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A substantial proportion of acute stroke patients fail to recover following successful endovascular therapy (EVT) and injury to the brain and vasculature secondary to reperfusion may be a contributor. Acute stroke patients were included with: i) large vessel occlusion of the anterior circulation, ii) successful recanalization, and iii) evaluable MRI early after EVT. Presence of hyperemia on MRI perfusion was assessed by consensus using a modified ASPECTS. Three different approaches were used to quantify relative cerebral blood flow (rCBF). Sixty-seven patients with median age of 66 [59–76], 57% female, met inclusion criteria. Hyperemia was present in 35/67 (52%) patients early post-EVT, in 32/65 (49%) patients at 24 hours, and in 19/48 (40%) patients at 5 days. There were no differences in incomplete reperfusion, HT, PH-2, HARM, severe HARM or symptomatic ICH rates between those with and without early post-EVT hyperemia. A strong association (R2 = 0.81, p

Introduction: A substantial proportion of acute stroke patients experience poor outcomes following successful endovascular therapy (EVT) and reperfusion injury to the brain and vasculature may be a contributor. The purpose of this pilot study was to characterize hyperemia following recanalization and test the association of hyperemia with evolution of the ischemic lesion. Methods: Acute stroke patients were included with: i) large vessel occlusion of the anterior circulation, ii) successful recanalization, and iii) evaluable MRI early after EVT. Presence of hyperemia on MRI perfusion was assessed by consensus using a modified ASPECTS. Three different approaches were used to quantify relative cerebral blood flow (rCBF) on early post-EVT, 24 hours, and 5 days. Two independent trained readers measured lesion volumes using a semi-automated validated approach. Results: Sixty-seven patients with median age of 66 [59-76], 57% female, met inclusion criteria. Hyperemia was present in 35/67 (52%) patients early post-EVT (Figure), in 32/65 (49%) patients at 24 hours, and in 19/48 (40%) patients at 5 days. In comparison to control regions, rCBF was elevated by greater than 50% in hyperemic regions. Patients with hyperemia had larger ischemic lesion volumes and early lesion growth. A strong association (R2=0.81, p Conclusions: Hyperemia is frequently observed after successful EVT and is associated with lesion growth at 24 hours. Evidence of hyperemia may identify patients that may benefit from adjunctive therapy to prevent reperfusion injury, edema, and lesion growth.

INTRODUCTION: Glioblastoma (GBM) represents the deadliest primary intracranial neoplasm. While many studies have focused on T cell responses to GBM, few have examined the role of humoral immunity. Here, we sought mechanistic insights into the anatomy and function of the humoral response against GBM. METHODS: C57BL/6J mice were injected stereotactically with syngeneic SB28 tumor cells. Humoral immunity was evaluated in the spleen and blood by high parameter flow cytometry and the meninges, brain, and spleen by confocal microscopy. The function importance of humoral immunity was interrogated using CD4-/- and IgA-/- mice and via transcranial application of the plasma cell survival factor, APRIL. RESULTS: High parameter flow cytometry revealed a significant increase of plasma B cells (CD19+, CD138+) in the spleen and blood of GBM mice within two weeks. Using blimp-1 reporter mice, we observed that IgA+ plasma cells localized to extrafollicular splenic regions and along meningeal dural venous sinuses near newly generated blood and lymphatic vessels that connected directly to the tumor. Decreased survival was observed in IgA-/- but not CD4-/- mice injected with SB28 cells, suggesting that a T helper independent IgA response plays a role in controlling GBM. This response was enhanced therapeutically by transcranial application of APRIL. CONCLUSION: Our results demonstrate that GBM elicits a systemic and meningeal humoral response, which plays in limiting GBM growth and can be transcranially enhanced via APRIL. The participation of IgA+ plasma cells in GBM immunity offers new possibilities for therapeutic intervention in this deadly disease.

