Alexandre P. Benechet

Lung‐resident memory CD8 T cells (TRM) are indispensable for optimal cross‐protection against pulmonary virus infection

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.

Visualizing T Cell Migration in situ

Mounting a protective immune response is critically dependent on the orchestrated movement of cells within lymphoid tissues. The structure of secondary lymphoid organs regulates immune responses by promoting optimal cell–cell and cell–extracellular matrix interactions. Naïve T cells are initially activated by antigen presenting cells in secondary lymphoid organs. Following priming, effector T cells migrate to the site of infection to exert their functions. Majority of the effector cells die while a small population of antigen-specific T cells persists as memory cells in distinct anatomical locations. The persistence and location of memory cells in lymphoid and non-lymphoid tissues is critical to protect the host from re-infection. The localization of memory T cells is carefully regulated by several factors including the highly organized secondary lymphoid structure, the cellular expression of chemokine receptors and compartmentalized secretion of their cognate ligands. This balance between the anatomy and the ordered expression of cell surface and soluble proteins regulates the subtle choreography of T cell migration. In recent years, our understanding of cellular dynamics of T cells has been advanced by the development of new imaging techniques allowing in situ visualization of T cell responses. Here, we review the past and more recent studies that have utilized sophisticated imaging technologies to investigate the migration dynamics of naïve, effector, and memory T cells.

T cell-intrinsic S1PR1 regulates endogenous effector T-cell egress dynamics from lymph nodes during infection

Significance The control of a microbial infection by effector T cells is intrinsically linked to their migration. However, little is known about the mechanisms that control effector T-cell egress after infection. Sphingosine-1-phosphate receptor-1 (S1PR1) is a G-coupled protein receptor that plays an important role in naive T-cell egress from lymph nodes. However, less is known about its role in regulating effector T-cell trafficking during infection. Here, we used an inducible mouse model with temporally disrupted S1PR1 signaling exclusively in endogenous effector CD8 T cells to demonstrate that, after infection, even in the absence of retention signals such as CC chemokine receptor 7 (CCR7), intrinsic S1PR1 signaling is the overriding factor that regulates effector T-cell egress kinetics from the draining lymph node. 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.

CD8 T Cells Enter the Splenic T Cell Zones Independently of CCR7, but the Subsequent Expansion and Trafficking Patterns of Effector T Cells after Infection Are Dysregulated in the Absence of CCR7 Migratory Cues.

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.

Intravital Microscopy Analysis of Hepatic T Cell Dynamics

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.

Determinants of hepatic effector CD8+ T cell dynamics

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.

Visualizing Endogenous Effector T Cell Egress from the Lymph Nodes

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.