Lymphotactin Mediates Antiviral T Cell Trafficking Within

LYMPHOTACTIN MEDIATES ANTIVIRAL T CELL TRAFFICKING WITHIN

THE CENTRAL NERVOUS SYSTEM DURING WEST NILE VIRUS

ENCEPHALITIS

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

Biological Sciences

Sharese Tronti

2019

SIGNATURE PAGE

THESIS: LYMPHOTACTIN MEDIATES ANTIVIRAL T CELL TRAFFICKING WITHIN THE CENTRAL NERVOUS SYSTEM DURING WEST NILE VIRUS ENCEPHALITIS

AUTHOR: Sharese Tronti

DATE SUBMITTED: Spring 2019

Department of Biological Sciences

Dr. Douglas Durrant Thesis Committee Chair Biological Sciences

Dr. Andrew Steele Biological Sciences

Dr. Jamie Snyder Biological Science

ABSTRACT

West Nile Virus (WNV), a neurotropic flavivirus, can cause neuroinvasive

disease in humans. After peripheral infection, WNV is able to enter the central nervous

system (CNS) and infect neurons causing neuronal injury and inflammation that

potentially may result in fatality. In order to restrict viral replication and pathogenesis within the CNS during WNV encephalitis, virus-specific CD8+ T cells are critically dependent on dendritic cell (DC) mediated reactivation at this site. However, the mechanism by which DCs are recruited to the brain to ensure their interaction with infiltrating virus-specific CD8+ T cells remains unknown. Previous studies have demonstrated that, upon activation, CD8+ T cells rapidly produce the chemokine lymphotactin when activated. The receptor for lymphotactin, XCR1, is exclusively

expressed on a subset of DCs, CD8+ DCs, which have been shown to be essential in

establishing protective peripheral immunity against viruses and intracellular bacteria. In

this study, we show that lymphotactin regulates the CNS entry of T lymphocytes,

potentially promoting virologic control within the CNS and limiting neuronal cell death.

Although, no apparent differences were obtained in survival and clinical disease

progression, WNV-infected mice, that had been treated with an XCL1-neutralizing

antibody, displayed increased parenchymal localization of T cells within the brain

compared to control mice. These data indicate that lymphotactin contributes to stabilizing

the cellular interactions between infiltrating T cells and CNS-localized DCs with may impart protective CNS inflammation by regulating the parenchymal entry of T cells during WNV encephalitis.

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Table of Contents

Signature Page ………………………………………………………………………… ii

Abstract ………………………………………………………………………………... iii

List of Tables ………………………………………………………………………….. vi

List of Figures …………………………………………………………………………. vii

Introduction ……………………………………………………………………………. 1

West Nile Virus ………………………………………………………………………… 1

West Nile Virus Neuroinvasive Disease ……………………………………………..3

West Nile Virus Immunity ……………………………………………………………. 5

Dendititic Cells and Lymphotactin …………………………………………………. 8

Hypothesis ……………………………………………………………………………... 10

Materials and Methods ………………………………………………………………… 12

Ethics Statement ……………………………………………………………………….. 12

Virus …………………………………………………………………………………….. 12

Mouse Infection ……………………………………………………………………….. 12

Lymphotactin Neutralization ………………………………………………………… 13

Tissue Collection and Preparation …………………………………………………. 13

RNA Isolation and qRT-PCR ………………………………………………………… 13

Lymphotactin Protein Analysis ……………………………………………………… 14

Immunohistochemistry ……………………………………………………………….. 14

Statistical Analysis ……………………………………………………….…………… 15

Results …………………………………………………………………………………. 16

Lymphotactin Expression and its Protective Role During WNV Neuroinvasive

Disease ……………………………………………………………………………...….. 16

Lymphotactin Is Effectively Neutralized In Peripheral Tissues via Anti-XCL1.. 18

Lymphotactin Plays a Role in Control of WNV Burden within the CNS .……... 19

Lymphotactin Plays a Protective Role Against Virus-Associated Neuronal Cell

Death ………………………………………………………………………………. ..… 19

Lymphotactin Impacts the Localization of T cells During WNV Encephalitis… 20

Discussion …………………………………………………………………………….. 31

References …………………………………………………………………………….. 37

LIST OF TABLES

Table 1: Health Status Assessment Results on day 5. ………………………………….22

LIST OF FIGURES

Figure 1: CD8+ T cells arrested within the perivascular space without antigen- recognition……………………………………………………………………………… 23

Figure 2: Lymphotactin levels during WNV encephalitis …………………………….. 24

Figure 3: Percent of weight change in mice from days -1 to 5 post West Nile infection..25

Figure 4: Percent of survival to day 5 post West Nile infection ………………………. 25

Figure 5: Lymphotactin is effectively neutralized in peripheral tissues via anti-

XCL1…………………………………………………………………………………… 26

Figure 6: Lymphotactin plays a role in control of WNV burden within the CNS. ...….. 27

Figure 7: Lymphotactin plays a protective role against virus-associated neuronal cell

death ……………………………………………………………………………….. ….. 28

Figure 8: Lymphotactin impacts the localization of T cells during WNV encephalitis.. 29

Figure 9: Lymphotactin-mediated recruitment of DCs to the perivascular space ……. 30

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INTRODUCTION

West Nile Virus (WNV), a mosquito-borne neurotropic flavivirus, has emerged

globally as a significant cause of viral encephalitis and meningitis. Due to human and

environmental factors, WNV has disseminated throughout the Western world and

continues to spread causing increased outbreaks and WNV disease in humans. Currently,

there remains no vaccine or specific therapy approved to treat or prevent WNV infection.

