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Live-cell imaging allows the study of apoptosis at cellular level, highlighting morphological hallmarks such as nuclear shrinkage, membrane blebbing, and cell disruption. Identifying the exact location and timing of this process is essential to foster the understanding of its spatial-temporal regulation. However, the analysis of live-cell imaging datasets is complex, whereas computational tools tailored to this task are yet scarce. Therefore, we developed ADeS, an Apoptosis Detection System based on deep learning and activity recognition. ADeS uses morpho-dynamic hallmarks to detect the exact location and timing of apoptotic events in different cell types, reaching an accuracy above 97% in the classification of our validation datasets acquiredin vitroandin vivo. Moreover, ADeS is the first successful implementation of a deep learning network for the automatic detection of apoptotic cells in full microscopy movies in an end-to-end fashion, outperforming human in the same task. As a case study, we employed ADeS for the analysis of cell survivalin vitro, and for tissue damage assessmentin vivo, showing its potential application for toxicity assays, treatments evaluation and measuring of tissue dynamics.

Protective immunity to cutaneous leishmaniasis is mediated by IFN-γ–secreting CD4+ Th1 cells. IFN-γ binds to its receptor on Leishmania-infected macrophages, resulting in their activation, production of NO, and subsequent destruction of parasites. This study investigated the role of Semaphorin 3E (Sema3E) in host immunity to Leishmania major infection in mice. We observed a significant increase in Sema3E expression at the infection site at different timepoints following L. major infection. Sema3E-deficient (Sema3E knockout [KO]) mice were highly resistant to L. major infection, as evidenced by significantly (p < 0.05–0.01) reduced lesion sizes and lower parasite burdens at different times postinfection when compared with their infected wild-type counterpart mice. The enhanced resistance of Sema3E KO mice was associated with significantly (p < 0.05) increased IFN-γ production by CD4+ T cells. CD11c+ cells from Sema3E KO mice displayed increased expression of costimulatory molecules and IL-12p40 production following L. major infection and were more efficient at inducing the differentiation of Leishmania-specific CD4+ T cells to Th1 cells than their wild-type counterpart cells. Furthermore, purified CD4+ T cells from Sema3E KO mice showed increased propensity to differentiate into Th1 cells in vitro, and this was significantly inhibited by the addition of recombinant Sema3E in vitro. These findings collectively show that Sema3E is a negative regulator of protective CD4+ Th1 immunity in mice infected with L. major and suggest that its neutralization may be a potential therapeutic option for treating individuals suffering from cutaneous leishmaniasis.

Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.

Intravital microscopy, such as 2-photon microscopy, is now a mainstay in immunological research to visually characterize immune cell dynamics during homeostasis and pathogen infections. This approach has been especially beneficial in describing the complex process of host immune responses to parasitic infections in vivo, such as Leishmania. Human-parasite co-evolution has endowed parasites with multiple strategies to subvert host immunity in order to establish chronic infections and ensure human-to-human transmission. While much focus has been placed on viral and bacterial infections, intravital microscopy studies during parasitic infections have been comparatively sparse. In this review, we will discuss how in vivo microscopy has provided important insights into the generation of innate and adaptive immunity in various organs during parasitic infections, with a primary focus on Leishmania. We highlight how microscopy-based approaches may be key to providing mechanistic insights into Leishmania persistence in vivo and to devise strategies for better parasite control.

Summary The long pentraxin 3 (PTX3) plays a critical role in inflammation, tissue repair, and wound healing. Here, we show that PTX3 regulates disease pathogenesis in cutaneous leishmaniasis (CL). PTX3 expression increases in skin lesions in patients and mice during CL, with higher expression correlating with severe disease. PTX3-deficient (PTX3−/−) mice are highly resistant to L. major and L. braziliensis infections. This enhanced resistance is associated with increases in Th17 and IL-17A responses. The neutralization of IL-17A abolishes this enhanced resistance, while rPTX3 treatment results in decrease in Th17 and IL-17A responses and increases susceptibility. PTX3−/− CD4+ T cells display increased differentiation to Th17 and expression of Th17-specific transcription factors. The addition of rPTX3 suppresses the expression of Th17 transcription factors, Th17 differentiation, and IL-17A production by CD4+ T cells from PTX3−/− mice. Collectively, our results show that PTX3 contributes to the pathogenesis of CL by negatively regulating Th17 and IL-17A responses.

