Myelomonocytic cell recruitment causes fatal CNS vascular injury during acute viral meningitis.

Lymphocytic choriomeningitis virus infection of the mouse central nervous system (CNS) elicits fatal immunopathology through blood-brain barrier breakdown and convulsive seizures. Although lymphocytic-choriomeningitis-virus-specific cytotoxic T lymphocytes (CTLs) are essential for disease, their mechanism of action is not known. To gain insights into disease pathogenesis, we observed the dynamics of immune cells in the meninges by two-photon microscopy. Here we report visualization of motile CTLs and massive secondary recruitment of pathogenic monocytes and neutrophils that were required for vascular leakage and acute lethality. CTLs expressed multiple chemoattractants capable of recruiting myelomonocytic cells. We conclude that a CD8(+) T-cell-dependent disorder can proceed in the absence of direct T-cell effector mechanisms and rely instead on CTL-recruited myelomonocytic cells.

ATP mediates rapid microglial response to local brain injury in vivo.

Parenchymal microglia are the principal immune cells of the brain. Time-lapse two-photon imaging of GFP-labeled microglia demonstrates that the fine termini of microglial processes are highly dynamic in the intact mouse cortex. Upon traumatic brain injury, microglial processes rapidly and autonomously converge on the site of injury without cell body movement, establishing a potential barrier between the healthy and injured tissue. This rapid chemotactic response can be mimicked by local injection of ATP and can be inhibited by the ATP-hydrolyzing enzyme apyrase or by blockers of G protein-coupled purinergic receptors and connexin channels, which are highly expressed in astrocytes. The baseline motility of microglial processes is also reduced significantly in the presence of apyrase and connexin channel inhibitors. Thus, extracellular ATP regulates microglial branch dynamics in the intact brain, and its release from the damaged tissue and surrounding astrocytes mediates a rapid microglial response towards injury.

The ABCs of artificial antigen presentation.

Artificial antigen presentation aims to accelerate the establishment of therapeutic cellular immunity. Artificial antigen-presenting cells (AAPCs) and their cell-free substitutes are designed to stimulate the expansion and acquisition of optimal therapeutic features of T cells before therapeutic infusion, without the need for autologous antigen-presenting cells. Compelling recent advances include fibroblast AAPCs that process antigens, magnetic beads that are antigen specific, novel T-cell costimulatory combinations, the augmentation of therapeutic potency of adoptively transferred T lymphocytes by interleukin-15, and the safe use of dendritic cell-derived exosomes pulsed with tumor antigen. Whereas the safety and potency of the various systems warrant further preclinical and clinical studies, these emerging technologies are poised to have a major impact on adoptive T-cell therapy and the investigation of T cell-mediated immunity.

Cognate antigen directs CD8+ T cell migration to vascularized transplants.

The migration of effector or memory T cells to the graft is a critical event in the rejection of transplanted organs. The prevailing view is that the key steps involved in T cell migration - integrin-mediated firm adhesion followed by transendothelial migration - are dependent on the activation of Gαi-coupled chemokine receptors on T cells. In contrast to this view, we demonstrated in vivo that cognate antigen was necessary for the firm adhesion and transendothelial migration of CD8+ effector T cells specific to graft antigens and that both steps occurred independent of Gαi signaling. Presentation of cognate antigen by either graft endothelial cells or bone marrow-derived APCs that extend into the capillary lumen was sufficient for T cell migration. The adhesion and transmigration of antigen-nonspecific (bystander) effector T cells, on the other hand, remained dependent on Gαi, but required the presence of antigen-specific effector T cells. These findings underscore the primary role of cognate antigen presented by either endothelial cells or bone marrow-derived APCs in the migration of T cells across endothelial barriers and have important implications for the prevention and treatment of graft rejection.

Migration of cytotoxic lymphocytes in cell cycle permits local MHC I-dependent control of division at sites of viral infection.

After virus infection, cytotoxic T lymphocytes (CTLs) divide rapidly to eradicate the pathogen and prevent the establishment of persistence. The magnitude of an antiviral 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. We demonstrate that antiviral CTLs remain in cell cycle while transiting to infected tissues. Up to one third of virus-specific CTLs within blood were found to be in cell cycle after infection with lymphocytic choriomeningitis virus or vesicular stomatitis virus. Using two-photon microscopy, we found that effector CTL divided rapidly upon arrest in the virus-infected central nervous system as well as in 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 the cell cycle. These results demonstrate that CTLs 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 laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites.

The mouse spinal cord is an important site for autoimmune and injury models. Skull thinning surgery provides a minimally invasive window for microscopy of the mouse cerebral cortex, but there are no parallel methods for the spinal cord. We introduce a novel, facile and inexpensive method for two-photon laser scanning microscopy of the intact spinal cord in the mouse by taking advantage of the naturally accessible intervertebral space. These are powerful methods when combined with gene-targeted mice in which endogenous immune cells are labeled with green fluorescent protein (GFP). We first demonstrate that generation of the intervertebral window does not elicit a reaction of GFP(+) microglial cells in CX3CR1(gfp/+) mice. We next demonstrate a distinct rostrocaudal migration of GFP(+) immune cells in the spinal cord of CXCR6(gfp/+) mice during active experimental autoimmune encephalomyelitis (EAE). Interestingly, infiltration of the cerebral cortex by GFP(+) cells in these mice required three conditions: EAE induction, cortical injury and expression of CXCR6 on immune cells.

Innate response to focal necrotic injury inside the blood-brain barrier.

We have studied the initial innate immune response to focal necrotic injury on different sides of the mouse blood-brain barrier by two-photon intravital microscopy. Transgenic mice in which the promoter of the myeloid isoform of lysozyme drives GFP were used to track granulocytes and monocytes. Necrotic injury in the meninges, but not the brain parenchyma, recruited GFP+ cells within minutes that fully surrounded the necrotic site within a day. Recently, it has been suggested that microglial cells and astrocytes cooperate to mount a distinct response to laser injury behind the blood-brain barrier. We followed the microglial response in heterozygous knockin mice in which GFP replaces CX3CR1 coding sequence. Prior to injury, microglial cell bodies were immobile over days, but moved to the laser injury site within 1 day. We followed astrocytes, which have been proposed to cooperate with microglial cells in response to focal injury, using transgenic mice in which glial fibrillary acidic protein promoter drives GFP expression. Before injury fine astrocyte processes permeate the parenchyma. Astrocytes polarized toward the injury in an ATP, connexin hemichannels, and intracellular Ca2+ -dependent process. The astrocytes network established a cytoplasmic Ca2+ gradient that preceded the microglial response. This is consistent with astrocyte-microglial collaboration to mount this innate response that excludes blood leukocytes.