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Summary Kupffer cells (KCs) are highly abundant, intravascular, liver-resident macrophages known for their scavenger and phagocytic functions. KCs can also present antigens to CD8+ T cells and promote either tolerance or effector differentiation, but the mechanisms underlying these discrepant outcomes are poorly understood. Here, we used a mouse model of hepatitis B virus (HBV) infection, in which HBV-specific naive CD8+ T cells recognizing hepatocellular antigens are driven into a state of immune dysfunction, to identify a subset of KCs (referred to as KC2) that cross-presents hepatocellular antigens upon interleukin-2 (IL-2) administration, thus improving the antiviral function of T cells. Removing MHC-I from all KCs, including KC2, or selectively depleting KC2 impaired the capacity of IL-2 to revert the T cell dysfunction induced by intrahepatic priming. In summary, by sensing IL-2 and cross-presenting hepatocellular antigens, KC2 overcome the tolerogenic potential of the hepatic microenvironment, suggesting new strategies for boosting hepatic T cell immunity., Graphical abstract, Highlights • KCs are required for in vivo reinvigoration of intrahepatically primed T cells by IL-2 • KCs respond to IL-2 and cross-present hepatocellular Ags • Single-cell RNA-seq identifies two distinct populations of KCs • KC2s have enriched IL-2 sensing machinery and Ag presentation capacity, De Simone et al. delineate the mechanisms by which hepatocellularly primed HBV-specific CD8+ T cells acquire antiviral effector functions following IL-2 administration. These mechanisms rely on KCs and, in particular, on a hitherto unidentified KC subset, referred to as KC2, that is poised to respond to IL-2 and cross-present viral antigens.

Viral clearance requires effector T-cell egress from the draining lymph node (dLN). The mechanisms that regulate the complex process of effector T-cell egress from the dLN after infection are poorly understood. Here, we visualized endogenous pathogen-specific effector T-cell migration within, and from, the dLN. We used an inducible mouse model with a temporally disrupted sphingosine-1-phosphate receptor-1 (S1PR1) gene specifically in endogenous effector T cells. Early after infection, WT and S1PR1(-/-) effector T cells localized exclusively within the paracortex. This localization in the paracortex by CD8 T cells was followed by intranodal migration by both WT and S1PR1(-/-) T cells to positions adjacent to both cortical and medullary lymphatic sinuses where the T cells exhibited intense probing behavior. However, in contrast to WT, S1PR1(-/-) effector T cells failed to enter the sinuses. We demonstrate that, even when LN retention signals such as CC chemokine receptor 7 (CCR7) are down-regulated, T cell intrinsic S1PR1 is the master regulator of effector T-cell emigration from the dLN.

CCR7 is an important chemokine receptor that regulates T cell trafficking and compartmentalization within secondary lymphoid organs. However, the T cell–intrinsic role of CCR7 during infection in the spleen is not well understood. This study was designed to understand how CCR7-dependent localization and migration of CD8+ T cells in different compartments of the spleen affected the primary and recall responses after infection. To this end, we used adoptive transfer of naive Ag-specific CD8 T cells (OT-I) that either lacked CCR7 or constitutively expressed CCR7 (CD2-CCR7) in mice that were subsequently infected i.v. with Listeria monocytogenes. We show that naive CCR7−/−CD8+ T cells failed to enter the T cell zone, whereas CD2-CCR7 OT-I cells were exclusively confined to the T cell zones of the spleen. Surprisingly, however, CCR7−/− OT-I cells entered the T cell zones after infection, but the entry and egress migratory pattern of these cells was dysregulated and very distinct compared with wild-type OT-I cells. Moreover, CCR7-deficient OT-I cells failed to expand robustly when compared with wild-type OT-I cells and were preferentially skewed toward a short-lived effector cell differentiation pattern. Interestingly, CCR7−/−, CD2-CCR7, and wild-type OT-I memory cells responded equally well to rechallenge infection. These results highlight a novel role of CCR7 in regulating effector CD8 T cell migration in the spleen and demonstrate differential requirement of CCR7 for primary and secondary CD8 T cell responses to infection.

Local anatomy of lymphoid tissues during infection has emerged as a critical regulator of immunity; thus, studying the cellular choreography in the context of an intact tissue environment in situ is crucial. Following an infection, the local pathogen-specific T cell migration and the subsequent egress of effector T cells from the draining lymph nodes are important and complex biological processes. The mechanisms that regulate this complex process can now be investigated by directly visualizing T cell dynamics in vivo using intravital two-photon (2P) microscopy. In addition, static whole-mount imaging technique can provide us with a comprehensive assessment of global changes in the distribution of cellular populations within an intact tissue. Thus, in this chapter, we detail methods to visualize the migration and egress of endogenous antigen-specific CD8 T cells following viral infection using two methods-intravital 2P microscopy and multicolor whole-mount in situ tetramer staining.