T cells actively migrate along reticular networks within lymphoid organs in search for cognate antigen, but how these behaviors impact HIV entry and infection is unclear. Here, we show that migratory T cells in 3D collagen matrix display significantly enhanced infection and integration by cell-free R5-tropic lab adapted and transmitted/founder molecular HIV clones in the absence of exogenous cytokines or cationic polymers. Using two different collagen matrices that either support or restrict T cell migration, we observe high levels of HIV fusion in migratory T cells, whereas non-motile T cells display low viral entry and integration. Motile T cells were less sensitive to combination antiretroviral drugs and were able to freely migrate into regions with high HIV densities, resulting in high infection rates. Together, our studies indicate that the environmental context in which initial HIV-T cell encounters occur modulates HIV-1 entry and integration efficiencies.

Summary Trafficking of cell-associated HIV-1 from the genital mucosa to lymphoid organs represents a critical first step toward systemic infection. Mature DCs capture and transmit HIV-1 to T cells, but insights into DC-to-T cell viral spread dynamics within a 3-dimensional environment is lacking. Using live-cell imaging, we show that mature DCs rapidly compartmentalize HIV-1 within surface-accessible invaginations near the uropod. HIV-1 capture did not interfere with DC migration toward lymph node homing chemo-attractants and their ability to enter lymphatic vessels. However, HIV-captured DCs engaged in prolonged contacts with autologous CD4+ T cells, which led to high T cell infection. Interestingly, we show that surface bound, virion-associated Env induced signal transduction in motile T cells that facilitated prolonged DC:T cell interactions, partially through high-affinity LFA-1 expression. Together, we describe a mechanism by which surface bound HIV-1 particles function as signaling receptors that regulate T cell motility, cell-cell contact dynamics, and productive infection., Graphical Abstract, Highlights • Mature DCs compartmentalize HIV particles near the uropodia via Siglec-1 receptor • HIV-captured DCs respond to lymph node-homing chemokines and access lymphatics • Prolonged contacts between HIV-captured DCs and CD4 T cells facilitate virus transfer • Surface-accessible HIV particles can induce T cell signaling via Env:CD4 engagement, Immunology; Virology

Protective immunity to cutaneous leishmaniasis is mediated by IFN-γ-secreting CD4

The PI 3-kinases (PI3K) are essential mediators of chemokine receptor signaling necessary for migration of chronic lymphocytic leukemia (CLL) cells and their interaction with tissue-resident stromal cells. While the PI3Kδ-specific inhibitor idelalisib shows efficacy in treatment of CLL and other B cell malignancies, the function of PI3Kγ has not been extensively studied in B cells. Here, we assess whether PI3Kγ has non-redundant functions in CLL migration and adhesion to stromal cells. We observed that pharmaceutical PI3Kγ inhibition with CZC24832 significantly impaired CLL cell migration, while dual PI3Kδ/γ inhibitor duvelisib had a greater impact than single isoform-selective inhibitors. Knockdown of PI3Kγ reduced migration of CLL cells and cell lines. Expression of the PI3Kγ subunits increased in CLL cells in response to CD40L/IL-4, whereas BCR cross-linking had no effect. Overexpression of PI3Kγ subunits enhanced cell migration in response to SDF1α/CXCL12, with the strongest effect observed within ZAP70 + CLL samples. Microscopic tracking of cell migration within chemokine gradients revealed that PI3Kγ functions in gradient sensing and impacts cell morphology and F-actin polarization. PI3Kγ inhibition also reduced CLL adhesion to stromal cells to a similar extent as idelalisib. These findings provide the first evidence that PI3Kγ has unique functions in malignant B cells.

The trafficking of cell-associated HIV from the genital mucosa to lymphoid organs represents a critical first step towards systemic infection. Mature DCs capture and transmit HIV to T cells, but insights into DC-to-T cell viral spread dynamics within a 3-dimensional environment is lacking. Using live-cell imaging, we show that mature DCs rapidly compartmentalize HIV within surface-accessible invaginations near the uropod. HIV capture did not interfere with DC migration towards lymph node homing chemo-attractants and their ability to enter lymphatic vessels. However, HIV-bearing DCs engaged in prolonged contacts with autologous CD4+ T cells which led to high T cell infection. Interestingly, we show that surface bound, virion-associated Env induced signal transduction in motile T cells that facilitated prolonged DC:T cell interactions, partially through high-affinity LFA-1 expression. Together, we describe a mechanism by which surface bound HIV particles function as signaling receptors that regulate T cell motility, cell-cell contact dynamics and productive infection.

