Stephan Halle

Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells

Mucosal vaccination via the respiratory tract can elicit protective immunity in animal infection models, but the underlying mechanisms are still poorly understood. We show that a single intranasal application of the replication-deficient modified vaccinia virus Ankara, which is widely used as a recombinant vaccination vector, results in prominent induction of bronchus-associated lymphoid tissue (BALT). Although initial peribronchiolar infiltrations, characterized by the presence of dendritic cells (DCs) and few lymphocytes, can be found 4 d after virus application, organized lymphoid structures with segregated B and T cell zones are first observed at day 8. After intratracheal application, in vitro–differentiated, antigen-loaded DCs rapidly migrate into preformed BALT and efficiently activate antigen-specific T cells, as revealed by two-photon microscopy. Furthermore, the lung-specific depletion of DCs in mice that express the diphtheria toxin receptor under the control of the CD11c promoter interferes with BALT maintenance. Collectively, these data identify BALT as tertiary lymphoid structures supporting the efficient priming of T cell responses directed against unrelated airborne antigens while crucially requiring DCs for its sustained presence.

Afferent lymph–derived T cells and DCs use different chemokine receptor CCR7–dependent routes for entry into the lymph node and intranodal migration

Little is known about the molecular mechanisms that determine the entry into the lymph node and intranodal positioning of lymph-derived cells. By injecting cells directly into afferent lymph vessels of popliteal lymph nodes, we demonstrate that lymph-derived T cells entered lymph-node parenchyma mainly from peripheral medullary sinuses, whereas dendritic cells (DCs) transmigrated through the floor of the subcapsular sinus on the afferent side. Transmigrating DCs induced local changes that allowed the concomitant entry of T cells at these sites. Signals mediated by the chemokine receptor CCR7 were absolutely required for the directional migration of both DCs and T cells into the T cell zone but were dispensable for the parenchymal entry of lymph-derived T cells and dendrite probing of DCs. Our findings provide insight into the molecular and structural requirements for the entry into lymph nodes and intranodal migration of lymph-derived cells of the immune system.

Lentiviral Vector Design and Imaging Approaches to Visualize the Early Stages of Cellular Reprogramming

Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iPSC, and imaged the conversion of fibroblasts to iPSC. We combined fluorescence microscopy with long-term single cell tracking, and used live-cell imaging to analyze the emergence and composition of early iPSC clusters. Applying our engineered lentiviral vectors, we demonstrate that vector silencing typically occurs prior to or simultaneously with the induction of an Oct4-EGFP pluripotency marker. Around 7 days post-transduction (pt), a subfraction of cells in clonal colonies expressed Oct4-EGFP and rapidly expanded. Cell tracking of single cell-derived iPSC colonies supported the concept that stochastic epigenetic changes are necessary for reprogramming. We also found that iPSC colonies may emerge as a genetic mosaic originating from different clusters. Improved vector design with continuous cell tracking thus creates a powerful system to explore the subtle dynamics of biological processes such as early reprogramming events.

Solitary Intestinal Lymphoid Tissue Provides a Productive Port of Entry for Salmonella enterica Serovar Typhimurium

ABSTRACT Oral infection of mice with Salmonella enterica serovar Typhimurium results in the colonization of Peyers patches, triggering a vigorous inflammatory response and immunopathology at these sites. Here we demonstrate that in parallel to Peyers patches a strong inflammatory response occurs in the intestine, resulting in the appearance of numerous inflammatory foci in the intestinal mucosa. These foci surround small lymphoid cell clusters termed solitary intestinal lymphoid tissue (SILT). Salmonella can be observed inside SILT at early stages of infection, and the number of infected structures matches the number of inflammatory foci arising at later time points. Infection leads to enlargement and morphological destruction of SILT but does not trigger de novo formation of lymphoid tissue. In conclusion, SILT, a lymphoid compartment mostly neglected in earlier studies, represents a major site for Salmonella invasion and ensuing mucosal pathology.

In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity

Summary According to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2–16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8+ T cell immunity.

Single cell detection of latent cytomegalovirus reactivation in host tissue

The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor-GFP reporter mice, we never observed reactivation originating from CX3CR1(+) monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b(+) and CX3CR1(+) subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.

Nodular inflammatory foci are sites of T cell priming and control of murine cytomegalovirus infection in the neonatal lung.