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Persistent immune activation remains the hallmark of HIV infection even in the context of successfully suppressed viremia. Its clinical significance is suggested by increased risk to develop non-AIDS defining illnesses such as cardiovascular disease. We previously have shown that human CD4 and CD8 T cells with effector memory and terminal effector memory phenotype expressed the protease activated receptor-1 (PAR1) and that expression was increased in CD8 T cells from HIV infected patients. Thrombin, in a PAR1 dependent fashion, enhanced CD8 T cell function measured by increased secretion of IFN-g and chemokinesis. PAR1 is the main receptor of thrombin and is also expressed on platelets and endothelial cells suggesting a cross-talk between the coagulation system and T cells at the site of vascular injury. To address the role of PAR1 in vivo, we used an infection model with the hepatotropic strain of lymphocytic choriomeningitis virus (LCMV-WE). To evaluate the role of PAR1 signaling in this model, we analyzed the T cells of littermate wild type control and PAR1−/−mice eight days following infection with LCMV-WE (2×105 pfu i.v.). In this model, we observed a similar expansion of total CD8 T cells in the spleens and livers of both WT and PAR1−/−mice at 8 days post infection. Despite similar expansion, PAR1 deficient mice showed impaired viral clearance at day 8 of infection. This lack of viral control was associated with a significant reduction in cytokine secretion by gp-33-specific CD8 T cells in the livers and spleens. These results suggest that PAR1 deficiency leads to an impair viral control that is in part mediated by a decrease ability to secrete cytokine by virus specific CD8 T cells.

Cerebral malaria is a potentially fatal complication associated with Plasmodium falciparum infection. Disease pathology includes blood brain barrier (BBB) breakdown, microvascular hemorrhage, edema and brainstem herniation. Studies in rodents (referred to as experimental cerebral malaria) have revealed that parasite-specific CD8+ T cells induce this pathology via engagement of cerebrovascular endothelial cells (ECs). In this study, we sought novel insights into the mechanisms underlying alterations in BBB structure and function during the development of ECM. We developed a novel transgenic reporter system that allowed us to simultaneously monitor calcium signaling in cerebrovascular ECs and parasite-specific CD8+ T cells. We then used intravital two-photon microscopy (TPM) to monitor BBB dynamics in real-time at the peak of disease. Within venous networks, we observed a significant increase in the magnitude and propagation of EC calcium signaling. Calcium wave propagation along lengthy stretches of cerebrovascular endothelium was associated with regions of parasite-specific CD8+ T cell engagement (both stable and dynamic). Recognition of cognate peptide-MHC on luminal and abluminal EC surfaces resulted in calcium fluxes within parasite-specific CD8+ T cells, which was followed temporally by calcium wave propagation in the associated vasculature. Importantly, this pathophysiological response was completely eliminated by depletion of T cells. We postulate that T cell-mediated induction of calcium waves along cerebral blood vessels promotes noncytopathic disruption of the BBB. Therapeutic manipulation of this activity might help restore BBB homeostasis and prevent neurological complications during ECM.

Pathogens that gain entry into the CNS elicit a uniquely tailored response from the immune compartment by modulating cytolytic and inflammatory functions to confer viral clearance with minimal brain damage. Neurotropic viruses such as vesicular stomatitis virus (VSV) infect neurons in the olfactory epithelial barrier and pass via sensory axons directly into the brain. Within the olfactory bulb, VSV-infected neurons contact a variety of CNS neurons; however, the infection rarely invades the brain. While type I interferon responses are crucial for initial viral control, little is known about how the local adaptive immune response prevents fatal viral neuroinvasion. In this study, we sought insights into how cytotoxic lymphocytes (CTL) prevent the distal spread of infection from the olfactory bulb. Without T cells, virus containment is compromised and VSV traverses caudally to hindbrain regions. Utilizing intravital two-photon microscopy and other techniques, we characterized the mechanism by which T cell interactions within the olfactory bulb facilitate barrier protection. Interestingly, chimeric mice deficient in MHC class I on brain-resident cells, as well as microglia-depleted mice, showed reduced antiviral T cell calcium signaling upon engagement of target cells, suggesting that microglia can cross-present viral antigen and indirectly facilitate viral clearance from adjacent virally infected neurons. This study has revealed a novel mechanism of T cell-mediated viral control in neurons that involves engagement of resident myeloid cells that acquire antigen but are not infected by the virus. This in turn provides barrier protection against a virus that attempts to enter the CNS via the nasal route.