Background Varicella-zoster virus (VZV), also known as chickenpox virus, constitutes a promising vector for a successful HIV vaccine. As an effort to scrutinize its potential, we are characterizing the susceptibility of VZV-specific CD4 T cells to HIV infection and the phenotypic profile of both CD4 and CD8 T cells. Methods Blood T cells isolated from a cohort of healthy Kenyan women with pre-immunity to VZV (NCT02514018) were stimulated in vitro using 15-mer peptides representing VZV glycoprotein E (gE) and VZV Open Reading Frame 4 (ORF4). CD4 and CD8 T cell memory phenotypes were characterized by flow cytometry based on the expression of CCR7/CD45RA. The activation status of VZV-specific CD4 T cells was measured by the expression of HLA-DR, CD69, and CD25 after 6-day stimulation with gE and ORF4 peptides. Susceptibility to HIV infection was assessed using in vitro infection with a CCR5-tropic virus. DMSO and CMV peptides were used as negative and positive controls, respectively. Results A similar frequency of central memory CD4 T cells (TCM) (median 24%, IQR 18%-32%) and effector memory CD4 T cells (TEM) (median 27%, IQR 20%-32%) was observed in our cohort. The predominant CD8 memory subtype was TEMRA (median 28%, IQR 21%-40%) followed by TEM cells (median 12%, IQR 8%-19%) (n=45). Preliminary results show our ability to expand VZV-specific cells in culture using gE and ORF4 as stimuli and that these cells highly express the marker HLA-DR. Their susceptibility to in vitro HIV infection is currently under investigation using CMV-specific cells as comparator. Conclusion A viral vector able to sustain CD8 TEM responses without fueling the immune system with HIV target cells constitutes an ideal candidate for an HIV vaccine. Hence, our study sheds light on key aspects of VZV-specific immunity that will help determining its future as a vector in an HIV vaccine. Disclosure No significant relationships.

Recent advances in intravital video microscopy have allowed the visualization of leukocyte behavior in vivo, revealing unprecedented spatiotemporal dynamics of immune cell interaction. However, state-of-the-art software and methods for automatically measuring cell migration exhibit limitations in tracking the position of leukocytes over time. Challenges arise both from the complex migration patterns of these cells and from the experimental artifacts introduced during image acquisition. Additionally, the development of novel tracking tools is hampered by the lack of a sound ground truth for algorithm validation and benchmarking. Therefore, the objective of this work was to create a database, namely LTDB, with a significant number of manually tracked leukocytes. Broad experimental conditions, sites of imaging, types of immune cells and challenging case studies were included to foster the development of robust computer vision techniques for imaging-based immunological research. Lastly, LTDB represents a step towards the unravelling of biological mechanisms by video data mining in systems biology.

Macrophages are susceptible to HIV infection and play an important role in viral dissemination through cell-cell contacts with T cells. However, our current understanding of macrophage-to-T cell HIV transmission is derived from studies that do not consider the robust migration and cell-cell interaction dynamics between these cells. Here, we performed live-cell imaging studies in 3-dimensional (3D) collagen that allowed CD4+ T cells to migrate and to locate and engage HIV-infected macrophages, modeling the dynamic aspects of the in situ environment in which these contacts frequently occur. We show that HIV+ macrophages form stable contacts with CD4+ T cells that are facilitated by both gp120-CD4 and LFA-1–ICAM-1 interactions and that prolonged contacts are a prerequisite for efficient viral spread. LFA-1–ICAM-1 adhesive contacts function to restrain highly motile T cells, since their blockade substantially destabilized macrophage-T cell contacts, resulting in abnormal tethering events that reduced cell-cell viral spread. HIV-infected macrophages displayed strikingly elongated podosomal extensions that were dependent on Nef expression but were dispensable for stable cell-cell contact formation. Finally, we observed persistent T cell infection in dynamic monocyte-derived macrophage (MDM)-T cell cocultures in the presence of single high antiretroviral drug concentrations but achieved complete inhibition with combination therapy. Together, our data implicate macrophages as drivers of T cell infection by altering physiological MDM-T cell contact dynamics to access and restrain large numbers of susceptible, motile T cells within lymphoid tissues. IMPORTANCE Once HIV enters the lymphoid organs, exponential viral replication in T cells ensues. Given the densely packed nature of these tissues, where infected and uninfected cells are in nearly constant contact with one another, efficient HIV spread is thought to occur through cell-cell contacts in vivo. However, this has not been formally demonstrated. In this study, we performed live-cell imaging studies within a 3-dimensional space to recapitulate the dynamic aspects of the lymphoid microenvironment and asked whether HIV can alter the morphology, migration capacity, and cell-cell contact behaviors between macrophages and T cells. We show that HIV-infected macrophages can engage T cells in stable contacts through binding of virus- and host-derived adhesive molecules and that stable macrophage-T cell contacts were required for high viral spread. Thus, HIV alters physiological macrophage-T cell interactions in order to access and restrain large numbers of susceptible, motile T cells, thereby playing an important role in HIV progression.