Due to the fact that WNV continues to pose a significant public health risk, there remains

a pressing need to understand the viral and host factors that determine viral pathogenesis

and outcome of WNV infection.

West Nile Virus

West Nile Virus (WNV) is spread by the bite of a mosquito and is maintained in a cycle between birds and mosquitoes, but can also be spread to humans and other mammals which serve as dead-end hosts (Lim et al, 2011). WNV is endemic in parts of

Africa, Asia, Europe, and the Middle East and since its emergence in the United States in

1999, reports have been made of its presence in the Caribbean, Mexico, and South

America (Dauphin et al, 2004). According to the CDC, there were 2544 cases of WNV infections in the US in 2018, 1594 of them being neuroinvasive resulting in 137 deaths

(CDC, 2019). West Nile infection in humans can range from a flu-like disease to encephalitis, acute flaccid paralysis, and death (Cho and Diamond, 2012). Prevention of

WNV infection is limited to the prevention of mosquito bites, including insect repellent, long clothing, and mosquito population control. There is currently no specific therapy or vaccine approved for human use (DeFilette et al, 2012).

WNV is a neurotropic flavivirus with a single-stranded positive-sense RNA

1 genome (Donadieu et al, 2013). The ssrRNA genome encodes for three structural proteins; including, the capsid protein (C), the envelope protein (E), and the membrane protein (prM), and at least seven non-structural proteins (NS1, NS2A, NS2B, NS3,

NS4A, NS4B, NS5) (Kramer et al, 2007). Upon infection, WNV initially targets primarily dendritic cells and endothelial cells as well as macrophages and monocytes

(Pierson et al, 2013). Viral dissemination occurs during the lytic infection of these cells, which leads to increased viremia. Although, the receptor necessary for virion attachment and entry have not yet been fully identified, it is known that WNV gains entry into cells by receptor-mediated endocytosis via clatherin-coated pits and delivery of the RNA genome occurs following membrane fusion within the early endosome (Chambers et al,

1990). After, the viral genome is then released into the cytoplasm where translation of the nonstructural protein occurs as well as RNA synthesis. The viral RNA then associates with the endoplasmic reticulum, with the aid of NS2A, NS2B, NS4A, and NS4B, where replication of the viral genome RNA occurs (Brinton, 2014). Genomic viral RNA is packaged into progeny virions which then bud into the endoplasmic reticulum to form enveloped immature virions. Further maturation occurs in the trans Golgi network, where the NS3-NS2B complex is required for efficient polyprotein processing. Finally, the virions are secreted into the extracellular space by exocytosis. West Nile virions begin to be released from infected cells 8 hours after infection and peak around 24 hours (Cho and

Diamond, 2012).

WNV is transferred after a mosquito intradermally inoculates a subject. The virus initially replicates in keratinocytes, newly recruited neutrophils, and skin dendritic cells

(Langerhans cells) which then migrate to the regional lymph nodes and the bloodstream

(Lim et al, 2011). This leads to primary viremia during days 3-4 after infection which can then lead to infection of the kidneys, liver, and spleen (Samuel and Diamond, 2006). In the periphery, infection is countered by the development of an early immune response including type I and II IFN production as was as the effector functions of innate immune cells such as IgM secreting B cells, NK cells, neutrophils, and macrophages (Cho and

Diamond, 2012). Within the draining lymph nodes, viral antigen presentation to naïve T cells, complement activation, and antiviral cytokine and chemokine expression occurs.

By the end of the first week post-infection, WNV is cleared from the peripheral organs and infection of the central nervous system (CNS) can be observed starting on day five after infection, with the probability of neuroinvasion correlated with the duration and level of viremia (Cho and Diamond, 2012). The mechanisms in which WNV enters the

CNS are not fully understood. Two major routes have been well supported: direct crossing of the blood brain barrier (BBB) after its integrity has been compromised

(hematogenous spread) or entry through the BBB via infected leukocytes. Retrograde axonal transport after infection of the peripheral nervous system has been suggested but is currently less supported (Donadieu et al, 2013).

