Immunosurveillance of the Liver by Intravascular Effector CD8 + T Cells

“Priming of adaptive immune responses during infection with intracellular bacteria or viruses results in extensive reprogramming of T cell trafficking so that effector cells can deal with pathogens that are located in peripheral compartments (Masopust et al., 2001).”

“(legend continued on next page) 2 Cell 161, 1–15, April 23, 2015 ª2015 Elsevier Inc. leukocyte trafficking in other organs (von Andrian and Mackay, 2000; Mueller, 2013).”

“…along physical structures—as described for naive T cells migrating along the fibroblastic reticular cell network in the T cell area of lymph nodes (Bajénoff et al., 2006) or for effector T cells migrating along the myeloid scaffold delineating hepatic granulomas (Egen et al., 2008)—or whether…”

“This is particularly relevant for the pathogenesis of viral infections such as those caused by HBV and HCV, noncytopathic pathogens that replicate selec- tively in the hepatocyte and cause acute or chronic liver disease that are triggered by virus-specific CD8 TE (Guidotti and Chisari, 2006).”

“The notion that circulating leukocytes can interact with he- patocytes through endothelial fenestrations has been proposed in previous studies (Ando et al., 1994; Geissmann et al., 2005; Warren et al., 2006).”

“Naive CD8 T cells from these TCR transgenic mice (referred to as Cor93 and Env28 CD8 T cells, respectively) could be differentiated in vitro into bona fide CD8 TE (data not shown) that, when transferred into HBV replication-competent transgenic mice (Guidotti et al., 1995), caused liver disease and inhibited viral rep…”

“…transgenic mice were chronically exposed to low doses of arsenite (Straub et al., 2007), a treatment that reduces liver porosity (i.e., number and size of sinusoidal endothelial cell fenestrae) recapitulating the sinusoidal defenestration that char- acterizes liver fibrosis (Friedman, 2004).”

“In vitro generation of CD8+ T cells (CD8 TE) was performed basically as described (Manjunath et al., 2001).”

“…and chemokines is relatively low in the liver of HBV repli- cation-competent transgenic mice prior to CD8 TE transfer (Figures S1A and S1B), consistent with the uninflamed environment seen in experimentally infected chimpanzees prior to HBV-specific CD8+ T cell arrival (Wieland and Chisari, 2005).”

“, 2008)—or whether noradrenergic neurotransmitters from sympathetic nerves can also modulate intrasinusoidal CD8 TE motility—as described for intrasinusoidal hepatic NKT cells (Wong et al., 2011)—remains to be determined.”

“Notably, platelets have been shown to form intrasinusoidal aggregates on the surface of bacterially infected Kupffer cells, possibly as a pathogen-trapping mechanism promoting immune-mediated clearance (Wong et al., 2013).”

“Understanding of the dynamic events leading to generation and expansion of effector CD8+ T cells (CD8 TE) within lymphoid organs has rapidly improved in recent years, particularly with the use of intravital mi- croscopy (IVM) (Germain et al., 2012).”

“…the T cell area of lymph nodes (Bajénoff et al., 2006) or for effector T cells migrating along the myeloid scaffold delineating hepatic granulomas (Egen et al., 2008)—or whether noradrenergic neu- rotransmitters from sympathetic nerves can also modulate intra- sinusoidal CD8 TE motility—as…”

“, 1995), caused liver disease and inhibited viral replication (data not shown) as much as previously reported polyclonal memory HBV-specific CD8 T cells (Iannacone et al., 2005).”

“The notion that circulating leukocytes can interact with he- patocytes through endothelial fenestrations has been proposed in previous studies (Ando et al., 1994; Geissmann et al., 2005; Warren et al., 2006).”

“Of note, CD44—expressed by CD8 TE (data not shown) and platelets (Figure S2B)—has the capacity to bind to hyaluronan on liver si- nusoids (McDonald et al., 2008).”

“, PSGL-1) expressed also by CD8 TE (Borges et al., 1997).”

“(mGPIbanull;hGPIbaTg mice) (Ware et al., 2000).”

“, 2008) or serotonin (Lang et al., 2008) were shown to be dispensable for these processes.”

“These processes were mediated by the extension of cellular protrusions through sinusoidal endothelial cell fenestrae by CD8 TE, producing contact sites with the hepatocyte membrane that are compatible with the establishment of an immunological synapse (Dustin and Groves, 2012).”

