It is the prevailing view that adaptive immune responses are initiated in secondary lymphoid organs. Studies using alymphoplastic mice have shown that secondary lymphoid organs are essential to initiate allograft rejection of skin, heart, and small bowel. The high immunogenicity of lungs is well recognized and allograft rejection remains a major contributing factor to poor outcomes after lung transplantation. We show in this study that alloreactive T cells are initially primed within lung allografts and not in secondary lymphoid organs following transplantation. In contrast to other organs, lungs are acutely rejected in the absence of secondary lymphoid organs. Two-photon microscopy revealed that recipient T cells cluster predominantly around lung-resident, donor-derived CD11c+ cells early after engraftment. These findings demonstrate for the first time that alloimmune responses following lung transplantation are initiated in the graft itself and therefore identify a novel, potentially clinically relevant mechanism of lung allograft rejection.

The behavior of antigen-specific CD4+ T lymphocytes during initial exposure to antigen probably influences their decision to become primed or tolerized, but this has not been examined directly in vivo. We have therefore tracked such cells in real time, in situ during the induction of oral priming versus oral tolerance. There were marked contrasts with respect to rate and type of movement and clustering between naive T cells and those exposed to antigen in immunogenic or tolerogenic forms. However, the major difference when comparing tolerized and primed T cells was that the latter formed larger and longer-lived clusters within mucosal and peripheral lymph nodes. This is the first comparison of the behavior of antigen-specific CD4+ T cells in situ in mucosal and systemic lymphoid tissues during the induction of priming versus tolerance in a physiologically relevant model in vivo.

Current optical microscope objectives of low magnification have low numerical aperture and therefore have too little depth resolution and discrimination to perform well in confocal and nonlinear microscopy. This is a serious limitation in important areas, including the phenotypic screening of human genes in transgenic mice by study of embryos undergoing advanced organogenesis. We have built an optical lens system for 3D imaging of objects up to 6 mm wide and 3 mm thick with depth resolution of only a few microns instead of the tens of microns currently attained, allowing sub-cellular detail to be resolved throughout the volume. We present this lens, called the Mesolens, with performance data and images from biological specimens including confocal images of whole fixed and intact fluorescently-stained 12.5-day old mouse embryos.DOI: http://dx.doi.org/10.7554/eLife.18659.001

Exocytosis of specialized endothelial cell secretory organelles, Weibel-Palade bodies (WPBs), is thought to play an important role in regulating hemostasis and intravascular inflammation. The major WPB core proteins are Von Willebrand factor (VWF) and its propolypeptide (Proregion), constituting more than 95% of the content. Although the composition of the WPBs can be fine-tuned to include cytokines and chemokines (eg, interleukin-8 [IL-8] and eotaxin-3), it is generally assumed that WPB exocytosis is inextricably associated with secretion of VWF. Here we show that WPBs can undergo a form of exocyto-sis during which VWF and Proregion are retained while smaller molecules, such as IL-8, are released. Imaging individual WPBs containing fluorescent cargo molecules revealed that during weak stimulation approximately 25% of fusion events result in a failure to release VWF or Prore-gion. The WPB membrane protein P-selectin was also retained; however, the membrane tetraspannin CD63 was released. Accumulation or exclusion of ex-tracellular fluorescent dextran molecules ranging from 3 kDa to 2 mDa show that these events arise due to the formation of a fusion pore approximately 12 nm in diameter. The pore behaves as a molecular filter, allowing selective release of WPB core and membrane proteins. WPB exocytosis is not inextricably associated with secretion of VWF. (Blood. 2008;111: 5282-5290)

Endothelial cells are reported to contain several distinct populations of regulated secretory organelles, including Weibel-Palade bodies (WPBs), the tissue plasminogen activator (tPA) organelle, and the type-2 chemokine-containing organelle. We show that the tPA and type-2 organelles in human endothelial cells represent a single compartment primarily responsible for unstimulated secretion of tPA or, in cells exposed to interleukin-1␤ (IL-1␤), the cytokines IL-8, IL-6, monocyte chemoattractant protein-1 (MCP-1), and growth-regulated oncogene-␣ (GRO-␣). This compartment was distinct from WPBs in that it lacked detectable von Willebrand factor, P-selectin, Rab27a, or CD63 immunoreactivity, displayed no time-dependent decrease in intragranule pH, underwent detectable unstimulated exocytosis, and was very poorly responsive to Ca 2؉ -elevating secretagogues. WPBs could also contain tPA, and in IL-1␤-treated cells, IL-8, IL-6, MCP-1, and GRO-␣, and were the primary source for histamine or ionomycin-stimulated secretion of these molecules. However, analysis of the storage efficiency of cytokines and tPA revealed that all were very poorly stored compared with von Willebrand factor. The nonmammalian, nonsecretory protein EGFP, when expressed in the secretory pathway, also entered WPBs and had a storage efficiency similar to tPA and the cytokines tested. Based on these data, we proposed a revised model for storage and secretion of cytokines and tPA. (Blood. 2010;116(12):2183-2191) IntroductionSeveral studies suggest that endothelial cells (ECs) possess several distinct populations of regulated secretory organelles (RSOs) in which different subsets of bioactive peptides and proteins are stored, trafficked, and rapidly secreted in response to physiologic stimuli. These include (1) Weibel-Palade bodies (WPBs) whose major cargo protein is von Willebrand factor (VWF) 1 ; (2) a small punctate organelle, morphologically distinct from WPBs, lacking endogenous VWF immunoreactivity but containing the anticoagulant protein tissue plasminogen activator (tPA; the tPA organelle) 2-4 ; and (3) a small punctate organelle, reported to specifically contain the small chemotactic cytokines growthregulated oncogene-␣ (GRO-␣) and monocyte chemoattractant protein 1 (MCP-1) and termed the type-2 granule. 5 The presence of distinct populations of RSOs within the same cell is not uncommon 6-8 and may allow the stimulated release of diverse bioactive molecules to be differentially controlled. If distinct populations of RSOs do exist in ECs, then a careful examination of their properties would provide insights into how trafficking and secretion of specific groups of bioactive molecules are regulated.WPBs are the best characterized RSO of ECs. After their formation at the trans-Golgi network (TGN), WPBs accumulate in the cytoplasm and can remain within the cell for long periods of time (1-2 days). 9,10 By these criteria, we define WPBs as true storage organelles. Under resting conditions, WPBs undergo a very slow process of basal exocytosis, 10 undete...