West Nile Virus Neuroinvasive Disease

Upon entry of the CNS, West Nile Virus is capable of causing neuroinvasive disease which can be irreversible or even fatal. Neuroinvasive disease symptoms include flaccid paralysis or encephalitis, which consists of cognitive impairment, muscle weakness, paralysis, and tremors. (Mostashari et al, 2001). In cases with neuroinvasive disease, the virus induces inflammatory lesions and neuronal damage in multiple regions of the brain including the anterior horn of the spinal cord, the brainstem, the cerebellum,

and the hippocampus (Kramer et al, 2007). The fatality rate for encephalitic cases is

approximately 10% and it has been shown that those with advanced age and

compromised immune systems are at an increased risk (Murray et al, 2006). A decline in

either innate or adaptive immune function has been shown to increase susceptibility as

IgM, CD4+ and CD8+ T cells all play vital roles in controlling infection. Collective

studies suggest that WNV-specific antibodies are responsible for reducing viremia while

CD8+ T cells, with the support of CD4+ T cells, play an important function in clearing

infection (Lim et al, 2011).

The mechanisms in which WNV infection induces neuronal injury and death are

still unclear as it is unknown whether it is primarily caused by viral infection (e.g. cell

death) or from the host inflammatory response and leukocyte infiltration into the CNS

which produce cytotoxic factors targeting neuronally infected cells. During WNV

infection, programmed cell death has an antiviral effect by targeting infected cells but this

can have pathological effects since it occurs in non-renewing neurons (Lim et al, 2011).

WNV induced cell death of infected neurons has been shown to be dependent of caspase-

3, which plays a role in regulating apoptosis (Samuel et al, 2007). The increased risk for immunocompromised patients seems to suggest that an intact immune system is essential for the control of WNV infection. However, this presents a problem since studies have

suggested that once the blood-brain-barrier has been compromised, immune cells may

enter the brain and cause immune mediated damage (Reijerkerk et al, 2008).

West Nile Virus Immunity

Following the bite of an infected mosquito, the early immune response to WNV

infection includes the effector functions of the innate immune cells, including dendritic

4 cells, macrophages, and natural killer cells. These cells are the producers of type I (IFN-

α/β) IFNs which have antiviral activity and type II (IFN-γ) IFNs which aid in macrophage activation (Cho and Diamond, 2012). The pathogen recognition receptors such as TLR, including TLR3, and RIG-I like receptors recognize nucleic acid intermediates of RNA virus replication and promote the activation of IRF-3 and IRF-7- mediated transcriptional programs which result in the activation of IFN-stimulated genes augmenting their anti-viral responses (Cho and Diamond, 2012). Macrophages provide protection against WNV infection by presenting antigen, producing reactive oxygen intermediates, and promoting proliferation of WNV-specific T cells (Donadieu et al,

2013). Infection is decreased in the peripheral tissues by IFN-α/β, IFN-γ, IgM, and CD8+

T cells (Wang et al, 2003). γ/δ T cells facilitate the maturation of DCs which in turn present antigen to CD8+ T cells, which have an essential role in viral clearance. IFN- stimulated genes have also been shown to inhibit WNV replication in neurons (Szretter et al, 2011).

After neuroinvasion, neurons infected with WNV initiate an inflammatory response by producing chemokines that recruit immune cells. Chemokines are a family of small peptides that control directional cell migration with their receptors (Lei and

Takahama, 2012). It has been shown that WNV infection of neurons induces the expression of CCL2, CCL3, CCL4, CCL5, CCL7, CXCL10, and CXCL12 (Zhang et al,

2008). CXCL10 expression promotes the trafficking of WNV-specific CD8+ T cells by binding to the receptor CXCR3 (Zhang et al, 2008). Expression of CCL3, CCL4, and

CCL5 results the CCR5-dependent trafficking of CD4+ and CD8+ T cells, NK cells, and macrophages (Zhang et al, 2008). CCL2, CCL7, and CXCL12 expression leads to the

trafficking of monocytes and T cells via the CCR2 receptor (Lim and Murphy, 2011).

This chemokine-mediated leukocyte recruitment appears necessary since T-cell-mediated

immunity is crucial in the control of WNV infection in the CNS and both T cells and

macrophages have been shown to participate in viral clearance in the brain (Wang et al,

2003) Not all trafficking of immune cells to the CNS is beneficial, however, as reduced

monocyte and neutrophil migration into the brain has been shown to correlate with

decreased viral load and mortality due to the fact that WNV-infected neutrophils further

shed virus once inside the brain (Wang et al, 2012).

T-cell mediated immunity is an essential part of the immune mediated protection

from WNV. Studies have indicated that cytotoxic CD8+ T cells control WNV infection in

the CNS via production of antiviral cytokines, such as IFN-γ, or by triggering cell death

of target cells (Cho and Diamond, 2012). It has been shown that infected neurons up-

regulate MHC class I molecules and are thus targeted by cytotoxic T cells (Chevalier et

al, 2011). CD8+ T cells control WNV infection in the CNS via multiple mechanisms

including through Fas-Fas ligand, perforin, or TRAIL-dependent pathways (Cho and

Diamond, 2012). Interactions between Fas on infected neurons and Fas L on CD8+ T

cells leads to the activation of a caspase apoptosis cascade (Shrestha et al, 2006).