Blood platelets are critical for hemostasis and thrombosis and play diverse roles during immune responses. Despite these versatile tasks in mammalian biology, their skills on a cellular level are deemed limited, mainly consisting in rolling, adhesion, and aggregate formation. Here, we identify an unappreciated asset of platelets and show that adherent platelets use adhesion receptors to mechanically probe the adhesive substrate in their local microenvironment. When actomyosin-dependent traction forces overcome substrate resistance, platelets migrate and pile up the adhesive substrate together with any bound particulate material. They use this ability to act as cellular scavengers, scanning the vascular surface for potential invaders and collecting deposited bacteria. Microbe collection by migrating platelets boosts the activity of professional phagocytes, exacerbating inflammatory tissue injury in sepsis. This assigns platelets a central role in innate immune responses and identifies them as potential targets to dampen inflammatory tissue damage in clinical scenarios of severe systemic infection. In addition to their role in thrombosis and hemostasis, platelets can also migrate to sites of infection to help trap bacteria and clear the vascular surface.

Local anatomy of lymphoid tissues during infection has emerged as a critical regulator of immunity; thus, studying the cellular choreography in the context of an intact tissue environment in situ is crucial. Following an infection, the local pathogen-specific T cell migration and the subsequent egress of effector T cells from the draining lymph nodes are important and complex biological processes. The mechanisms that regulate this complex process can now be investigated by directly visualizing T cell dynamics in vivo using intravital two-photon (2P) microscopy. In addition, static whole-mount imaging technique can provide us with a comprehensive assessment of global changes in the distribution of cellular populations within an intact tissue. Thus, in this chapter, we detail methods to visualize the migration and egress of endogenous antigen-specific CD8 T cells following viral infection using two methods-intravital 2P microscopy and multicolor whole-mount in situ tetramer staining.

Background & Aims Besides secreting pro-inflammatory cytokines, chemokines and effector molecules, effector CD8 + T cells that arise upon acute infection with certain viruses have been shown to produce the regulatory cytokine interleukin (IL)-10 and, therefore, contain immunopathology. Whether the same occurs during acute hepatitis B virus (HBV) infection and role that IL-10 might play in liver disease is currently unknown. Methods Mouse models of acute HBV pathogenesis, as well as chimpanzees and patients acutely infected with HBV, were used to analyse the role of CD8 + T cell-derived IL-10 in liver immunopathology. Results Mouse HBV-specific effector CD8 + T cells produce significant amounts of IL-10 upon in vivo antigen encounter. This is corroborated by longitudinal data in a chimpanzee acutely infected with HBV, where serum IL-10 was readily detectable and correlated with intrahepatic CD8 + T cell infiltration and liver disease severity. Unexpectedly, mouse and human CD8 + T cell-derived IL-10 was found to act in an autocrine/paracrine fashion to enhance IL-2 responsiveness, thus preventing antigen-induced HBV-specific effector CD8 + T cell apoptosis. Accordingly, the use of mouse models of HBV pathogenesis revealed that the IL-10 produced by effector CD8 + T cells promoted their own intrahepatic survival and, thus supported, rather than suppressed liver immunopathology. Conclusion Effector CD8 + T cell-derived IL-10 enhances acute liver immunopathology. Altogether, these results extend our understanding of the cell- and tissue-specific role that IL-10 exerts in immune regulation. Lay summary: Interleukin-10 is mostly regarded as an immunosuppressive cytokine. We show here that HBV-specific CD8 + T cells produce IL-10 upon antigen recognition and that this cytokine enhances CD8 + T cell survival. As such, IL-10 paradoxically promotes rather than suppresses liver disease.

T cells play critical roles in controlling hepatotropic viral infections and liver tumors. The protective capacity of these cells is mediated by antigen-experienced effector cells and depends on their ability to migrate to and traffic within the liver, recognize pathogen- or tumor-derived antigens, get activated and deploy effector functions.While some of the rules that characterize T cell behavior in the healthy and cancerous antigen-expressing liver have been characterized at the population level, we have only limited knowledge of the precise dynamics of T cell interactions with different kinds of liver cells at the single-cell level. Here, we describe in detail an intravital microscopy technique that allows the analysis of T cell dynamic behavior in the liver of anesthetized mice at high spatial and temporal resolution. A detailed understanding of the spatiotemporal dynamics of T cells within the liver is important for the rational design of targeted immunotherapeutic approaches for chronic liver infections and tumors.