Neonates, including mice and humans, are highly susceptible to cytomegalovirus (CMV) infection. However, many aspects of neonatal CMV infections such as viral cell tropism, spatio-temporal distribution of the pathogen as well as genesis of antiviral immunity are unknown. With the use of reporter mutants of the murine cytomegalovirus (MCMV) we identified the lung as a primary target of mucosal infection in neonatal mice. Comparative analysis of neonatal and adult mice revealed a delayed control of virus replication in the neonatal lung mucosa explaining the pronounced systemic infection and disease in neonates. This phenomenon was supplemented by a delayed expansion of CD8+ T cell clones recognizing the viral protein M45 in neonates. We detected viral infection at the single-cell level and observed myeloid cells forming “nodular inflammatory foci” (NIF) in the neonatal lung. Co-localization of infected cells within NIFs was associated with their disruption and clearance of the infection. By 2-photon microscopy, we characterized how neonatal antigen-presenting cells (APC) interacted with T cells and induced mature adaptive immune responses within such NIFs. We thus define NIFs of the neonatal lung as niches for prolonged MCMV replication and T cell priming but also as sites of infection control.

Mck2-dependent infection of alveolar macrophages promotes replication of MCMV in nodular inflammatory foci of the neonatal lung

Infection with cytomegalovirus (CMV) shows a worldwide high prevalence with only immunocompromised individuals or newborns to become symptomatic. The host’s constitution and the pathogen’s virulence determine whether disease occurs after infection. Mouse CMV (MCMV) is an appreciated pathogen for in vivo investigation of host–pathogen interactions. It has recently been reported that a single base pair deletion can spontaneously occur in the open reading frame of MCMV-encoded chemokine 2 (MCK2), preventing the expression of the full-length gene product. To study the consequences of this mutation, we compared the Mck2-defective reporter virus MCMV-3D with the newly generated repaired Mck2+ mutant MCMV-3DR. Compared with MCMV-3D, neonatal mice infected with MCMV-3DR showed severe viral disease after lung infection. Viral disease coincided with high viral activity in multiple organs and increased virus replication in previously described nodular inflammatory foci (NIF) in the lung. Notably, MCMV-3DR showed tropism for alveolar macrophages in vitro and in vivo, whereas MCMV-3D did not infect this cell type. Moreover, in vivo depletion of alveolar macrophages reduced MCMV-3DR replication in the lung. We proposed an Mck2-mediated mechanism by which MCMV exploits alveolar macrophages to increase replication upon first encounter with the host’s lung mucosa.

Mechanisms and Dynamics of T Cell-Mediated Cytotoxicity In Vivo.

Cytotoxic T lymphocytes (CTLs) are critical in the elimination of infected or malignant cells and are emerging as a major therapeutic target. How CTLs recognize and kill harmful cells has been characterized in vitro but little is known about these processes in the living organism. Here we review recent insights into CTL-mediated killing with an emphasis on in vivo CTL biology. Specifically, we focus on the possible rate-limiting steps determining the efficiency of CTL-mediated killing. We also highlight the need for cell-based datasets that permit the quantification of CTL dynamics, including CTL location, migration, and killing rates. A better understanding of these factors is required to predict protective CD8 T cell immunity in vivo and to design optimized vaccination protocols.

Shared and Unique Features Distinguishing Follicular T Helper and Regulatory Cells of Peripheral Lymph Node and Peyer’s Patches

Follicular helper (TFH) and regulatory (TFR) cells are critical players in managing germinal center (GC) reactions that accomplish effective humoral immune responses. Transcriptome analyses were done comparing gene regulation of TFH and TFR cells isolated from Peyer’s Patches (PP) and immunized peripheral lymph nodes (pLNs) revealing many regulatory patterns common to all follicular cells. However, in contrast to TFH cells, the upregulation or downregulation of many genes was attenuated substantially in pLN TFR cells when compared to those of PP. Additionally, PP but not pLN TFR cells were largely unresponsive to IL2 and expressed Il4 as well as Il21. Together with fundamental differences in gene expression that were found between cells of both compartments this emphasizes specific adaptations of follicular T cell functions to their micro-milieu. Moreover, although GL7 expression distinguishes matured follicular T cells, GL7+ as well as GL7− cells are present in the GC.