Perforin-mediated control occurs through the granzyme-dependent granule exocytosis pathway, which leads to apoptosis of infected neurons (Shrestha and Diamond, 2007).

Trail binding to the death receptor DR5 on neurons triggers also leads to a caspase- dependent apoptosis cascade (Cho and Diamond, 2012). While T cell responses are important for viral clearance, they can also cause irreversible damage to the host as the infected neurons that are targeted for apoptosis are largely non-renewable.

Recruitment of leukocytes, particularly cytotoxic T cells, into the brain parenchyma is necessary to clear WNV infection. To achieve this, immune cells must first cross the blood brain barrier (BBB). The BBB is composed of the endothelial cell layer and the glia limitans, which is a barrier of astrocyte foot processes that wrap around the brain microvasculature, and the perivascular space in between these two layers

(Shechter et al, 2013). The BBB is maintained mechanically by tight junctions at the endothelial cell layer (Zlokovic, 2008) and is designed to prevent the entry of macromolecules and pathogens into the brain parenchyma from the blood that may potentially damge non-renewable and vulnerable neurons. Cellular transmigration across the blood-brain barrier (BBB) involves two processes: transmigration of the vascular wall into the perivascular space and progression across the glia limitans into the parenchyma

(Owens et al, 2008). Activated lymphocytes have been shown to cross the vascular wall for immunosurveillance but do not progress through the glia limitans (Ladeby et al,

2005). This first step of transmigration is controlled by adhesion molecules in the inflamed endothelium and chemokine gradients which play a role in diapedesis, particularly CXCL12 expression (Lim et al, 2011) (Engelhardt and Ransohoff, 2005).

Antigen recognition appears to be required for T cells to perform the second step of migration across the BBB during experimental allergic encephalomyelitis, a neuroinflammatory disease mouse model (Trans et al, 1998) (Fig. 1). During WNV infection of the brain, infected neurons release chemokines such as CXCL10, that helps recruit CXCR3 expressing T cells to the CNS (Hosking and Lane, 2010). Through

CXCL12/CXCR4 interactions, these T cells are able to cross the first barrier, the microvascular endothelial cell layer, into the perivascular space (Hosking and Lane,

2010). In order for these cells to transmigrate past the second barrier, the glial limitans, and into the CNS parenchyma, these cells need to be fully reactivated (Owens et al,

2008), which has been shown to be achieved through the help of perivascular-localized dendritic cells (DCs). Dendritic cell-mediated reactivation of antigen-specific CD8+ T cells within the perivascular space is crucial for the transmigration into the WNV- infected parenchyma resulting in viral control and reduced neuronal injury. However, the mechanisms that ensure the co-localization of Ag-specific T cells and antigen-bearing

DCs within this space remains to be defined.

Dendritic Cells and Lymphotactin

Initiation of an adaptive immune response occurs due to the interaction between antigen-bearing dendritic cells (DCs) and T cells within the lymph nodes. DCs present antigen to naïve T cells and prime them for immune function, which leads to T cell differentiation and proliferation (Brewitz et al, 2017). Once T cells are activated, they are released into the periphery where chemokine-mediated chemotaxis occurs to migrate T cells to areas of infection. Lymphotactin, also called XCL1, is a C class chemokine and is expressed by activated CD8+ T cells, CD4+ T cells, NK cells, thymic medullary epithelial cells, and γδ T cells (Lei and Takahama, 2012). C class chemokines only have two cysteines, opposed to the normal four, and consists only of lymphotactin in the mouse genome. The expression of lymphotactin has been detected in the intestines, lungs, peripheral blood leukocytes, spleen, and thymus and is most highly expressed by CD8+ T cells in the blood and thymus once activated in association with a Th-1 type immune response (Kennedy et al, 1995). Chemokine receptors are G-protein-coupled seven- transmembrane receptors which trigger intracellular signals that lead to directional

8 cellular migration (Allen et al, 2007). The receptor for lymphotactin is XCR1, which is expressed by a specific subset of DCs, CD8+ DCs (Dorner et al, 2009). The CD8+ DC subset is more specifically involved in antigen cross-presentation in which exogenous antigen in antigen in shunted to the MHC class 1 pathway (Dorner et al, 2009). This process allows CD8+ DCs to present exogenous antigen to CD8+ T cells without becoming virally infected. In the presence of lymphotactin, XCR1 expressing cells display calcium mobilization and chemotactic response (Yoshida et al, 2009), thus lymphotactin expressed by activated CD8+ T cells exclusively chemoattracts CD8+ DCs

(Dorner et al, 2009). Additionally, lymphotactin has been shown to increase early CD8+

T cell responses to intracellular infection indicating that DC subsets that express XCR1 are uniquely capable of activating an effective CD8+ T cell mediated defense against intracellular pathogens. (Crozat et al, 2010).