The long Pentraxin 3 (PTX3), a soluble pattern recognition molecule, plays a critical role in inflammation, tissue repair and wound healing. Here, we show that PTX3 regulates disease pathogenesis in cutaneous leishmaniasis (CL). PTX3 expression is increased in active skin lesions in patients and mice during CL, with higher levels being expressed in individuals with severe disease. PTX3 deficient (PTX3-/-) mice were highly resistant toL. majorinfection and the enhanced resistance was associated with increased IL-17 response. Neutralization of IL-17A abolished this enhanced resistance while treatment with recombinant PTX3 resulted in reduced IL-17A response and increased susceptibility toL. majorinfection. Naïve CD4+T cells from PTX3-/-mice displayed increased differentiation into Th17 cells, which was reversed in the presence of recombinant PTX3. The enhanced Th17 response observed in PTX3-/-cells was associated with increasedLeishmaniaspecific IL-6 production from dendritic cells along with enhanced expression of Th17-specific transcription factors including RORγt, AhR and STAT3. Addition of recombinant PTX3 significantly inhibited the expression of Th17-specific transcription factors and dramatically reduced the frequency of Th17 cells in Th17-polarizing cultures of PTX3-/-CD4+T cells. Collectively, our results show that PTX3 contributes to the pathogenesis of CL by suppressing Th17 differentiation and IL-17A production.Author SummaryCutaneous leishmaniasis (CL) is caused by several species ofLeishmania. Currently, there is no approved vaccine against human CL because of the poor understanding of the mechanisms that regulate disease pathogenesis and correlates of protective immunity. Because the long pentraxin 3 (PTX3, a soluble pattern recognition molecule that forms an integral part of the host innate immunity), regulates inflammation and tissue repair, which are critical physiological events associated with resolution of skin lesions during CL, we investigated its role in disease pathogenesis.Here, we show that PTX3 levels were elevated in skin-lesions in patients and mice during CL. Using a loss of function approach, we showed that PTX3 contributes to pathogenesis, and this was associated with increased IL-17A responses. Neutralization and recombinant cytokine treatment studies showed that the increased resistance of PTX3 deficient mice toL. majoris due to enhanced Th17 response in these mice. We further show that PTX3 negatively regulates IL-6 production by dendritic cells and the expression of IL-17A-specific transcription factors (including RORγT, STAT3, IRF4, BATF and AhR) in CD4+T cells. Collectively, these findings show that PTX3 is a negative regulator of Th17 response and protective immunity duringL. majorinfection.

The long pentraxin 3 (PTX3) plays a critical role in inflammation, tissue repair, and wound healing. Here, we show that PTX3 regulates disease pathogenesis in cutaneous leishmaniasis (CL). PTX3 expression increases in skin lesions in patients and mice during CL, with higher expression correlating with severe disease. PTX3-deficient (PTX3

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.

HIV-1 Env mediates fusion of viral and target cell membranes, but it can also mediate fusion of infected (producer) and target cells, thus triggering the formation of multinucleated cells, so-called syncytia. Large, round, immobile syncytia are readily observable in cultures of HIV-1-infected T cells, but these fast growing “fusion sinks” are largely regarded as cell culture artifacts. In contrast, small HIV-1-induced syncytia were seen in the paracortex of peripheral lymph nodes and other secondary lymphoid tissue of HIV-1-positive individuals. Further, recent intravital imaging of lymph nodes in humanized mice early after their infection with HIV-1 demonstrated that a significant fraction of infected cells were highly mobile, small syncytia, suggesting that these entities contribute to virus dissemination. Here, we report that the formation of small, migratory syncytia, for which we provide further quantification in humanized mice, can be recapitulated in vitro if HIV-1-infected T cells are placed into 3D extracellular matrix (ECM) hydrogels rather than being kept in traditional suspension culture systems. Intriguingly, live-cell imaging in hydrogels revealed that these syncytia, similar to individual infected cells, can transiently interact with uninfected cells, leading to rapid virus transfer without cell-cell fusion. Infected cells were also observed to deposit large amounts of viral particles into the extracellular space. Altogether, these observations suggest the need to further evaluate the biological significance of small, T cell-based syncytia and to consider the possibility that these entities do indeed contribute to virus spread and pathogenesis.

HIV vaginal transmission accounts for the majority of newly acquired heterosexual infections. However, the mechanism by which HIV spreads from the initial site of viral entry at the mucosal surface of the female genital tract to establish a systemic infection of lymphoid and peripheral tissues is not known. Once the virus exits the mucosa it rapidly spreads to all tissues, leading to CD4+ T cell depletion and the establishment of a viral reservoir that cannot be eliminated with current treatments. Understanding the molecular and cellular requirements for viral dissemination from the genital tract is therefore of great importance, as it could reveal new strategies to lengthen the window of opportunity to target the virus at its entry site in the mucosa where it is the most vulnerable and thus prevent systemic infection. Using HIV vaginal infection of humanized mice as a model of heterosexual transmission, we demonstrate that blocking the ability of leukocytes to respond to chemoattractants prevented HIV from leaving the female genital tract. Furthermore, blocking lymphocyte egress from lymph nodes prevented viremia and infection of the gut. Leukocyte trafficking therefore plays a major role in viral dissemination, and targeting the chemoattractant molecules involved can prevent the establishment of a systemic infection.

