1. Program in Molecular Pathogenesis, Helen L. and Martin S. Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York 10016, USA
2. Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California 92037, USA
3. These authors contributed equally to this work.

Correspondence to: Michael L. Dustin¹Dorian B. McGavern² Correspondence and requests for materials should be addressed to M.L.D. (Email: dustin@saturn.med.nyu.edu) and D.B.M. (Email: mcgad@scripps.edu).

To examine the dynamics of lymphocytic choriomeningitis virus (LCMV)-specific CTLs, we transferred 1  10⁵ naive green fluorescent protein (GFP)-tagged T-cell receptor (TCR) transgenic CD8⁺ T cells (GFP⁺ P14 cells) into B6 mice one day before intracerebral (i.c.) inoculation with LCMV Armstrong. Naive CD8⁺ T cells obtained from GFP⁺ P14 transgenic mice express GFP under the -actin promoter and express a TCR that recognizes the LCMV glycoprotein (amino acids 33–41) presented in H-2D^b. Two-photon microscopy (TPM) was performed through a thinned skull window to visualize the meninges overlying the visual cortex in asymptomatic (day 5) and symptomatic (day 6) mice (Fig. 1 and Supplementary Movie 1). In contrast to the few P14 cells observed on day 5 (Fig. 1a), the number of GFP⁺ P14 cells was notably increased in the meninges and perivascular regions on day 6 (Fig. 1b, c). To determine whether GFP⁺ P14 cells were engaging in antigen-specific interactions, we analysed their motion in the presence of control antibody (immunoglobulin G, IgG) or a blocking monoclonal antibody to D^b (anti-class I) introduced into the subarachnoid space through a small craniotomy (Fig. 1d–i). P14 speed averaged 3.41  0.27 m min^-1 (mean  s.e.m.) in the absence of the craniotomy and 3.04  0.33 m min^-1 in the presence of the craniotomy and IgG (Fig. 1j). The anti-class I antibody significantly increased (*P < 0.0001) the speed of P14 cells to 5.16  0.46 m min^-1 (Fig. 1j, k) and decreased the arrest coefficient (Fig. 1l), but did not influence the speed of CTLs specific for an irrelevant antigen (Supplementary Fig. 1). This significant change in P14 cell speed and arrest after anti-class I treatment was observed in all mice examined and did not depend on CTL abundance (Supplementary Fig. 2). GFP⁺ P14 CTL migration appeared random (Supplementary Fig. 3), with confined motion at longer times that was reversed by anti-class I antibodies. Comparison of the speed distributions showed that the entire population shifts after anti-class I treatment (Fig. 1k), suggesting that all GFP⁺ P14 cells encountered antigen. Despite this high frequency of antigen encounter, CTLs rarely synapsed with any one target for >10 min (Fig. 1g, m). These intravital observations raised questions about the infected target population and the CTL effector mechanisms used during fatal meningitis.

Figure 1: CTL localization and dynamics in the meninges of LCMV-infected mice.

a, b, A representative three-dimensional reconstruction of an intravital two-photon z-stack viewed through a thinned skull is shown for representative mice at day 5 (a) and day 6 (b) post-infection. Few GFP⁺ P14 CTLs (green) were observed in the meninges at day 5 post-infection. At day 6 post-infection, GFP⁺ P14 CTLs were confined to a 50-m meningeal space between the undersurface of the skull bone (blue) and the pial surface. Very few CTLs entered the parenchyma. Grid scale is 19.7 m. c, A representative three-dimensional reconstruction shows that GFP⁺ P14 CTLs (green) localized preferentially along meningeal vasculature (red) at day 6. Skull bone is blue. Grid scale is 119 m. d–i, Representative xy-plane thinned skull images of GFP⁺ P14 cells at day 6 post-infection (grey scale; d–f); corresponding 30-min time-lapse cell tracks (coloured lines; g–i) are shown below each image. (Representative xz-plane images are shown in Supplementary Fig. 1.) Note the highly dynamic motion of the CTLs in the meningeal surface at baseline (BL; d, g) and after injection of 10 g ml^-1 IgG isotype control (IgG; e, h) or anti-H-2D^b antibody (Class I; f, i). Grid scale is 29.6 m. j–m, Compared with baseline and IgG isotype control, class I inhibition induced a statistically significant (*P < 0.0001) change in average speed (j), arrest coefficient (l) and arrest duration (m) for GFP⁺ P14 CTLs. Primary velocity data shown in j are plotted as histograms using 0.5 m min^-1 bins and a Gaussian curve fit (k). See corresponding Supplementary Movie 1 and Supplementary Fig. 1.