“One possibility is that they invade hepatocytes, enter endosomal/ lysosomal compartments and are degraded, a process of suicidal emperipolesis that was recently described for naive CD8 T cells undergoing primary activation in the liver (Benseler et al., 2011).”

“…treatment, we infected arse- nite-treated mice with LCMV, a virus whose tropism in the liver is mostly restricted to intravascular Kupffer cells (Guidotti et al., 1999) and where, therefore, subsequently transferred LCMV-specific CD8 TE should recognize infected cells independent of…”

“…HBV-specific CD8 TE into wild-type (WT) mice, previously injected with a reporter adenovirus (Ad-HBV-GFP) rendering HBV-replicating hepatocytes fluorescent (Sprinzl et al., 2001), re- vealed that CD8 TE adhere to liver sinusoids regardless of the location of HBV Ag-producing hepatocytes (Movie S1).”

“CD8 TE Arrest within Liver Sinusoids Independently of Ag Recognition To study howCD8 TE traffic within the liver and recognize Ag, we made use of two transgenic mouse strains whose CD8 T cells express H2- or H2-restricted T cell receptors (TCRs) specific for the HBV nucleocapsid (Cor) or envelope (Env) proteins, respe…”

“…to vascular beds in most organs—where a continuous endothelial cell layer and a basal membrane physically separate parenchymal cells from circulating leukocytes—LSEC lack tight junctions as well as a basal membrane and contain numerous fenestrae of up to 200 nm in diameter (Jacobs et al., 2010).”

“The notion that circulating leukocytes can interact with he- patocytes through endothelial fenestrations has been proposed in previous studies (Ando et al., 1994; Geissmann et al., 2005; Warren et al., 2006).”

“…(80 mg of clone 7-88 + 80 mg of clone 7-106/mouse, both provided by S. Jalkanen), anti-CD44 (clone KM81, blocking CD44 binding to hyaluronan [Zheng et al., 1995]; Cedarlane; 20 mg/mouse), anti-CD44 (clone IM7, not interfering with CD44 binding to hyaluronan [Zheng et al., 1995]; BioXCell;…”

“Using reconstitution experiments and genetic approaches to identify molecules thatmightmediate the capacity of platelets to support early hepatic CD8 TE (legend continued on next page) Cell 161, 1–15, April 23, 2015 ª2015 Elsevier Inc. 5 accumulation, we found that platelet-derived CD44 (but not platelet-derived P-sel…”

“These results, together with the notion that Kupffer cell depletion does not affect hepatic CD8 TE accumulation in these mouse models (Sitia et al., 2011), indicate that platelet-Kupffer cell interaction does not play a role in the hepatic homing of CD8 TE targeting hepatocellular Ags.”

“…of intrasinusoidal CD8 TE crawling is dictated by proximity to HBV Ag-expressing hepatocytes and by capacity to recognize cognate Ag, Cor93 and Env28 CD8 TE were co-transferred into a lineage of HBV transgenic mice that express HBcAg (but not HBsAg) in 100% of hepatocytes (Guidotti et al., 1994).”

“To test the biological significance of CD8 TE-mediated Ag recognition through sinusoidal fenestrae, HBV replicationcompetent transgenic mice were chronically exposed to low doses of arsenite (Straub et al., 2007), a treatment that reduces liver porosity (i.”

“) with 20 U/g hyaluronidase type-IV (Sigma-Aldrich), as described (Johnsson et al., 1999).”

“These results are consistent with recent reports showing that HLA class I restricted HBV epitopes are exclusively expressed by human hepatocytes during natural HBV infection (Ji et al., 2012).”

“Cell Isolation and Flow Cytometry Single-cell suspensions of livers, spleens, and lymph nodes were generated as described (Iannacone et al., 2005; Tonti et al., 2013).”

“As a general rule, CD8 TE are thought to recognize pathogen-infected parenchymal cells and perform effector functions in the brain, skin, or gut following extravasation from post-capillary venules mediated by different combinations of inflammation-regulated selectins, integrins, and chemokines (Mueller, 2013).”

“Dynamic imaging revealed that CD8 TE not adjacent to Ag-expressing hepatocytes crawl upstream and downstream in the liver sinusoids at an average speed of 10 mm/min (note that blood flows in liver sinusoids at 100–400 mm/s) (Sironi et al., 2014).”

“CD44 is an important adhesion molecule for recruitment of leukocytes to liver (Guidotti et al., 2015; McDonald et al., 2008; Shi et al., 2010).”