During WNV infection, DCs play roles in the dissemination of virus and in the development of the immune response. WNV initially replicates in skin DCs and then migrate into secondary lymphoid tissues, or draining lymph nodes. During this time, DCs mature and reach their activation state, which allows them to present antigen to T cells within the lymph node and secrete pro-inflammatory cytokines and other antiviral molecules (Freer and Matteucci, 2009). The chemoattraction of lymphotactin for DCs to migrate to T cells within secondary lymphoid tissues for antigen presentation is vital in the development of an adaptive immune response against WNV (Lei and Takahama,

2011). In fact, previous studies have shown that CD8+ T cells secrete lymphotactin in a dose-dependent manor 8-36 hours after encountering antigen on CD8+DCs (Dorner et al,

2009). The amount of lymphotactin secreted has been found to be correlated with the

9 strength of the antigenic signal, whereas the initial time point of lymphotactin secretion was found not to be dependent of antigen levels (Dorner et al, 2009). These experiments indicate that activated CD8+ T cells secrete lymphotactin over the entire period of time in which stable T cell-DC interactions are known to occur. These T cell-DC interactions then in turn increase the differentiation, expansion, and survival of CD8+ T cells, which are necessary for an appropriate immune response (Dorner et al, 2009). These studies suggest that T cells orchestrate the development of an optimal priming environment upon their initial identification of an antigen bearing DC.

Hypothesis

As previously discussed, viral infections of the CNS require efficient T cell responses to promote viral clearance and limit immunopathology. During WNV encephalitis, activated virus-specific T lymphocytes can cross the endothelial layer of the

BBB where they accumulate within the perivascular space. Within this space, DCs regulate their ability to cross the glial limitans layer into the WNV-infected CNS parenchyma by mediating their reactivation at this site (Hosking and Lane, 2010) (Trans et al, 1998). While it has been shown that lymphotactin attracts DCs and also stabilizes T cell interactions with Ag-bearing DCs within peripheral lymph nodes (Lei and Takahama,

2011); however, the mechanisms that accomplish DC recruitment and cellular interactions at this site is unknown. Within secondary lymphoid organs, such as draining lymph nodes, it has been shown that lymphotactin attracts DCs and also stabilizes T cell interactions with AG-bearing DCs to permit an effective and appropriate immune response (Lei and Takahama, 2011). Perivascular spaces within the BBB have similar properties to secondary lymphoid tissues in that this region governs the trafficking and

effector functions of CNS-infiltrating leukocytes; nevertheless, it remains unknown

whether lymphotactin has a similar role within the perivascular space during West Nile

Encephalitis (Fig. 1). Potentially, activated antigen-specific T cells arrested in the perivascular space secrete lymphotactin to ensure an optimal reactivation environment via cellular interactions with CNS-localized DCs. In response, T cells may overcome retention at the endothelial barriers and migrate into the WNV-infected parenchyma during WNV encephalitis. We hypothesize that T cells within the CNS produce lymphotactin in order to attract and stabilize their interaction with DCs to guarantee their proper activation, migration, and subsequent protection from

WNV-induced neurological damage during WNV encephalitis. To test this

hypothesis, lymphotactin expression was measured over the course of infection within

both peripheral and CNS tissues following infection with WNV. Then survival, clinical

disease, viral burden, and neuronal cell death were measured following lymphotactin

depletion via an XCL1 neutralizing antibody in a well-established murine model for

WNV encephalitis. In addition, we utilized immunohistochemical staining techniques to determine the role of lymphotactin in the localization of T cells within the WNV-infected brain during WNV encephalitis. Through these studies, the cellular mechanisms crucial for immunoprotective responses within the CNS during viral encephalitis has been partially illuminated, particularly the role of lymphotactin during WNV encephalitis.

These results have clear implications for understanding viral pathogenesis within the

CNS and in considering potential targets for future therapies.

MATERIALS AND METHODS

Ethics Statement

The experiments were performed according to the Animal Care and Use

Committee (ACUC) guidelines under protocol: 16.014 Intracerebral DCs modulate

immune responses in the CNS during viral encephalitis. The protocol was approved by

the California State Polytechnic University, Pomona committee.

Virus

Kunjin West Nile Virus (KUNV) (isolate MRM16), received from the CDC

isolated from Culex annulirostris mosquito in Australia. was used throughout these

experiments and was propagated using C6/36 cells derived from whole Aedes albopictus

mosquito larvae cultured in DMEM containing 10% FBS.

Mouse Infections

C57BL/6 mice were obtained commercially (The Jackson Laboratory). All

mice were housed in the pathogen-free animal facilities of the California Sate Polytechnic

University, Pomona. 4-5-week old mice were inoculated subcutaneously via footpad injection (50 µl) or intracranially (60 µl) with either 103 or 2.4 x 103 PFU of WNV- kunjin, respectively as previously described (May et al, 2011). Infected mice were monitored daily for weight loss and presentation of clinical signs of disease, including activity level (A), ability to lay down/get up (L), breathing (B), hair coat (C) including ruffled hair, and weight loss (W). Scores ranged from 0 to 3, with a score of 0 identifying no change while a score of 3 representing a severe change in the designated parameter. In activity level mice were scored a 1 if weak or reluctant to move, a 2 if hunched over, or a

3 if immobile. In lay down/get up mice were scored a 1 if slow to lay down or get up, a 2

12 if very weak, or a 3 if not able to right itself. In breathing mice were scored a 1 if exhibiting an increased/decreased rate of breathing, a 2 if breathing with an open mouth, or a 3 if gasping. In hair coat mice were scored a 1 if exhibiting piloerection, a 2 if exhibiting a lack of grooming, or a 3 if exhibiting coat staining or nasal/ocular discharge.