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We identified the LCMV-infected cells through immunohistochemical studies (Fig. 2, Supplementary Fig. 4 and Supplementary Movie 2). The main LCMV-infected population in the meninges and around meningeal vasculature was ER-TR7⁺ stromal cells. LCMV infection was occasionally observed in CD45⁺ infiltrating leukocytes and astrocytic foot processes that comprise the glial limitans (Supplementary Fig. 4). Infection of endothelium, smooth muscle cells and pericytes was never observed (Supplementary Fig. 4). ER-TR7⁺ stromal cells support rapid migration of CD8⁺ and CD4⁺ T cells in lymph nodes⁵ and may provide strong chemokinetic signals that can overwhelm synapse forming stop signals⁶. This might explain the paucity of antigen-specific arrest (Fig. 1).

Figure 2: LCMV infection of ER-TR7⁺stromal cells in the meninges.

a–h, A representative two-dimensional image (a–d) and a maximal projection of a three-dimensional z-stack (e–h) are shown for a mouse at day 6 post-infection. Both images were captured using a one-photon confocal microscope. Note the localization of LCMV (red) to ER-TR7⁺ fibroblast-like cells (green) that line the meninges and meningeal vasculature. A cross section of a meningeal blood vessel is denoted with a white asterisk in d. e–h depict a top-down view of a large meningeal blood vessel in which the network of fibroblast processes are clearly visible and infected by LCMV. i, A three-dimensional reconstruction of a meningeal blood vessel cross section (centre denoted with a white asterisk) is shown to illustrate the degree to which LCMV infects fibroblast-like cells that completely surround meningeal blood vessels. Grid scale is 19.5 m. Cell nuclei are shown in blue in all merged panels. ER-TR7 is shown in green and LCMV in red. See corresponding Supplementary Movie 2 and Supplementary Fig. 3.

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Because CD8⁺ T cells are essential for pathology⁴, we evaluated several CTL effector mechanisms using genetic knockout and mutant mice (Fig. 3a). Surprisingly, mice with single deficiencies in all major CTL effector pathways—interferon- receptor, tumour necrosis factor-, Fas, granzymes, perforin and the degranulation pathway (Jinx, also known as Unc13d, mutant)—succumbed to the convulsive seizures observed after LCMV infection of wild-type mice. The delay in disease onset observed in perforin-knockout mice was recently attributed to slower CTL recruitment into the central nervous system (CNS)⁷. These data supported the imaging studies in suggesting that CTL effector functions might not be responsible for rapid-onset disease.

Figure 3: Analysis of mononuclear cell infiltrate and effector mechanisms during LCMV-induced meningitis.

a, Survival was monitored in mice deficient in all major CTL effector pathways. All infected knockout mice developed convulsive seizures and succumbed to LCMV-induced meningitis. A slight extension (13 h, P = 0.029) in survival was observed in tumour necrosis factor- (Tnf) knockout and Jinx mice when compared to wild type C57BL/6 (B6) controls. As reported previously, perforin (Prf1)-deficient mice survived until day 9 (P = 0.029). No extension in survival was observed in mice deficient in granzymes (Gzm), interferon- receptor (Ifngr) and Fas. b, The composition of the CNS mononuclear cell infiltrate was examined flow cytometrically at the denoted time points after LCMV infection. A massive influx of CTLs (CD45⁺Thy1.2⁺CD8⁺) and peripheral monocytes (CD45^hiThy1.2^-CD11b⁺Gr-1^int) into the CNS was observed only at day 6 post-infection. Low numbers of neutrophils (CD45^intThy1.2⁺CD11b⁺Gr-1^hi), B cells (CD45⁺Thy1.2⁺CD19⁺) and CD4 T cells (CD45⁺Thy1.2⁺CD4⁺) were also observed in the CNS at day 6 post-infection. See Supplementary Fig. 5 for examples of flow cytometric data. c, d, Injection of low-dose anti-Gr-1 antibody (RB6-8C5, 125 g intraperitoneally on day 4) achieved depletion of neutrophils from the CNS (c), but did not improve survival (d) compared to control mice treated with rat IgG. e, f, CCR2 deficiency did not improve survival (f) after i.c. LCMV infection when compared to wild-type B6 controls; however, flow cytometric studies revealed a compensatory increase in the number of CNS neutrophils (e) in CCR2-knockout mice at day 6. Survival was significantly extended (P = 0.029) in LCMV-infected CCR2-deficient mice that received high-dose anti-Gr-1 antibody (f). The frequency of P14 CTLs gated on CD45^hi cells was not significantly reduced after anti-Gr-1 treatment (data not shown). For all studies described, n = 4 mice per group were used. Asterisks denote statistical significance (P < 0.05). Bar graph data are represented as the mean and s.d.