Adoptive cell transfer (ACT) immunotherapy can result in complete responses in the treatment of metastatic solid cancers, however the majority of patients eventually progress. Although many immune regulatory programs are context-dependent, the in vivo behavior of adoptively-transferred CD8+ T cells over time remains unknown. Using the Pmel-1 model, we performed intravital microscopy of subcutaneous tumors in mice to visualize the in vivo motility of transferred tumor-specific CD8+ T cells. This revealed that CD8+ T cell motility arrest increased over time after adoptive transfer. By ex vivo analyses, behavioral change coincided with a reduction in IFN-γ production and glucose uptake. As the transferred cells strongly expressed PD-1, we treated mice with both ACT and anti-PD-1 blocking antibody to determine the effect of immune checkpoint blockade on cellular motility. In mice treated with anti-PD-1 blocking antibody, transferred intratumoral CD8+ T cell motility arrest diminished, as compared with controls. This corresponded with an increase in CD8+ T cell glucose uptake. These data demonstrate that intratumoral CD8+ T cell behavior in ACT is dynamic and that greater motility is associated with increased functionality and bioenergetic demand. Furthermore, concurrent PD-1 blockade exerts a cell-intrinsic effect on transferred CD8+ T cells suggesting synergy between ACT and anti-PD-1 immunotherapy and the potential for their combinatorial use in the treatment of cancer

Classic (M1) and alternative (M2) polarization of macrophages has been broadly studied in vitro but its relevance in the context of meningeal inflammation is unknown. The meningeal and perivascular spaces of the central nervous system (CNS) are inhabited by specialized macrophages. To gain novel insights into these cells, we analyzed the naive and virally infected brain using intravital two-photon microscopy (TPM) and flow cytometry. Examination of the naive brain by TPM revealed that meningeal and perivascular macrophages are highly dynamic and constantly survey their immediate surroundings similar to microglia. Under steady state conditions, we uncovered that they are maintained in a M2 state, which likely facilitates brain homeostasis. Interestingly, during the development of fatal viral meningitis induced by lymphocytic choriomeningitis virus, these cells were directly engaged by infiltrating virus-specific CD8+ T cells following acquisition of viral antigen and conversion into an inflammatory M1 phenotype. Mechanistically, the M2 to M1 transition occurred in the absence of CD8+ T cells and relied instead on innate cytokine release. These data indicate that meningeal and perivascular macrophages are highly plastic cells that can rapidly convert from a M2 to M1 phenotype and participate in the antiviral defense against an invading pathogen. This rapid inflammatory transition may explain why most CNS immune responses first develop in the meningeal and perivascular spaces.

Type I interferons play a critical role in host defense against most viral infections. This is achieved in part through induction of innate antiviral proteins like tetherin (BST-2). BST-2 was identified based on its ability to “tether” viral proteins to infected cells and decrease virion release in vitro. How this protein functions in the host defense against a persistent viral infection in vivo is not entirely understood. To gain novel insights into BST-2 function, we examined its role during the establishment of a persistent lymphocytic choriomeningitis virus (LCMV) infection. Transgenic overexpression of BST-2 in cell culture decreased release of LCMV from infected cells. We uncovered in vivo that BST-2 is significantly upregulated early after viral infection in plasmacytoid / myeloid dendritic cells, monocytes / macrophages, and brain-resident microglia. Deletion of BST-2 enabled a persistence-prone strain of LCMV to transition more efficiently from the splenic marginal zone to the white pulp following intravenous inoculation. This resulted in enhanced T cell exhaustion, which was evidenced by reduced cytokine production and altered motility. Interestingly, these early alterations in antiviral immunity translated into delayed control of LCMV in peripheral tissues and lifelong persistence in the brain. These data demonstrate that expression of single innate antiviral protein can influence adaptive immune cell function and dictate the fate of a viral infection.

The goal of anti-viral therapy is to promote pathogen clearance without causing tissue injury. Therapeutic administration of anti-viral T cells (adoptive immunotherapy) has shown promise in clinical studies for the treatment of CMV, EBV, and adenovirus. Our laboratory models adoptive immunotherapy by transferring anti-viral memory T cells into mice persistently infected from birth with lymphocytic choriomeningitis virus (LCMV). Here we demonstrate that brain-resident myeloid cells (microglia), in addition to neurons, are persistently infected with LCMV in carrier mice. Therapeutic memory T cells can completely purge virus from persistently infected microglia and neurons without causing blood brain barrier breakdown or cellular injury. This is accomplished through a tailored release of chemoattractants that recruit adaptive immune cells, but few pathogenic innate immune cells. In addition, memory T cells enlist the support of nearly all microglia by converting them into CD11c-expressing antigen-presenting cells (APCs). We observed similarly converted myeloid cells in the brains of humans infected with JC virus and HIV-1. Using two-photon microscopy, we revealed that during clearance of the LCMV-infected brain anti-viral CD8 T cells form stable, noncytopathic interactions with CD11c+ microglia, whereas CD4 T cells interact transiently. These data demonstrate that brain-resident APCs can thwart the cytolytic functions of anti-viral T cells while allowing viral clearance.