Humanized mouse models have, over the past few years, seen dramatic improvements, including the colonization of both lymphoid and nonlymphoid tissues with all major immune cell lineages, the development of T cells with human major histocompatibility complex restriction, and the ability to mount functional adaptive immune responses to human pathogens, as documented in some instances. This has greatly increased the range of questions related to the biology of human immunodeficiency virus (HIV) infection that can be usefully addressed through experimental approaches utilizing small animal models. Among these approaches is in vivo imaging, and specifically multiphoton intravital microscopy (MP-IVM), which allows for the investigation of dynamic biological processes at cellular and subcellular resolution in the tissues of live animals. We have recently begun to use MP-IVM in lymph nodes of humanized mice in order to examine HIV infectious spread in vivo at the tissue and cellular level. Here, we provide a short perspective on the close link between the patterns of immune cell migration and the mechanisms of viral dissemination, and summarize the results of our initial studies.

The deposition of immune complexes (IC) in tissues induces a "type III hypersensitivity" that results in tissue damage and underlies the pathogenesis of many autoimmune diseases. The neutrophil is the first immune cell recruited into sites of IC deposition and plays a critical role in shaping the overall tissue response. However, the mechanism by which IC initiate and propagate neutrophil infiltration into tissue is not known. Here, using intravital multiphoton joint imaging of IC-induced arthritis in live mice, we found that the complement C5a receptor (C5aR) was the key initiator of neutrophil adhesion on joint endothelium. C5a presented on joint endothelium induced β2 integrin-dependent neutrophil arrest, facilitating neutrophil spreading and transition to crawling, and subsequent leukotriene B4 receptor (BLT1)-mediated extravasation of the first neutrophils. The chemokine receptor CCR1 promoted neutrophil crawling on the joint endothelium while CXCR2 amplified late neutrophil recruitment and survival once in the joint. Thus, imaging arthritis has defined a new paradigm for type III hypersensitivity where C5a directly initiates neutrophil adhesion on the joint endothelium igniting inflammation.

In earlier studies we provided evidence that vaccinia virus (VACV) phosphorylation-activation of host cell signaling effectors is critical for subsequent viral replication. In this report, using mass spectrometry-based proteomics, we have identified 387 host cell proteins that co-immunoprecipitate with VACV in infected, permissive PM1.CCR5 human T cells. Among these, glomulin was distinguishable based on its known interaction with a tyrosine kinase receptor, c-Met, its ability to become tyrosine-phosphorylated, and its association with signaling effectors. siRNA knockdown of glomulin expression in PM1.CCR5 T cells reduces VACV infection. Glomulin interacts with the inactive, nonphosphorylated form of c-MET. We demonstrate that treatment of PM1.CCR5 T cells with a c-Met phosphorylation inhibitor leads to a significant reduction in VACV infectivity. Additionally, inhibition of phosphorylation of c-Met abrogates VACV-inducible phosphorylation of Erk 1/2 and IRS-2, signaling effectors identified as critical for VACV infection. These data identify glomulin as a permissivity factor for VACV infection and as a potential therapeutic target for inhibition of VACV infection.

Summary HIV-1 primarily infects T lymphocytes and uses these motile cells as migratory vehicles for effective dissemination in the host. Paradoxically, the virus at the same time disrupts multiple cellular processes underlying lymphocyte motility, seemingly counterproductive to rapid systemic infection. Here we show by intravital microscopy in humanized mice that perturbation of the actin cytoskeleton via the lentiviral protein Nef, and not changes to chemokine receptor expression or function, is the dominant cause of dysregulated infected T cell motility in lymphoid tissue by preventing stable cellular polarization required for fast migration. Accordingly, disrupting the Nef hydrophobic patch that facilitates actin cytoskeletal perturbation initially accelerates systemic viral dissemination after female genital transmission. However, the same feature of Nef was subsequently critical for viral persistence in immune-competent hosts. Therefore, a highly conserved activity of lentiviral Nef proteins has dual effects and imposes both fitness costs and benefits on the virus at different stages of infection.