In weight loss mice were scored a 1 if experiencing 0-10% weight loss from pre-infection weight, a 2 if experiencing 10-20% weight loss from pre-infection, and a 3 if experiencing >20% weight loss from pre-infection. Mice were given a score of 0 in all categories if they showed no signed of illness or abnormality. Scores for all categories were then added to derive a sum (S) to assess the overall health of the mouse.

Lymphotactin Neutralization

150 µl of 1 mg/ml Rat α-Mouse XCL1 Rat IgG, or PBS as a control, was injected intracranially (Matsumoto et al, 2017) on day -1 WNV infection to neutralize lymphotactin.

Tissue Collection and Preparation

All tissues harvested from mice for subsequent analysis were extracted following

extensive cardia perfusion with 30 ml of sterile PBS. Mice were first anesthetized with a

Ketamine/Xylazine cocktail at a dose of 0.1 ml per 10 g mouse via intraperitoneal

injection. Approximately 0.5 ml of blood was collected via retro-orbital bleed and the

serum separated via centrifugation. Spleen and brain tissues were collected and all tissue

was weighed. Tissue used for histological studies were immersion fixed in 4%

paraformaldehyde.

RNA Isolation and qRT-PCR

Total RNA was prepared using the RNeasy kit (QIAGEN) according to the

13 manufacturer’s instructions. Levels are shown as relative fold changes in gene expression compared to the housekeeping gene GAPDH.

Lymphotactin Protein Analysis

Lymphotactin expression in brain and spleen homogenates was measured using a lymphotactin ELISA kit (R&D systems DY486) according to the manufacturer’s instructions. 96 well plates were coated with lymphotactin capture antibody overnight.

After washing with wash buffer, reagent diluent was added for 1 hour and washed again.

Standards and samples were left for 2 hours and washed. Detection antibody was left for

2 hours and washed followed by the addition of substrate solution for 20 min. Lastly stop solution was added and samples were immediately read using a microplate reader

(Beckman Coulter Allegra X-30R Centrifuge) set to 450 nm. All procedures and incubations were done at room temperature.

Immunohistochemistry

Brains collected on days 2 and 5 post WNV-infection were immersion fixed in

4% paraformaldehyde for at least 24 hours. Frozen tissue was then sectioned to 8 µm.

Antibody staining was performed using rabbit α- human CD3 (DAKO) at 1:500 and rat

α- mouse (BD) at 1:20 dilution. Alexa Flour 555 goat α- rabbit (Fisher) and Alexa Flour

488 goat α- rat (Fisher) were used at 1:200 dilution as fluorotags, along with 4’, 6-

Diamindino-2-Phynylindole, Dihydrochloride (DAPI) (Fisher) at 1:10,000 dilution.

WNV stain was performed using mouse α-West Nile/Kunjin E (Millipore) at 1:20 dilution and goat α-mouse AF488 (Fisher) at 1:200 dilution. TUNEL staining was done using a kit (Invitrogen C10617). Brain imaging was conducted on a Nikon Eclipse

TE2000-U light microscope linked to a computer with NIS Elements BR 3.0 software or

14 a Nikon Eclipse Ti-E inverted microscope system linked to a computer system with NIS-

Elements Imaging Software. Quantitative analysis was performed by counting the number of positive cells per image.

Statistical Analysis

Graphs were made and statistical analysis was performed via computerized software (Prism). Depending on the data, an unpaired t test, f test, or one-way ANOVA was performed, with P < 0.05 considered to be significant.

RESULTS

Lymphotactin Expression and its Protective Role During WNV Neuroinvasive Disease

Lymphotactin has been shown to be elevated during bacterial, viral, and parasitic

infection (Lei and Takahama, 2012). However, the expression of lymphotactin during

WNV encephalitis remains unknown. In order to begin investigating the precise role of

lymphotactin during anti-WNV immunity, the expression levels of lymphotactin in

peripheral and CNS tissues were examined in 4-5-week-old C57BL/6 mice after footpad

inoculation of 103 PFU of WNV-Kunjin strain (May et al, 2011). In the spleen, transcript

levels of lymphotactin peaked at day 4 after infection and then returned to background

levels by day 6, and then reversed course to rise again at day 8 and 10 after infection (Fig.

2). In the CNS, transcript levels of lymphotactin peaked at day 6 after infection, a time

point at which infectious virus can be recovered from the WNV-infected CNS (Fig. 2 and

Samuel and Diamond, 2012). These results indicate that lymphotactin appears to be

produced in response to WNV infection both in the periphery and in the CNS.