Experimental autoimmune encephalomyelitis is a murine model for multiple sclerosis in which autoreactive CD4+ T-helper cells infiltrate the central nervous system (CNS) and initiate autoimmune inflammation. Previous studies have shown that EAE induction via the adoptive transfer of CNS antigen-specific Th17 cells requires T-cell-intrinsic expression of the transcription factor T-bet, but the potential pathogenic role of T-bet expression in non-T-helper cells remains unclear. Using T-bet-ZsGreen reporter mice, we confirmed that multiple non-T-helper cell populations express T-bet in the CNS during active EAE. To assess the pathogenic contribution of T-bet in non-Th cells, we transferred 2D2-transgenic (expressing a CNS antigen-specific TCR) T-bet-/- or WT Th17 cells into T-bet-/- or WT recipient mice. Consistent with previous reports, 2D2 T-bet-/- Th17 cells did not induce significant levels of EAE disease in either T-bet-/- or WT recipient mice, supporting the role for T-bet in CD4+ Th cells. Surprisingly, WT Th17 cells, while fully pathogenic in WT hosts, were unable to induce EAE in T-bet-/- hosts. Our preliminary data suggest that 2D2 WT Th17 cells were unable to breach the blood-brain barrier and infiltrate the CNS parenchyma due to ineffective local re-activation of 2D2 WT cells in the CNS of T-bet-/- recipients. Ongoing studies will seek to identify the role of T-bet expression in non-Th cell populations and their interactions with 2D2 Th17 cells in EAE induction.

Lymphocytes are essential for control of neurotropic viruses such as HSV, HIV, and JC virus. Understanding the mechanisms involved in the regulation of these cells is essential for the development of therapeutic interventions to treat central nervous system (CNS) infections. Using two-photon microscopy, we observed that CD8 T cells responding to a viral infection interact with meningeal stromal cells (MSCs), a population of specialized fibroblasts that comprise a dense, collagenous fiber network within the lining of the brain. Little is known about the immunological functions of MSCs. To gain novel insights into these cells, we conducted genomic analyses of purified MSCs at different time points following CNS infection with lymphocytic choriomeningitis virus (LCMV). Our analyses revealed that MSCs responded to virus infection through induction of a robust immunological program that included genes associated with antigen presentation, chemotaxis, immune regulation, among others. We also observed in vivo that MSCs were able to present viral-peptide MHC I complexes, which corroborated our intravital imaging studies showing direct, stable interactions between MSCs and anti-viral T cells. MSCs possess the capacity to regulate these interactions through expression of multiple immune regulators such as PD-L1, galectin-9, and PVR. Our data demonstrate that MSCs are a novel CNS-resident antigen presenting cell with the ability to modulate antiviral T cells following infection.

Many viruses can establish persistence and cause disease in humans. Studies have established a role for T cells in resolving persistent viral infections; however, the importance of a humoral response is less clear. In this study, we sought novel insights into the importance of humoral immunity in resolving a persistent infection by utilizing the lymphocytic choriomeningitis virus (LCMV) clone 13 model. Infection with aggressive LCMV variants results in a chronic viral infection during which antiviral T cells pass through a state of functional exhaustion, but the infection is nevertheless controlled over time. We postulated that humoral immunity was required for LCMV control and could be harnessed as an immunotherapy. We observed dramatic increases in splenic plasma cells as early as one week following LCMV infection, which coincided with the emergence of non-neutralizing antibodies. B cell depletion starting at the peak of T cell exhaustion (day 15) resulted in a failure to control virus. Moreover, adoptive transfer of LCMV immune B cells into mice with an active infection dramatically accelerated viral clearance. We found using two-photon microscopy that therapeutic B cells home to the infected recipient spleen where donor antibody secreting cells proliferate to amplify antiviral humoral responses. These data demonstrate that humoral immunity can contribute to the resolution of a persistent viral infection and that B cells can be harnessed for adoptive immunotherapy.

