Semaphorin 3E Promotes Susceptibility to Leishmania major Infection in Mice by Suppressing CD4+ Th1 Cell Response.

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.

The transcription factor NFAT exhibits signal memory during serial T cell interactions with antigen-presenting cells.

Interactions with antigen-presenting cells (APCs) interrupt T cell migration through tissues and trigger signaling pathways that converge on the activation of transcriptional regulators, including nuclear factor of activated T cells (NFAT), which control T cell function and differentiation. Both stable and unstable modes of cognate T cell-APC interactions have been observed in vivo, but the functional significance of unstable, serial contacts has remained unclear. Here we used multiphoton intravital microscopy in lymph nodes and tumors to show that while NFAT nuclear import was fast (t(1/2 max)∼1 min), nuclear export was slow (t(1/2)∼20 min) in T cells. During delayed export, nuclear NFAT constituted a short-term imprint of transient TCR signals and remained transcriptionally active for the T cell tolerance gene Egr2, but not for the effector gene Ifng, which required continuous TCR triggering for expression. This provides a potential mechanistic basis for the observation that a predominance of unstable APC interactions correlates with the induction of T cell tolerance.

CXCR3 chemokine receptor-ligand interactions in the lymph node optimize CD4+ T helper 1 cell differentiation.

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.

HIV Infection Stabilizes Macrophage-T Cell Interactions To Promote Cell-Cell HIV Spread.

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.

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

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-infected T cells are migratory vehicles for viral dissemination.

After host entry through mucosal surfaces, human immunodeficiency virus-1 (HIV-1) disseminates to lymphoid tissues to establish a generalized infection of the immune system. The mechanisms by which this virus spreads among permissive target cells locally during the early stages of transmission and systemically during subsequent dissemination are not known. In vitro studies suggest that the formation of virological synapses during stable contacts between infected and uninfected T cells greatly increases the efficiency of viral transfer. It is unclear, however, whether T-cell contacts are sufficiently stable in vivo to allow for functional synapse formation under the conditions of perpetual cell motility in epithelial and lymphoid tissues. Here, using multiphoton intravital microscopy, we examine the dynamic behaviour of HIV-infected T cells in the lymph nodes of humanized mice. We find that most productively infected T cells migrate robustly, resulting in their even distribution throughout the lymph node cortex. A subset of infected cells formed multinucleated syncytia through HIV envelope-dependent cell fusion. Both uncoordinated motility of syncytia and adhesion to CD4(+) lymph node cells led to the formation of long membrane tethers, increasing cell lengths to up to ten times that of migrating uninfected T cells. Blocking the egress of migratory T cells from the lymph nodes into efferent lymph vessels, and thus interrupting T-cell recirculation, limited HIV dissemination and strongly reduced plasma viraemia. Thus, we have found that HIV-infected T cells are motile, form syncytia and establish tethering interactions that may facilitate cell-to-cell transmission through virological synapses. Migration of T cells in lymph nodes therefore spreads infection locally, whereas their recirculation through tissues is important for efficient systemic viral spread, suggesting new molecular targets to antagonize HIV infection.

HIV-1 infection impairs regulatory T-cell suppressive capacity on a per-cell basis.

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.

Intravital microscopy in BLT-humanized mice to study cellular dynamics in HIV 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.

An HIV-1 CRISPR-Cas9 membrane trafficking screen reveals a role for PICALM intersecting endolysosomes and immunity.

HIV-1 hijacks host proteins involved in membrane trafficking, endocytosis, and autophagy that are critical for virus replication. Molecular details are lacking but are essential to inform on the development of alternative antiviral strategies. Despite their potential as clinical targets, only a few membrane trafficking proteins have been functionally characterized in HIV-1 replication. To further elucidate roles in HIV-1 replication, we performed a CRISPR-Cas9 screen on 140 membrane trafficking proteins. We identified phosphatidylinositol-binding clathrin assembly protein (PICALM) that influences not only infection dynamics but also CD4+ SupT1 biology. The knockout (KO) of PICALM inhibited viral entry. In CD4+ SupT1 T cells, KO cells exhibited defects in intracellular trafficking and increased abundance of intracellular Gag and significant alterations in autophagy, immune checkpoint PD-1 levels, and differentiation markers. Thus, PICALM modulates a variety of pathways that ultimately affect HIV-1 replication, underscoring the potential of PICALM as a future target to control HIV-1.