Background: Interferon (IFN)-β is an effective therapy for relapsing-remitting multiple sclerosis, yet its mechanism of action remains ill-defined. Objectives: Our objective was to characterize the role of IFN-β in immune regulation in experimental autoimmune encephalomyelitis (EAE). Methods: IFN-β +/+ and IFN-β—/— mice were immunized with myelin oligodendrocyte glycoprotein peptide in the presence or absence of IFN-β, to induce EAE. Disease pathogenesis was monitored in the context of incidence, time of onset, clinical score, and immune cell activation in the brains, spleens and lymph nodes of affected mice. Results: Compared with IFN-β+/+ mice, IFN-β—/ — mice exhibited an earlier onset and a more rapid progression of EAE, increased numbers of CD11b+ leukocytes infiltrating affected brains and an increased percentage of Th17 cells in the central nervous system and draining lymph nodes. IFN-β treatment delayed disease onset and reduced disease severity. Ex vivo experiments revealed that the lack of IFN-β results in enhanced generation of autoreactive T cells, a likely consequence of the absence of IFN-β-regulated events in both the CD4+ T cells and antigen-presenting dendritic cells. Gene expression analysis of IFN-β-treated bone marrow macrophages (CD11b +) identified modulation of genes affecting T cell proliferation and Th17 differentiation. Conclusions: We conclude that IFN-β acts to suppress the generation of autoimmune-inducing Th17 cells during the development of disease as well as modulating pro-inflammatory mediators.

In an earlier report, we provided evidence that expression of CCR5 by primary human T cells renders them permissive for vaccinia virus (VACV) replication. This may represent a mechanism for dissemination throughout the lymphatic system. To test this hypothesis, wild-type CCR5+/+and CCR5 null mice were challenged with VACV by intranasal inoculation. In time course studies using different infective doses of VACV, we identified viral replication in the lungs of both CCR5+/+and CCR5−/−mice, yet there were diminished viral loads in the spleens and brains of CCR5−/−mice compared with CCR5+/+mice. Moreover, in association with VACV infection, we provide evidence for CD4+and CD8+T-cell as well as CD11c+and F4/80+cell infiltration into the lungs of CCR5+/+but not CCR5−/−mice, and we show that the CCR5-expressing T cells harbor virus. We demonstrate that this CCR5 dependence is VACV specific, since CCR5−/−mice are as susceptible to intranasal influenza virus (A/WSN/33) infection as CCR5+/+mice. In a final series of experiments, we provide evidence that adoptive transfer of CCR5+/+bone marrow leukocytes into CCR5−/−mice restores VACV permissiveness, with evidence of lung and spleen infection. Taken together, our data suggest a novel role for CCR5 in VACV dissemination in vivo.

The multistep, coordinated process of T-cell chemotaxis requires chemokines, and their chemokine receptors, to invoke signaling events to direct cell migration. Here, we examined the role for CCL5-mediated initiation of mRNA translation in CD4+ T-cell chemotaxis. Using rapamycin, an inhibitor of mTOR, our data show the importance of mTOR in CCL5-mediated T-cell migration. Cycloheximide, but not actinomycin D, significantly reduced chemotaxis, suggesting a possible role for mRNA translation in T-cell migration. CCL5 induced phosphorylation/activation of mTOR, p70 S6K1, and ribosomal protein S6. In addition, CCL5 induced PI-3′K–, phospholipase D (PLD)–, and mTOR-dependent phosphorylation and deactivation of the transcriptional repressor 4E-BP1, which resulted in its dissociation from the eukaryotic initiation factor-4E (eIF4E). Subsequently, eIF4E associated with scaffold protein eIF4G, forming the eIF4F translation initiation complex. Indeed, CCL5 initiated active translation of mRNA, shown by the increased presence of high-molecular-weight polysomes that were significantly reduced by rapamycin treatment. Notably, CCL5 induced protein translation of cyclin D1 and MMP-9, known mediators of migration. Taken together, we describe a novel mechanism by which CCL5 influences translation of rapamycin-sensitive mRNAs and “primes” CD4+ T cells for efficient chemotaxis.

The introduction of multiphoton microscopy has dramatically broadened the scope of intravital imaging studies and has allowed researchers to validate and refine basic mechanistic concepts in many areas of biology within the context of physiologically relevant tissue microenvironments. This has also led to new insights into the behavior of immune cells at steady state, and how their behaviors are altered during an immune response. At the same time, advances in the humanized mouse model have allowed for in vivo studies of strictly human pathogens, such as HIV-1. Here, we describe in detail an intravital microscopy approach to visualize the dynamic behavior of HIV-infected T cells within the lymph nodes of live, anesthetized humanized mice.