Viral infection of the central nervous system (CNS) routinely induces immune-mediated vascular damage that compromises the integrity of the blood brain barrier (BBB) and induces neurological complications. We have shown previously that cytotoxic lymphocytes massively recruit monocytes and neutrophils to the brain following infection with lymphocytic choriomeningitis virus (LCMV). These cells promote vascular leakage through synchronous extravasation. Here, we focus on identifying the mechanism by which innate myelomonocytic cells injure brain vasculature. Quantitative analyses revealed that 6 metalloproteinases (MMP-2, 3, 8, 12, 13, and 19) and 1 disintegrin (ADAM-8) were highly expressed in symptomatic mice with LCMV meningitis. Among these, the collagenase, MMP-8, was expressed greater than 2000-fold in neutrophils and 200-fold in monocytes. We therefore postulated that vascular collagenase activity during viral meningitis promotes myelomonocytic cell extravasation and vessel leakage. This theory was tested experimentally by administering a specific MMP-8 antagonist to LCMV-infected mice prior to the development of symptoms. Our results revealed that MMP-8 antagonism significantly reduced vascular damage, BBB breakdown, and the development of symptoms in infected mice. These data demonstrate that inhibition of collagenases might be a viable therapeutic approach to lessen the pathogenic effects of innate inflammatory cells following CNS viral infection.

Several viruses can infect the mammalian nervous system, some with devastating consequences, and others with little or no overt pathology. Adoptive immunotherapy is an approach that has shown promise in clinical studies for the treatment of CMV, EBV and adenovirus infections. Our laboratory models immunotherapy by transferring memory T cells into mice persistently infected from birth with lymphocytic choriomeningitis virus. Here, we demonstrate that memory T cells can completely purge the brain of persistently infected mice without causing blood brain barrier breakdown or severe tissue damage. This is accomplished by a tailored release of chemoattractants that recruit adaptive immune cells, but few pathogenic innate immune cells such as neutrophils and inflammatory monocytes. Interestingly, memory T cells enlist the support of nearly all brain resident myeloid cells (microglia) by converting them into CD11c-expressing antigen-presenting cells (APC). Two-photon imaging studies revealed that anti-viral CD8 T cells are more likely to decelerate and form stable interactions with brain-resident APC than CD4 T cells. Importantly, microglia do not undergo cell death following T cell engagement and appear to protect themselves by upregulating serine protease inhibitors like Spi-6. We propose that non-cytopathic viral clearance from the brain by therapeutic memory T cells results from tailored chemoattractant production and interactions with resident myeloid cells protected by Spi-6.

Antigen presenting cells (APCs) upon activation break down proteins into peptides that are presented on major histocompatibility complexes (MHC). APCs help shape immune responses at sites of viral infection and can foster the development of immunopathology. Intracerebral infection of mice with lymphocytic choriomeningitis virus (LCMV) induces a CD8 T cell-dependent fatal meningitis. Because little is known about the dynamics of antigen-presentation in the brain, we set out to identify the APCs that influence CD8 T cell motility and function during the development of meningitis. This was accomplished by generating a LCMV recombinant expressing ovalbumin (OVA), which enabled us to quantify in vivo presentation of OVA257-264 (SIINFEKL) peptides by H-2Kb. We also used two-photon (2P) microscopy to study interactions between virus-specific CD8 T cells and different APC populations in the virally infected brain. Surprisingly, our results revealed that all brain resident APCs (dendritic cells, macrophages, and microglia) contained LCMV antigen during the development of meningitis, but only microglia had detectable H-2Kb-OVA257-264 complexes on their cell surface. The presentation capacity of microglia was confirmed by 2P data showing extensive CD8 T cell interactions and clustering around microglia. During LCMV meningitis, our findings suggest that CD8 T cell interactions with brain resident microglia are favored over those with peripherally-derived professional APCs.

Persistent viral infections are often very difficult to treat in humans. Adoptive immunotherapy is an approach that involves administration of anti-viral T cells and has shown some promise in the clinic. Our laboratory models adoptive immunotherapy by transferring anti-viral memory T cells into mice persistently infected from birth with lymphocytic choriomeningitis virus (LCMV). Here, we demonstrate that memory T cells can completely purge the brain of persistently infected mice without causing immunopathology or blood brain barrier breakdown. Memory T cells accomplish this by inducing a tailored release of chemoattractants that recruit adaptive immune cells, but few pathogenic innate immune cells (e.g. neutrophils and inflammatory monocytes) into the nervous system. Memory T cells also enlist the support of nearly all brain resident myeloid cells (referred to as microglia) by converting them into CD11c-expressing antigen-presenting cells (APCs). Following conversion, CD11c+ microglia release CCL5 and promote interactions with therapeutic memory T cells in the brain parenchyma. Our two photon imaging studies revealed that anti-viral CD8 T cells are more likely than CD4 T cells to decelerate and form stable interactions with brain-resident APCs. We propose that non-cytopathic viral clearance from the brain by therapeutic memory T cells results from tailored chemoattractant production and conversion of resident myeloid cells into CD11c+ APCs.