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To investigate other effectors, we temporally examined the composition of the CNS infiltrate after i.c. LCMV infection (Fig. 3b and Supplementary Fig. 5). Baseline populations prevailed until day 6, at which point monocytes and macrophages were massively recruited into the CNS. A low number of these cells preceded the arrival of CTLs by 2 days. At day 6 a small increase in the number of neutrophils, CD4⁺ T cells and B cells was also observed; however, the latter two populations are not required for disease^{8, 9}. It should be noted that our methodology accounts primarily for extravasated leukocytes, because cells arrested in the vasculature (for example, neutrophils) are expunged during intracardiac saline perfusions. Nevertheless, our results demonstrate a minimal innate cellular response to the virus alone and massive recruitment of myelomonocytic cells that coincided with the arrival of CTLs at day 6.

We next asked whether monocytes and/or neutrophils were required for the seizure-induced death on day 6 (Fig. 3c–f). Neutrophil depletion with low-dose anti-Gr-1 (also known as anti-Ly6g) antibody¹⁰ (Fig. 3c, d) or monocyte infiltration blockade using chemokine receptor 2 (CCR2)-deficient mice¹¹ (Fig. 3e, f) had no effect on the nature or kinetics of death. Therefore, we hypothesized that both populations might have the potential to induce CNS injury. To test this hypothesis, we depleted monocytes and neutrophils simultaneously by administering high-dose anti-Gr-1 to CCR2-knockout mice (Fig. 3e, f). When both cell populations were depleted, seizure-induced death at day 6 was averted and survival was extended by 3 days (Fig. 3e, f), despite a normal frequency of virus-specific CTLs on day 6 (data not shown). These data suggested that myelomonocytic cells were highly pathogenic and were responsible for the rapid-onset seizure-induced death observed at day 6.

During TPM analyses of GFP⁺ P14 cells, we often noted that the vasculature appeared ragged and displayed plasma leakage tracked with intravascular-injected quantum dots (Fig. 4a–d and Supplementary Movie 3). We considered that the seizure-induced death at day 6 might be induced by vascular leakage caused by myelomonocytic cells. To test this possibility, we conducted TPM in LCMV-infected lysozyme M (LysM)–GFP mice, in which neutrophils and monocytes were labelled with GFP, to detect the relationship between myelomonocytic extravasation and vascular leakage. There was a tight correlation between locally synchronized LysM–GFP⁺ cell extravasation and vascular leakage on day 6 (Fig. 4e–h and Supplementary Movie 4).

Figure 4: Recruitment of myelomonocytic cells into CNS and the relationship to meningeal vascular injury.