Antigen-specific effector CD8+ T cells play a critical role in controlling hepatic infections, such as the one caused by hepatitis B virus (HBV). We review here recent results where we coupled advanced dynamic imaging with dedicated mouse models of HBV pathogenesis to show that circulating effector CD8+ T cells aimed at viral clearance initially arrest in liver sinusoids by preferentially docking onto platelets that have previously adhered to liver sinusoids. Upon detachment from platelets, effector CD8+ T cells crawl within the sinusoids irrespective of bloodstream direction, and probe underlying hepatocytes for the presence of antigen by extending filopodia-like protrusions through the sinusoidal fenestrae. Effector CD8+ T cells recognize hepatocellular antigen and perform effector functions (i.e., IFN-γ production and hepatocyte killing) while still in the intravascular space. They later extravasate in the parenchyma. Finally, we provide our perspective on how, in the next few years, intravital microscopy might shed new light on yet unresolved issues with particular regard to identifying the determinants of hepatic effector CD8+ T cell trafficking, antigen recognition and effector functions during hepatocellular carcinoma and understanding the mechanisms whereby intrahepatic T cell priming induces functionally defective T cell responses. A better understanding of how adaptive immunity mediates pathogen clearance and tumor elimination may lead to improved vaccination and treatment strategies for immunotherapy of infectious diseases and cancer.

Previous studies have shown that some respiratory virus infections leave local populations of tissue TRM cells in the lungs which disappear as heterosubtypic immunity declines. The location of these TRM cells and their contribution to the protective CTL response have not been clearly defined. Here, fluorescence microscopy is used to show that some CD103+ TRM cells remain embedded in the walls of the large airways long after pulmonary immunization but are absent from systemically primed mice. Viral clearance from the lungs of the locally immunized mice precedes the development of a robust Teff response in the lungs. Whereas large numbers of virus-specific CTLs collect around the bronchial tree during viral clearance, there is little involvement of the remaining lung tissue. Much larger numbers of TEM cells enter the lungs of the systemically immunized animals but do not prevent extensive viral replication or damage to the alveoli. Together, these experiments show that virus-specific antibodies and TRM cells are both required for optimal heterosubtypic immunity, whereas circulating memory CD8 T cells do not substantially alter the course of disease.

The spleen is an important site for generating protective immune responses against pathogens. Immune cells in the spleen undergo rapid reorganization to initiate and maintain local inflammatory responses against pathogens. How the spatial dynamics of cellular communication are regulated in the spleen after infection remains unclear. Respectively, CD169+ macrophages are a population of tissue resident macrophages that are positioned in the marginal zone to rapidly encounter invading pathogens. However, the role of splenic CD169+ macrophages during infections is not well understood. Here we show that splenic CD169+ macrophages serve as a primary cellular host to blood-borne bacteria Listeria monocytogens (Lm) and are also essential for the clearance of other bacterial and viral pathogens. In addition to controlling initial bacterial growth, CD169+ macrophages orchestrated a second phase of innate protection by mediating the transport of Lm to the splenic T cell zones and driving the subsequent reorganization of neutrophils, monocytes, and NK cells into hierarchical clusters. Specifically our study revealed that bacterial transport to the T cell zones was mediated by trans-infection of closely interacting CD8α+ DCs with Lm-infected CD169+ macrophages. Although, CD8α+ DCs are required for a productive Lm infection in the spleen, to our surprise mice that lacked both CD8α+ DCs and CD169+ macrophages were unable to control Lm infection. These results demonstrated that CD169+ macrophages regulated bacterial access to the pathogen favorable CD8α+ DC niche, ultimately providing a rationale for reassessing the current paradigm regarding the temporal role of CD8α+ DCs during Lm infection.

The responses of CD8(+) T cells to hepatotropic viruses such as hepatitis B range from dysfunction to differentiation into effector cells, but the mechanisms that underlie these distinct outcomes remain poorly understood. Here we show that priming by Kupffer cells, which are not natural targets of hepatitis B, leads to differentiation of CD8(+) T cells into effector cells that form dense, extravascular clusters of immotile cells scattered throughout the liver. By contrast, priming by hepatocytes, which are natural targets of hepatitis B, leads to local activation and proliferation of CD8(+) T cells but not to differentiation into effector cells; these cells form loose, intravascular clusters of motile cells that coalesce around portal tracts. Transcriptomic and chromatin accessibility analyses reveal unique features of these dysfunctional CD8(+) T cells, with limited overlap with those of exhausted or tolerant T cells; accordingly, CD8(+) T cells primed by hepatocytes cannot be rescued by treatment with anti-PD-L1, but instead respond to IL-2. These findings suggest immunotherapeutic strategies against chronic hepatitis B infection.

SummaryIntestinal microbial metabolites are conjectured to affect mucosal integrity through an incompletely characterized mechanism. Here we showed that microbial-specific indoles regulated intestinal barrier function through the xenobiotic sensor, pregnane X receptor (PXR). Indole 3-propionic acid (IPA), in the context of indole, is a ligand for PXR in vivo, and IPA downregulated enterocyte TNF-α while it upregulated junctional protein-coding mRNAs. PXR-deficient (Nr1i2−/−) mice showed a distinctly “leaky” gut physiology coupled with upregulation of the Toll-like receptor (TLR) signaling pathway. These defects in the epithelial barrier were corrected in Nr1i2−/−Tlr4−/− mice. Our results demonstrate that a direct chemical communication between the intestinal symbionts and PXR regulates mucosal integrity through a pathway that involves luminal sensing and signaling by TLR4.