Viral infections of brain usually trigger inflammatory responses that give rise to a wide range of pathological outcomes. To gain novel insights into how the brain responds to a virus as it establishes persistence, we utilized genomic and two-photon imaging approaches to study a pure innate immune response to a lymphocytic choriomeningitis virus (LCMV) infection. This was accomplished by monitoring temporal gene expression patterns and myeloid cells dynamics in the brain of CX3CR1-GFP mice lacking LCMV-specific T cells. Within the first two weeks of infection, brain resident myeloid cells were highly activated and responded directly by sequestering DsRed-labeled viral antigen. At the genomic level, this triggered massive changes in gene expression that were attenuated over time (likely due to inhibition by viral nucleoprotein) and were all directly linked to type I interferon (IFN-I) signaling. Surprisingly, in the absence of IFN-I signaling, no genes were differentially expressed in the nervous system despite the presence of persistent virus. In addition, two-photon imaging studies revealed that IFN-I receptor deficient innate immune sentinels were completely unresponsive to the establishment of a persistent viral infection. These data demonstrate that IFN-I drives all innate immune activity in the brain following LCMV infection, and deficiency in IFN-I signaling renders LCMV invisible in its murine host.

Restoration of T cell function mediated by PD-1 blockade is a finding of great clinical relevance for the treatment of persistent infections and tumors. Presently, it is not known how PD-1 blockade improves the function of exhausted T cells. Infection of mice with an immunosuppressive strain of lymphocytic choriomeningitis virus (LCMV) is a well described model for T cell exhaustion. Here, we set out to gain novel insights into the mechanisms that underlie T cell exhaustion by imaging virus-specific CD8 and CD4 T cell responses to an acute versus persistent LCMV infection in the spleen using two-photon microscopy. Analysis of T cell dynamics in these two distinct environments revealed that T cell exhaustion during viral persistence is mediated by prolonged motility paralysis in the splenic marginal zone and red pulp - two regions where an abundance of viral antigen and PD-L1 was found. Importantly, we observed that intravenous injection anti-PD-1 enhanced anti-viral T cell motility in 30 minutes, which coincided with increased phosphorylation of ZAP-70 and ERK. Once T cells were released from infected targets, IFN-γ production significantly increased (within 1 hr), viral loads dropped by 70% (within 16 hrs), and mice ultimately developed fatal immunopathology (with 2-4 days). These data indicate that the PD-1 disables anti-viral CD8 and CD4 T cell signaling and function by stabilizing the synapse and holding them in a state of motility paralysis.

Many viruses can establish persistence and cause disease in humans. Studies have established a role for T cells in resolving persistent viral infections; however, the importance of a humoral response is less clear. Emerging evidence suggests that a coordinated effort between T and B cells is required to resolve some persistent infections. In this study, we sought novel insights into the importance of humoral immunity in resolving a persistent infection by utilizing the lymphocytic choriomeningitis virus (LCMV) model. Infection with aggressive LCMV variants results in a chronic viral infection during which anti-viral T cells pass through a state of functional exhaustion, but the infection is nevertheless controlled over time. We postulated that humoral immunity was required for LCMV control and could be harnessed as an immunotherapy. We observed plasma B cells in the repertoire as early as one week following LCMV infection, which coincided with the emergence of anti-viral antibodies (non-neutralizing). Depletion of B cells with anti-CD79 antibodies starting at the peak of T cell exhaustion (day 15) resulted in a failure of mice to control virus. Moreover, B cells harvested from mice that had controlled infection and transferred into mice with active infection resulted in accelerated viral control. These data demonstrate that humoral immunity can contribute to the resolution of a persistent viral infection and that B cells can be harnessed for adoptive immunotherapy.