A close up view of retrovirus spreading Viral infections typically begin with a small number of viral particles gaining access to the host at a specific tissue site. But how do viruses that cause systemic infections, such as HIV, spread more widely? Sewald et al. visualized how the retroviruses murine leukemia virus (MLV) and HIV spread within lymph nodes in mice (see the Perspective by Hope). Specific macrophages that line the lymph-draining sinuses in lymph nodes first captured the virus using the carbohydrate-binding protein CD169. These macrophages subsequently transferred virus to the B1 subclass of B lymphocytes, which migrated further into the lymph node, disseminating the virus more widely. Science , this issue p. 563 ; see also p. 511

Initial events after exposure determine HIV-1 disease progression, underscoring a critical need to understand host mechanisms that interfere with initial viral replication. Although associated with chronic HIV-1 control, it is not known whether interleukin-21 (IL-21) contributes to early HIV-1 immunity. Here we take advantage of tractable primary human lymphoid organ aggregate cultures to show that IL-21 directly suppresses HIV-1 replication, and identify microRNA-29 (miR-29) as an antiviral factor induced by IL-21 in CD4 T cells. IL-21 promotes transcription of all miR-29 species through STAT3, whose binding to putative regulatory regions within the MIR29 gene is enriched by IL-21 signalling. Notably, exogenous IL-21 limits early HIV-1 infection in humanized mice, and lower viremia in vivo is associated with higher miR-29 expression. Together, these findings reveal a novel antiviral IL-21-miR-29 axis that promotes CD4 T-cell-intrinsic resistance to HIV-1 infection, and suggest a role for IL-21 in initial HIV-1 control in vivo., HIV-infected patients who maintain undetectable virus levels possess elevated plasma concentrations of IL-21. Here, Adoro et al. show that IL-21 inhibits early viral infection in humanized mice and suppresses HIV-1 replication in vitro by upregulating a microRNA via the regulatory protein STAT3.

The expression of viral envelope genes can cause HIV-1-infected cells to fuse with CD4-receptor and coreceptor-expressing uninfected cells, leading to the formation of syncytia, or so-called multinucleated giant cells. Early histological studies have reported the presence of HIV-induced syncytia in tissues of infected individuals, and evidence for the involvement of both dendritic cells and lymphocytes in syncytia formation has been put forward.1,2 The fusion of uninfected CD4+ T cells has been suggested to contribute to their depletion, ultimately leading to a generalized immunodeficiency and increased susceptibility to AIDS-related illnesses. More recently, however, syncytia formation has generally been discounted as a major mechanism of T cell death because of their low frequency within infected tissues, and because of mounting evidence in favor of alternative mechanisms of T cell depletion, such as through persistent immune activation and abortive T cell infection. Thus, although the emergence of CXCR4-tropic, syncytia-inducing strains of HIV-1 in the circulation of infected individuals is associated with a more pronounced CD4+ T cell decline and progression to AIDS, the pathophysiological significance of HIV-induced syncytia formation remains unclear. We have recently begun to investigate the behavior of HIV virions and of HIV-infected immune cells in vivo through their visualization in the BLT humanized mouse model of HIV infection using fluorescent viral reporter strains and multiphoton intravital microscopy. Our initial studies, in which we explored the migratory behavior of T cells productively infected with CCR5-tropic, GFP-expressing HIV strains in the lymph node, revealed that the formation of T cell-derived syncytia occurred early (within 2 days) after initial infection. However, these were distinct from syncytia observed in vitro in that they were highly motile, and that individual nuclei moved in different directions, yet remained tethered to each other by fine membrane bridges.3 Additionally, early syncytia, although stretching out over distances of more than 100 μm, typically contained no more than four to five discernible nuclei. Here we report that only several days later, on days 5–6 after infection, some syncytia had dramatically increased in size, with volumes exceeding by 50-fold over those of individual, infected T cells (Fig. 1). They also adopted new morphological characteristics, such as a more massive cell body and an overall “dendritic” appearance, but nevertheless remained dynamic through the movements of their extensions. These changes may simply reflect their larger size compared to earlier syncytia, or could be the result of fusion with cells other than T cells to form mixed syncytia, similar to those observed in dendritic cells and T cell cocultures in vitro.4 FIG. 1. Large syncytium with multiple dendritic extensions in the lymph node of a BLT humanized NOD-SCID mice. The footpad was injected with 1×105 IU of HIV-GFP and the draining popliteal lymph node was prepared for intravital microscopy at day 6 postinfection. ... We have shown that HIV-induced syncytia remain viable and motile in the lymph nodes of infected BLT mice. Using our intravital imaging approaches, we hope to address additional questions, including (1) how and from which cells they are formed, (2) how their formation is affected by changes in viral envelope fusogenicity, and (3) whether syncytia formation enhances viral replication and spread in some way, or is merely a viral “accident” and dead-end of viral replication.