To assess the role of lymphotactin in WNV infection and neuroninvasive disease, the survival rates in mice that had lymphotactin depleted via monoclonal anti-XCL1 (α-

XCL1 + WNV) (Matsumoto et al, 2017) were compared to mock treated (PBS + WNV)

control mice following WNV infection. 4-5-week-old C57BL/6 wild-type (WT) mice

were infected via intracranial injection with 2.4 x 103 PFU of WNV-Kunjin that had been

intracranially administered 150 µl lymphotactin-neutralizing antibody (WNV + αXCL1)

at day -1 prior to infection. Following infection, mice were monitored daily for survival

and development of clinical signs of disease. By day 5 after infection, all mice that had

been inoculated with 2.4 x 103 PFU of WNV-KUNV exhibited accelerated and uniform

mortality compared to uninfected (naïve) and mock-treated (PBS + WNV) controls

(Table 1 & Fig. 4). Increased mortality in the WNV-infected mice was accompanied by

earlier and more severe development of clinical signs of disease burden (Table 1). Any

mice found to be moribund were euthanized and considered to have died on that day. By

day 4 post-infection, weight loss could be observed in WNV-infected mice with 18% of

the mock-treated (PBS + WNV) control mice deceased and 50% of the lymphotactin

neutralized (αXCL1 + WNV) mice deceased. The WNV infected mice began to show

substantial weight loss on day 4 but there was no significant difference in weight loss

between the lymphotactin neutralized (αXCL1 + WNV) and mock-treated groups (PBS +

WNV) (Fig. 3). There was, however, a statistical difference in weight loss on day 4

between the WNV infected mice and the uninfected mice. These data suggest that the

viral burden, and most likely the initial inoculation, was too high in order to effectively

assess the role of lymphotactin in the restriction of WNV infection or in its ability to

control viral replication within the CNS

To determine if the increased susceptibility of WNV-infected mice was caused by

viral load, to injection-induced microtrauma, or to antibody (Ab)-induced inflammation

within the brain, 4-5-week-old C57BL/6 mice were either intracranially (i.c.) infected

with the same dosage of WNV (2.4 x 103 PFU) as previously performed in the absence of neutralizing AB (WNV), or were injected, i.c., with lymphotactin-neutralizing Ab alone

(αXCL1) or with PBS alone (PBS) and then monitored daily for survival and development of disease. Differences in survival rates or in the development in disease were not observed between the αXCL1-treated (αXCL1) and the PBS-treated (PBS) mice up to day 8 after inoculation (Fig. 3). However, mice that had been solely inoculated with

2.4 x 103 PFU of WNV succumbed universally (0% survival) by day 5 after infection

(Fig. 4). These results suggest that the increased susceptibility as was as the increased

morbidity was a direct result of the high viral inoculation and not from any externally-

derived inflammation due to the intracranial inoculation with the αXCL1 neutralizing

antibody.

Lymphotactin Is Effectively Neutralized In Peripheral Tissues Via Anit-XCL1

Neutralizing Antibody

Since lymphotactin expression increased in both peripheral and CNS tissue

following WNV infection (Fig. 2) and, due to the fact, that XCL1 neutralization did not

impact on susceptibility to WNV infection or on clinical neuroinvasive disease

progression (Fig. 3), the neutralization efficiency of the αXCL1 antibody, and therefore

the function of XCL1, was evaluated. In order to determine if lymphotactin was effectively neutralized via i.c. administration of XCL1-neutralizing antibody, the protein levels of lymphotactin were determined by performing an enzyme-linked immunosorbent assay (ELISA) in peripheral and CNS tissues after i.c. inoculation of 2.4 x 103 PFU WNV in 4-5-week-old mice. In the spleen, lymphotactin levels were significantly lower at day 2 after WNV infection in mice that had been i.c. inoculated with the αXCL1 neutralizing

antibody (αXCL1 + WNV) compared to mice that had been mock-treated (PBS + WNV)

(Fig. 5). By day 5 post infection, a time point one day after peak expression levels of

lymphotactin were detected within peripheral tissues (Fig. 2), lymphotactin levels

remained low in the αXCL1 neutralizing Ab treated mice (αXCL1 + WNV) but failed to

reach levels of significance when compared to mock-treated (PBS + WNV) or naïve

controls (P = 0.1601)(Fig. 5). In the CNS, protein levels of lymphotactin were detected at

day 2 and 5 post infection, but these levels did not significantly change in response to

XCL1 neutralization via αXCL1 antibody treatment compared to mock-treated controls

(PBS + WNV) (Fig. 5). Since expression levels of lymphotactin peak at ay 6 post

infection within the WNV-infected CNS (Fig. 2), it remains unclear whether αXCL1

antibody treatment would have had any impact on the CNS-specific anti-viral immune

response during WNV encephalitis. Overall, these findings suggest that treatment via i.c.

injection with XCL1-neutralizing antibody was more effective within peripheral tissues

than within tissues that were isolated from the CNS at early time points following WNV

infection.