a–d, Two representative two-photon images at early (t = 0 min; a, c) and late (t = 30 min; b, d) points in the time-lapse sequence show the position of GFP⁺ P14 CTLs (green) in relation to meningeal vascular changes (red). GFP⁺ CTLs were typically found in perivascular regions. Severe disruption of vascular integrity as evidenced by extravascular quantum dot (Qdot) signal (red) is shown in a and b. Note the ghost outlines of large cells near the ragged vessels that do not correspond to P14 CTLs. In other areas, P14 CTLs remained near perivascular areas that had preserved blood vessel integrity (c, d). Scale bar, 50 m. See corresponding Supplementary Movie 3. e–h, A representative two-photon 30 min time-lapse sequence of vascular leakage of quantum dots (red) and extravasation of LysM–GFP⁺ myelomonocytic cells (green) is shown for a symptomatic mouse at day 6 (d6) post-infection. Note that myelomonocytic cells roll and arrest inside the meningeal vessel before penetrating through the vascular wall and extravasating into the meningeal space. The extravasation of myelomonocytic cells is associated with severe vascular injury and quantum dot leakage. Grid scale is 19.7 m. See corresponding Supplementary Movie 4. No similar extravasation events were observed in asymptomatic control mice at day 5 post-infection (data not shown). i–l, A representative 30 min time-lapse is shown for a symptomatic LysM–GFP mouse depleted of neutrophils but not monocytes using low-dose anti-Gr-1 antibody. Note that LysM–GFP⁺ myelomonocytic cells (green) in the absence of neutrophils localize perivascularly and are associated with transient vascular leakage (red). Scale, 25 m. See corresponding Supplementary Movie 5. m–o, The fluorescent ratio (FR) of extravascular (e.v.) to intravascular (i.v.) GFP and quantum dot fluorescence signal was calculated at each frame, normalized to the baseline ratio at time point 0 (FR_t0), and plotted against time. Data are represented as mean  s.e.m. P14 CTL extravasation or positioning was not associated with quantum dot leakage (o). Myelomonocytic cell extravastion correlated (r = 0.99; P < 0.0001) with sustained vascular leakage only in the presence of neutrophils (m). In neutrophil-depleted LysM–GFP mice, perivascular myelomonocytic cells were associated with transient quantum dot leakage (n). p–r, Representative three-dimensional reconstructions of two-photon z-stacks depicting skull bone (blue), quantum dot (red) and LysM–GFP⁺ myelomonocytic cells (green) are shown for an asymptomatic mouse at day 5 (p), a symptomatic LysM–GFP mouse at day 6 (q), and a symptomatic LysM–GFP mouse at day 6 depleted of neutrophils (r). At day 5, vasculature showed smooth borders, no quantum dot (red) leakage and few LysM–GFP⁺ cells (green). In symptomatic mice at day 6, LysM–GFP⁺ cells were mostly observed extravasating from meningeal vasculature with some cells accompanying the vascular leakage down into the parenchyma (white arrow). In mice depleted of neutrophils (-PMN), LysM–GFP cells were observed in perivascular meningeal spaces. Grid scale for panels p–r is 19.7 m. See corresponding Supplementary Movies 4 and 5.

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To determine the relative contribution of neutrophils versus monocytes to vascular injury, we imaged LysM–GFP mice injected with low-dose anti-Gr-1 antibody, which depletes only neutrophils (Fig. 3c and Supplementary Fig. 6). Interestingly, synchronous extravasation of LysM–GFP⁺ cells was not observed in low-dose Gr-1-depleted mice (Fig. 4i–l and Supplementary Movie 5), suggesting that synchronously extravasating LysM–GFP⁺ cells are neutrophils. In neutrophil-depleted LysM–GFP mice, we observed perivascular LysM–GFP⁺ cells (that is, monocytes and macrophages) in areas of transient vascular leakage. Unlike neutrophils that display intravascular accumulation followed by explosive extravasation with vascular leakage, the monocytes accumulated more gradually in vascular sites that nonetheless displayed leakage. Statistically, sustained vascular leakage was only correlated (r = 0.99; P < 0.0001) with neutrophils (Fig. 4m). The presence of intra- or extra-vascular P14 CTLs was not associated with either pattern of vascular leakage (Fig. 4o). Quantum dot leakage was not observed on day 5 post-infection (Fig. 4p), despite low numbers of infiltrating monocytes (Fig. 3b). Vascular injury occurred only at day 6 post-infection and extended into the brain parenchyma (Fig. 4q). Myelomonocytic cells were restricted to the meninges on day 6 (Fig. 4q, r and Supplementary Fig. 10).

The impact of myelomonocytic cells on vascular injury was further assessed by quantifying leakage of Evans blue dye into the brain (Supplementary Fig. 7). Only mice depleted of both monocytes and neutrophils showed significant (P < 0.001) preservation of vascular integrity at day 6 post-infection (Supplementary Fig. 7e, f). Depletion of monocytes (Supplementary Fig. 7d, f) or neutrophils (Supplementary Fig. 7c, f) alone failed to prevent Evans blue leakage. Interestingly, in untreated wild-type mice at day 6 post-infection, we observed substantial leakage of Evans blue from meningeal blood vessels into the brain parenchyma (Supplementary Fig. 8), reflecting disrupted blood–brain barrier (BBB) integrity, which has the potential to cause severe seizures¹².

