078/d1st46
title: Immunofluorescence images: Inflammasome activation leads to cDC1-independent cross-priming of CD8 T cells by epithelial cell derived antigen

description: The innate immune system detects pathogens and initiates adaptive immune
 responses. Inflammasomes are central components of the innate immune
 system, but whether inflammasomes provide sufficient signals to activate
 adaptive immunity is unclear. In intestinal epithelial cells (IECs),
 inflammasomes activate a lytic form of cell death called pyroptosis,
 leading to epithelial cell expulsion and the release of cytokines. Here we
 employed a genetic system to show that simultaneous antigen expression and
 inflammasome activation specifically in IECs is sufficient to activate
 CD8+ T cells. By genetic elimination of direct T cell priming by
 IECs, we found that IEC-derived antigens are cross-presented to
 CD8+ T cells. However, activation of CD8+ T cells by
 IEC-derived antigen only partially depended on IEC pyroptosis. In the
 absence of inflammasome activation, cross-priming of CD8+ T cells
 required Batf3+ dendritic cells (cDC1), whereas
 cross-priming in the presence of pyroptosis required
 a Batf3-independent cDC population. These data suggest the
 existence of parallel pyroptosis-dependent and pyroptosis-independent but
 cDC1-dependent pathways for cross-presentation of IEC-derived antigens.
description: Mice were fed a single day pulse of tamoxifen chow and euthanized two days from start of the chow feeding. Approximately 2.5 cm pieces were taken from the proximal and distal ends of the small intestine. These pieces were flushed and fixed in PLP buffer (0.05 M phosphate buffer containing 0.1 M L-lysine [pH 7.4], 2 mg/mL NaIO<sub>4</sub>, and 1% PFA) overnight at 4 °C. The following day, tissues were washed 2x in phosphate buffer and placed in 30% sucrose overnight at 4 °C. Tissue was frozen in Tissue-Tek® OCT (VWR; 25608-930), cut on a Leica cryostat, and sections were placed on Fisherbrand™ Tissue Path Superfrost™ Plus Gold Slides (Fisher Scientific; 15-188-48).  For staining, slides were allowed to warm to room temperature, traced with an ImmEdge Hydrophobic Barrier Pen (Vector Labs; H-4000), washed 3× in 1× PBS with 0.5% Tween-20, and blocked with 10% normal donkey serum (Sigma; D9663) in 0.5% Tween-20, 100 mM TrisHCl [pH 7.5], 150 mM NaCl, 0.5% blocking reagent (Perkin Elmer; FP1020) for 30 minutes. Tissues were then stained with 1:300 GFP polyclonal antibody (Invitrogen; A-6455) overnight at 4 °C. Slides were washed 3X and stained with donkey anti-rabbit Alexa Fluor 488 (Jackson Immunoresearch; 711-545-152) for 60 minutes at RT, followed by 150 nM Acti-stain™ 555 phalloidin (Cytoskeleton, Inc; PHDH1-A) and 100 mM DAPI (D1306) for 30 minutes at RT. Slides were then washed 2X in H<sub>2</sub>0 and sealed under glass coverslips prior to imaging. All antibody dilutions were done in 100 mM TrisHCl [pH 7.5], 150 mM NaCl, 0.5% blocking reagent; all washes were done in 1X PBS with 0.5% Tween-20. Slides were imaged on a Zeiss LSM710 at the CNR Biological Imaging Facility at the University of California, Berkeley.
description: .czi image files can be opened with Fiji (ImageJ)