Transformer-based spatial-temporal detection of apoptotic cell death in live-cell imaging.

Intravital microscopy has revolutionized live-cell imaging by allowing the study of spatial-temporal cell dynamics in living animals. However, the complexity of the data generated by this technology has limited the development of effective computational tools to identify and quantify cell processes. Amongst them, apoptosis is a crucial form of regulated cell death involved in tissue homeostasis and host defense. Live-cell imaging enabled the study of apoptosis at the cellular level, enhancing our understanding of its spatial-temporal regulation. However, at present, no computational method can deliver robust detection of apoptosis in microscopy timelapses. To overcome this limitation, we developed ADeS, a deep learning-based apoptosis detection system that employs the principle of activity recognition. We trained ADeS on extensive datasets containing more than 10,000 apoptotic instances collected both in vitro and in vivo, achieving a classification accuracy above 98% and outperforming state-of-the-art solutions. ADeS is the first method capable of detecting the location and duration of multiple apoptotic events in full microscopy timelapses, surpassing human performance in the same task. We demonstrated the effectiveness and robustness of ADeS across various imaging modalities, cell types, and staining techniques. Finally, we employed ADeS to quantify cell survival in vitro and tissue damage in mice, demonstrating its potential application in toxicity assays, treatment evaluation, and inflammatory dynamics. Our findings suggest that ADeS is a valuable tool for the accurate detection and quantification of apoptosis in live-cell imaging and, in particular, intravital microscopy data, providing insights into the complex spatial-temporal regulation of this process.

Identifying physiological tissue niches that support the HIV reservoir in T cells.

Successful antiretroviral therapy (ART) can efficiently suppress Human Immunodeficiency Virus-1 (HIV-1) replication to undetectable levels, but rare populations of infected memory CD4+ T cells continue to persist, complicating viral eradication efforts. Memory T cells utilize distinct homing and adhesion molecules to enter, exit, or establish residence at diverse tissue sites, integrating cellular and environmental cues that maintain homeostasis and life-long protection against pathogens. Critical roles for T cell receptor and cytokine signals driving clonal expansion and memory generation during immunity generation are well established, but whether HIV-infected T cells can utilize similar mechanisms for their own long-term survival is unclear. How infected, but transcriptionally silent T cells maintain their recirculation potential through blood and peripheral tissues, or whether they acquire new capabilities to establish unique peripheral tissue niches, is also not well understood. In this review, we will discuss the cellular and molecular cues that are important for memory T cell homeostasis and highlight opportunities for HIV to hijack normal immunological processes to establish long-term viral persistence.

Antigen recognition reinforces regulatory T cell mediated Leishmania major persistence.

Cutaneous Leishmania major infection elicits a rapid T cell response that is insufficient to clear residually infected cells, possibly due to the accumulation of regulatory T cells in healed skin. Here, we used Leishmania-specific TCR transgenic mice as a sensitive tool to characterize parasite-specific effector and immunosuppressive responses in vivo using two-photon microscopy. We show that Leishmania-specific Tregs displayed higher suppressive activity compared to polyclonal Tregs, that was mediated through IL-10 and not through disrupting cell-cell contacts or antigen presentation. In vivo expansion of endogenous Leishmania-specific Tregs resulted in disease reactivation that was also IL-10 dependent. Interestingly, lack of Treg expansion that recognized the immunodominant Leishmania peptide PEPCK was sufficient to restore robust effector Th1 responses and resulted in parasite control exclusively in male hosts. Our data suggest a stochastic model of Leishmania major persistence in skin, where cellular factors that control parasite numbers are counterbalanced by Leishmania-specific Tregs that facilitate parasite persistence.