Following virus infection, cytotoxic T lymphocytes (CTL) divide rapidly to eradicate the pathogen and prevent the establishment of persistence. The magnitude of an anti-viral CTL response is thought to be controlled by the initiation of a cell cycle program within lymphoid tissues. However, it is presently not known whether this division program proceeds during migration or is influenced locally at sites of viral infection. Here we demonstrate that anti-viral CTL remain in cell cycle while transiting to infected tissues. One third of virus-specific CTL within blood were found to be in cell cycle following infection with lymphocytic choriomeningitis virus (LCMV). Using two-photon microscopy, we revealed that effector CTL divided rapidly upon arrest in the virus-infected central nervous system (CNS) as well as meningeal blood vessels. We also observed that MHC I-dependent interactions (but not costimulation) influenced the division program by advancing effector CTL through stages of cell cycle. These results demonstrate that CTL are poised to divide in transit and that their numbers can be influenced locally at the site of infection through interactions with cells displaying cognate antigen.

Two-photon intravital imaging through thinned skull was used to visualize the dynamics of LCMV specific P14 effector T cells on day 6 LCMV meningitis. Most P14 were motile without forming stable synapses with infected stroma before the mice succumb to immunopathology. To determine if a subset of effector T cells can form more stable interactions with professional antigen presenting cells (APCs), we visualized the dynamics of CFP+ P14 T cells in CD11c-YFP transgenic or CX3CR1GFP/+ mice. p14 formed synapses with CD11c+ dendritic cells, found mostly on the meninges and perivascular spaces in the layer 1 cortex, and with activated CX3CR1+ microglia in the brain parenchyma and perivascular areas. Long contacts were rare with ramified CX3CR1+ microglia and small CD11c+ cells. P14 motility increased upon infusion of anti-MHC class I and anti-LFA-1 blocking antibodies. Fluo-Red dye infusion revealed Ca2+ flux in both P14 and tissue cells. Unexpectedly, during extensive imaging, we noted division of P14. P14 underwent divisions after contacting CD11c+ and away from these cells during the imaging period. We conclude that while the dominant behavior of P14 effector T cells during LCMV meningitis is continuous motility, a subset arrests with professional APCs. These prolonged interactions were sometimes terminated by cell division. Other instances of cell division suggest that there is significant division of anti-viral effector cells at the site of infection.

The recent demonstration of improved anti-viral T cell function following PD-1 blockade is a finding of great clinical importance that can benefit patients with persistent infections and cancer. Presently, it is not clear how blockade of this inhibitory pathway improves the function of exhausted T cells. Infection of mice with an immunosuppressive strain of LCMV is a well described paradigm to study T cell exhaustion. In this study we sought novel insights into the mechanisms of T cell exhaustion and recovery during persistent infection by simultaneously imaging CD8 and CD4 T cell responses using two-photon microscopy in the spleen. To define the motility parameters associated with immune exhaustion, we compared anti-viral T cell dynamics during an acute versus a persistent LCMV infection. Analyses of T cells in these two distinct environments revealed that exhaustion during viral persistence is associated with prolonged TCR signaling and cellular arrest in the splenic marginal zone and red pulp - two regions with abundant LCMV antigen and elevated levels of PD-L1.

Many viruses can infect the mammalian central nervous system (CNS), some with devastating consequences, and others with little or no overt pathology. Immunotherapy is a successful approach to treat persistent infections that has shown promise in humans. Our laboratory studies a murine model of persistent lymphocytic choriomeningitis virus (LCMV) infection to understand the mechanism(s) by which immunotherapy with virus-specific memory T cells cleanses the infected CNS with minimal cellular damage and blood brain barrier breakdown. Using two-photon microscopy, we observed that therapeutic memory T cells invade the meninges and brain parenchyma, scanning both areas systemically for viral antigen. Interestingly, T cell surveillance of the persistently infected brain was not associated with massive recruitment of peripheral myelomonocytic cells - innate cells known to cause severe vascular pathology during LCMV meningitis. Instead, we revealed using CD11c-YFP reporter mice that immunotherapeutic T cells transformed microglia into a DC phenotype, inducing CD11c expression on microglia throughout the brain parenchyma. Microglia also participated in immunotherapy by directly releasing chemokines such as CCL5. We propose that successful clearance of a persistently infected brain can be achieved without induction of severe tissue injury by tailoring chemokine release to limit myelomonocytic cell recruitment and by converting microglia into local antigen presenting cells.