CCL5 (RANTES (regulated on activation normal T cell expressed and secreted)) and its cognate receptor, CCR5, have been implicated in T cell activation. CCL5 binding to glycosaminoglycans (GAGs) on the cell surface or in extracellular matrix sequesters CCL5, thereby immobilizing CCL5 to provide the directional signal. In two CCR5-expressing human T cell lines, PM1.CCR5 and MOLT4.CCR5, and in human peripheral blood-derived T cells, micromolar concentrations of CCL5 induce apoptosis. CCL5-induced cell death involves the cytosolic release of cytochrome c, the activation of caspase-9 and caspase-3, and poly(ADP-ribose) polymerase cleavage. CCL5-induced apoptosis is CCR5-dependent, since native PM1 and MOLT4 cells lacking CCR5 expression are resistant to CCL5-induced cell death. Furthermore, we implicate tyrosine 339 as a critical residue involved in CCL5-induced apoptosis, since PM1 cells expressing a tyrosine mutant receptor, CCR5Y339F, do not undergo apoptosis. We show that CCL5-CCR5-mediated apoptosis is dependent on cell surface GAG binding. The addition of exogenous heparin and chondroitin sulfate and GAG digestion from the cell surface protect cells from apoptosis. Moreover, the non-GAG binding variant, (44AANA47)-CCL5, fails to induce apoptosis. To address the role of aggregation in CCL5-mediated apoptosis, nonaggregating CCL5 mutant E66S, which forms dimers, and E26A, which form tetramers at micromolar concentrations, were utilized. Unlike native CCL5, the E66S mutant fails to induce apoptosis, suggesting that tetramers are the minimal higher ordered CCL5 aggregates required for CCL5-induced apoptosis. Viewed altogether, these data suggest that CCL5-GAG binding and CCL5 aggregation are important for CCL5 activity in T cells, specifically in the context of CCR5-mediated apoptosis.

Vaccinia virus, a poxvirus, produces structurally distinct forms of virions for which the immediate events following cell entry are ill-defined. We provide evidence that intracellular mature virus (IMV) enters both permissive and nonpermissive T-cell lines and that introduction of CCR5 into nonpermissive mouse fibroblasts or human primary T cells renders the cells permissive for vaccinia replication. Notably, T cells expressing CCR5 in which tyrosine 339 in the intracellular region is replaced by phenylalanine no longer support virus replication or virus-inducible activation of specific host cell signaling effectors IRS-2, Grb2, and Erk1/2. We show that following IMV entry into the cell, the intact but not the tyrosine-deficient CCR5 is rapidly internalized and colocalizes with virus. This colocalization precedes virus-inducible signaling and replication.

In vitro studies in primary or immortalized cells continue to be used to elucidate the essential principles that govern the interactions between HIV-1 and isolated target cells. However, until recently, substantial technical barriers prevented this information from being efficiently translated to the more complex scenario of HIV-1 spread in the host in vivo, which has limited our understanding of the impact of host physiological parameters on the spread of HIV-1. In this Review, we discuss the recent development of imaging approaches to visualize HIV-1 spread and the adaptation of these approaches to organotypic ex vivo models and animal models. We focus on new concepts, including the mechanisms and in vivo relevance of cell-cell transmission for HIV-1 spread and the function of the HIV-1 pathogenesis factor Nef, which have emerged from the application of these integrative approaches in complex cell systems.

The impact of CD4+ regulatory T cells (Tregs) on human immunodeficiency virus type 1 (HIV-1) pathogenesis remains incompletely understood. Although it has been shown that Tregs can be infected with HIV-1, the consequences of infection on a per-cell basis are still unknown. In vitro HIV-GFP infected and noninfected Tregs were isolated by flow-based cell-sorting to investigate Treg suppressive capacity and gene expression profiles. Our data show that HIV-1-infected Tregs were significantly less suppressive than noninfected Tregs and demonstrated down-regulation of genes critical to Treg function. This impaired function may have detrimental consequences for the control of generalized immune activation and accelerate HIV disease progression.

Summary Differentiation of naive CD4 + T cells into T helper (Th) cells is a defining event in adaptive immunity. The cytokines and transcription factors that control Th cell differentiation are understood, but it is not known how this process is orchestrated within lymph nodes (LNs). Here we have shown that the CXCR3 chemokine receptor was required for optimal generation of interferon-γ (IFN-γ)-secreting Th1 cells in vivo. By using a CXCR3 ligand reporter mouse, we found that stromal cells predominately expressed the chemokine ligand CXCL9 whereas hematopoietic cells expressed CXCL10 in LNs. Dendritic cell (DC)-derived CXCL10 facilitated T cell-DC interactions in LNs during T cell priming while both chemokines guided intranodal positioning of CD4 + T cells to interfollicular and medullary zones. Thus, different chemokines acting on the same receptor can function locally to facilitate DC-T cell interactions and globally to influence intranodal positioning, and both functions contribute to Th1 cell differentiation.