Nonoptimal bacteria species induce neutrophil-driven inflammation and barrier disruption in the female genital tract.

Neutrophil recruitment and activation within the female genital tract are often associated with tissue inflammation, loss of vaginal epithelial barrier integrity, and increased risk for sexually transmitted infections, such as HIV-1. However, the direct role of neutrophils on vaginal epithelial barrier function during genital inflammation in vivo remains unclear. Using complementary proteome and immunological analyses, we show high neutrophil influx into the lower female genital tract in response to physiological surges in progesterone, stimulating distinct stromal, immunological, and metabolic signaling pathways. However, despite the release of extracellular matrix-modifying proteases and inflammatory mediators, neutrophils contributed little to physiological mucosal remodeling events such as epithelial shedding or re-epithelialization during transition from diestrus to estrus phase. In contrast, the presence of bacterial vaginosis-associated bacteria resulted in a rapid and sustained neutrophil recruitment, resulting in vaginal epithelial barrier leakage and decreased cell-cell junction protein expression in vivo. Thus, neutrophils are important mucosal sentinels that rapidly respond to various biological cues within the female genital tract, dictating the magnitude and duration of the ensuing inflammatory response at steady state and during disease processes.

HPV and the Risk of HIV Acquisition in Women.

The risk of HIV acquisition is low on a per-contact basis but increased by transmission co-factors such as other sexually transmitted infections (STIs). Human papillomavirus (HPV) is a prevalent STI that most individuals will acquire HPV in their lifetime. Current HPV vaccines can prevent newly acquired infections, but are largely ineffective against established HPV, complicating worldwide eradication efforts. In addition to being the causative agent of cervical cancer, accumulating evidence suggests that HPV infection and/or accompanying cervical inflammation increase the risk of HIV infection in men and women. The fact that immunological features observed during HPV infection overlap with cellular and molecular pathways known to enhance HIV susceptibility underscore the potential interplay between these two viral infections that fuel their mutual spread. Here we review current insights into how HPV infection and the generation of anti-HPV immunity contribute to higher HIV transmission rates, and the impact of HPV on mucosal inflammation, immune cell trafficking, and epithelial barrier function.

T cell migration potentiates HIV infection by enhancing viral fusion and integration.

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.

MiRNA-103 downmodulates CCR5 expression reducing human immunodeficiency virus type-1 entry and impacting latency establishment in CD4+ T cells.

Activated-to-memory transitioning CD4+ T cells display elevated expression of the HIV-1 co-receptor CCR5 and are more prone to HIV-1 latent infection. Here, we show that p53-regulated miRNA-103 downmodulates CCR5 levels in CD4+ T lymphocytes. We reveal that miRNA-103 mimics, as well as Nutlin-3, an inhibitor of Mdm2-mediated p53 degradation, decrease CCR5-dependent HIV-1 infection. Using a dual-reporter virus, we subsequently validate that in transitioning CD4+ T cells, Nutlin-3 treatment decreases the frequency of both productively and latently infected cells via upregulation of miRNA-103. Importantly, we provide evidence that CD4+ T cells from HIV-1 elite controllers express less CCR5 than those from antiretroviral therapy-naïve progressors, an effect linked to a significant increase in miRNA-103 levels. By contributing to the control of CCR5 expression in CD4+ T cells, miRNA-103 is likely to play a key role in countering the establishment of latent HIV-1 reservoirs in vivo.

Visualizing the In Vivo Dynamics of Anti-Leishmania Immunity: Discoveries and Challenges.

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.

Mouse Papillomavirus L1 and L2 Are Dispensable for Viral Infection and Persistence at Both Cutaneous and Mucosal Tissues.

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.

Role for CCR5 in dissemination of vaccinia virus in vivo.

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.

CCL5-mediated T-cell chemotaxis involves the initiation of mRNA translation through mTOR/4E-BP1.

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.