Viral infections of central nervous system (CNS) often trigger an inflammatory response that gives rise to a wide range of pathological outcomes. The CNS is equipped with an elaborate network of innate immune sentinels that routinely serve as first responders to these infections. The meninges and perivascular spaces are inhabited by specialized macrophages, and the brain parenchyma contains microglia - the most abundant CNS myeloid cell. To gain novel insights into how myeloid cells respond to an acute CNS viral infection, we utilized genomics and two-photon imaging approaches to study a pure innate response to LCMV infection. This was accomplished by monitoring temporal gene expression patterns and myeloid cell dynamics in the brains of CX3CR1-GFP mice lacking LCMV-specific T cells. Two-photon imaging studies revealed that CNS myeloid sentinels responded significantly to acute infection by sequestering DsRed-labeled virus. Microglia but not peripheral myeloid cells proliferated following acute infection and responded directly to viral antigen by reducing process complexity, suggesting a localized response to the presence of virus. However, at the genomic level, acute infection triggered massive changes in CNS gene expression with the majority of genes being linked to innate viral control and the type I interferon system. These data suggest that localized detection of virus by CNS myeloid sentinels can translate into engagement of an extensive anti-viral program.

Following virus infection, cytotoxic T lymphocytes (CTL) divide rapidly to eradicate the pathogen and prevent the establishment of persistence. The magnitude of an anti-viral CTL response is thought to be controlled by the initiation of a cell cycle program within lymphoid tissues. However, it is presently not known whether this division program proceeds during migration or is influenced locally at sites of viral infection. Here we demonstrate that anti-viral CTL remain in cell cycle while transiting to infected tissues. One third of virus-specific CTL within blood were found to be in various stages of cell cycle following infection with lymphocytic choriomeningitis virus (LCMV). Using two-photon microscopy, we revealed that effector CTL divided within the virus-infected central nervous system (CNS) as well as meningeal blood vessels. Within the CNS, MHC I-dependent interactions with antigen presenting cells influenced the division program by pushing effector CTL into cell cycle. These results demonstrate that CTL are poised to divide in transit and that their numbers can be influenced at the site of infection through interactions with local APCs.

Many viruses can cause severe neurological complications in the central nervous system (CNS) through initiation of immunopathology. A case in point is the fatal meningitis induced by lymphocytic choriomeningitis virus (LCMV) - a disease associated with vascular breakdown and seizures. Although LCMV-specific CNS-infiltrating cytotoxic lymphocytes (CTL) are essential for disease, it is not known precisely how CTL mediate fatal immunopathology. Recently, it has become possible to directly observe immunity and immunopathology using two photon laser scanning microscopy (TPLSM). We initiated our real time studies of LCMV-induced meningitis by visualizing LCMV-specific CTL dynamics in the meninges during the onset of symptoms. Our findings yielded a novel and unexpected mode of pathogenesis defined by CTL trafficking into the meningeal space followed by a massive secondary recruitment of pathogenic myelomonocytic cells (i.e., monocytes and neutrophils). Importantly, myelomonocytic cells were responsible for severe vascular leakage and rapid onset disease during meningitis, and CTL directly participated in their recruitment by releasing myelomonocytic cell chemoattractants. Our results demonstrate that a CD8+ T cell dependent disorder can proceed in the absence of CTL effector mechanisms and rely instead on secondarily recruited myelomonocytic cells.

Highly pathogenic strains of influenza virus can result in high mortality rates due to severe tissue damage caused by direct injury induced by the virus or by the immune response against the virus (immunopathology). Immune-mediated pulmonary tissue damage is a result of cytokine/chemokine release and the antiviral activity by cells of the innate and adaptive immune responses. Here we report that intratracheal treatment with a sphingosine analog, AAL-R, results in the inhibition of dendritic cell (DC) activation and antigen presentation to T cells in the mediastinal lymph nodes and the lung. Diminished DC function restricts virus-specific T cell proliferation causing a subsequent reduction in the accumulation of T cells in the lungs. The immunosuppressive effect involves signaling through S1P receptors on DC, but is independent of S1P1, S1P2 and S1P3. Inhibition of the accumulation of virus-specific T cells in the lungs occurs without impinging the production of anti-viral neutralizing antibodies. AAL-R treatment hinders cytokine/chemokine production which leads to a reduction in polymorphonuclear leukocyte and macrophage infiltration resulting in reduced lung injury. Importantly, local AAL-R administration into the lung was effective in controlling CD8+ T cell accumulation in the lungs when given four days following influenza virus infection. Administration of sphingosine analogs during influenza virus infection offers a potentially powerful therapeutic for alleviating pulmonary immunopathology.