To examine a potential mechanism by which CTLs attract myelomonocytic cells, we used gene arrays to quantify differentially regulated transcripts in the brains of mock- versus day 6 LCMV-infected mice. Our results revealed a statistically significant increase (P < 0.05) in six chemokines (CCL2, 7.3-fold; CCL3, 8.1-fold; CCL4, 1.6-fold; CCL5, 5.6-fold; CCL7, 4.2-fold; CXCL2, 2.8-fold) and two chemokine receptors (CCR1, 2.6-fold; CCR2, 3.6-fold) that can recruit myelomonocytic cells into the CNS. Because it was reported that none of these chemokines were observed in the CNS of T-cell-deficient mice infected i.c. with LCMV¹³, we next used flow cytometry to examine which of these chemokines were produced by virus-specific P14 cells (Supplementary Fig. 9). Our flow cytometric analyses of CNS and splenic P14 CTLs at day 6 revealed that CCL3, CCL4 and CCL5 were all produced at the protein level, which was confirmed using gene arrays^{14, 15}. Both CCL3 and CCL4 required GP_33–41 peptide stimulation for maximum synthesis, whereas CCL5 was produced on differentiation from naive to effector cells and was not further upregulated on peptide stimulation.

It is well known that CD8⁺ T cells are required for LCMV-induced meningitis⁴ and vascular leakage¹⁶. Our results revealed that P14 CTLs produce three of the chemokines with the potential to attract the myelomonocytic cells responsible for vascular injury (Fig. 4 and Supplementary Fig. 7) and rapid-onset seizure-induced death (Fig. 3). To establish a direct link between CD8⁺ T cells and CNS myelomonocytic cell recruitment, we infected mice with LCMV and administered anti-CD8 antibody at days 4 and 5 post-infection—after CTL priming. This treatment, which reduced the number of CD8⁺ T cells in the CNS by 94%, prevented the rapid onset of seizures at day 6 post-infection (data not shown), and significantly reduced the number of monocytes and neutrophils in the CNS (Supplementary Fig. 10). These data indicate that virus-specific CTLs can contribute to the recruitment of pathogenic myelomonocytic cells either by directly releasing chemoattractants or possibly by inducing other cells to release chemoattractants.

The requirement for CD8⁺ T cells in the pathogenesis of LCMV meningitis led to the proposal that CTLs are directly responsible for tissue injury and death¹⁷. We propose that CTL activation through transient interactions with infected cells leads to massive recruitment of myelomonocytic cells, which compromise vascular integrity and initiate fatal convulsive seizures³. It is likely that once seizure-induced death is averted, infected mice ultimately succumb to another pathogenic mechanism possibly mediated by CTLs¹⁸.

It is well established that neutrophil extravasation can be linked to vascular leakage^{19, 20, 21}, and this process usually depends on signalling between leukocytes and endothelial cells²². Neutrophil extravasation causes tissue injury in many models including reperfusion injury and sepsis^{23, 24}. Using TPM we directly visualized this classical process in LCMV meningitis. Monocytes have been associated with atherosclerosis²⁵ and facilitation of the trafficking of neutrophils²⁶. Our results suggest that monocytes also contribute vascular leakage, possibly through a mechanism linked to adherence to the blood vessels²⁷ and/or chemokine release²⁸. Recognizing the complementary pathogenic functions of neutrophils and monocytes is critical for devising therapeutic approaches in CD8⁺ T-cell-mediated pathology.

It is not clear why CD8⁺ T cells recruit myelomonocytic cells to a site of viral infection. CD4⁺ Th17 cells produce interleukin (IL)-17 to coordinate neutrophil recruitment, but anti-viral CD8⁺ T cells express transcription factors that suppress this program²⁹, and we observed no IL-17 production by peptide-stimulated P14 cells (data not shown). Therapies directed at reducing myelomonocytic activation are obvious treatment candidates to prevent the mode of immunopathology we observed, but are challenging because of their numerous effector mechanisms, fast turnover and acute importance in host defence. A more tractable approach might be to target the chemotactic mechanisms used by CTLs to attract myelomonocytic cells, or, alternatively, to enhance CTL-mediated killing of relevant targets by improving immunological synapse formation or stability³⁰. The latter approach might break the feedback to the pathogenic myelomonocytic arm and improve survival as well as immunity in viral infections of the CNS.

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Supplementary Information

Supplementary information accompanies this paper.

Acknowledgements

This work was supported by National Institutes of Health grants AI070967-01 (D.B.M.), AI055037 (M.L.D.), a grant from The Burroughs Wellcome Fund (D.B.M.) and the Dana Foundation (M.L.D.). S.S.K. was supported by a National Institutes of Health training grant NS041219-06 and is presently supported by a National Research Service Award (NS061447-01), and J.V.K. is supported by a Multiple Sclerosis Society Center Grant. We thank C. Yau for technical support and the Scripps DNA Array core for their assistance with the gene array experiment.

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