Lymphotactin Promotes Control of WNV Burden within the CNS

Due to high viral inoculum, which resulted in universal mortality between the

separate experimental groups (Fig. 4), it is possible that the immunoprotective effects of lymphotactin were obscured at the organism level in the WNV-infected mice. In order to

investigate whether lymphotactin has a role in controlling WNV infection at the cellular

level, viral burden was analyzed within the neuronal cells of the CNS following i.c.

inoculation with WNV. Following lymphotactin neutralization, WNV-infected (WNV-

Ag+) cells were found to be increased in the XCL1-neutralizing Ab treated mice (αXCL1

+ WNV) compared to mock-treated (PBS + WNV) control mice (Fig. 6), however, this

increase in WNV-infected cells did not reach levels of statistical significance (P = 0.28).

These data suggest that lymphotactin may have an impact in restricting WNV replication

but due to the high inoculum, its protective effects were hidden within the CNS.

Lymphotactin Plays a Protective Role Against Virus-Associated Neuronal Cell Death

High viral loads in the CNS have previously been associated with extensive

19 neuronal apoptosis (Samuel and Diamond, 2006). In order to further determine whether lymphotactin had an immunoprotective role at the cellular level, apoptosis of neuronal cells was analyzed within the brain tissue isolated from i.c. inoculated animals during

WNV encephalitis. After lymphotactin neutralization treatment in WNV-infected mice

(αXCL1 + WNV), TUNEL+ cells were in abundance when compared to the mock-treated control mice (PBS + WNV), indicating an increase in the neuronal cell death (Fig. 7).

However, as was seen previously with WNV burden in the CNS (Fig. 6), this increase did not reach levels of significance (P = 0.3744). These data suggest that lymphotactin may play a protective role against virus-associated mortality and may have a slight role in the control of viral proliferation and thus the neuronal death caused by viral infection. These results also further support the conclusion that the high viral innoculum is most likely the cause for the uniform death of the WNV-infected mice.

Lymphotactin Impacts the Localization of T cells during WNV Encephalitis

Previous studies have demonstrated that the parenchymal entry of WNV-specific lymphocytes is important for clearing WNV infection from the CNS and that these Ag- specific T lymphocytes require full reactivation through a DC-dependent mechanism

(McCandless E, 2008 and Durrant, 2013). Since lymphotactin has been shown to have a mediatory role in the recruitment and establishment of DC-T cell interactions ensuring effective T cell priming, activation, and proliferation within secondary lymphoid tissue

(Dorner et al, 2009), the role of lymphotactin in the recruitment and establishment of DC-

T cell interactions within the CNS was examined by determining the localization of CNS- infiltrating T lymphocytes during WNV encephalitis. In order to determine whether lymphotactin impacts on the full reactivation of viral-specific T cells and subsequently

20 their ability to migrate out of the perivascular space of the BBB and into the parenchyma, we evaluated the trafficking of T lymphocytes within the brains of WNV-infected XCL1- neutralizing AB treated and mock-treated mice. The immunohistochemical detection of

CD3, a T cell marker, in relation to CD31, an endothelial cell marker, was measured within brain tissues collected day 2 and 5 post-infection (Figure 8). Analysis of infiltrating T cells at day 5 post-infection revealed increased number of CD3+ lymphocytes within the CNS tissue and WNV-infected, lymphotactin-neutralizing AB

(αXCL1 + WNV) treated mice compared to those of their similarly infected, mock- treated (PBS + WNV) counterparts (Fig. 8). Despite the increased numbers of infiltrating

T lymphocytes in the Ab-treated mice, these data did not reach statistical significance (P

= 0.2661). Further evaluation of the location of CD3+ T cells within the brain of WNV-

infected Ab-treated and mock-treated mice with respect to the CD31+ microvasculature

showed that parenchymal entry of T cells at day 5 post-infection was significantly

reduced in the WNV-infected Ab-treated (αXCL1 + WNV) mice compared with similarly

infected mock-treated (PBS + WNV) mice (Figure 8) suggesting that lymphotactin

neutralization led to a decrease in the percentage of T cells that are found in the

perivascular space. These data suggest that lymphotactin is required to stimulate T cell

entry during WNV encephalitis.

Tables

Table I: Health Status Assessment Results on Day 5. 4-week-old mice were either administered 150 µl lymphotactin neutralizing antibody (α-XCL1 + WNV) or phosphate buffered saline (PBS + WNV) intracranially on day -1 post infection and then inoculated intracranially with 2400 PFU of WNV. Mice were then monitored daily and given scores on a scale from 0-3 in each category. The scores were then added to produce a total score to assess the overall health of the mouse. Data provided is the mean ± standard deviation for all mice within each treatment group. WNV A B C L W S 2 ± 1.0 1 ± 0.0 1 ± 1.0 1.5 ± 0.5 3 ± 0.0 8.5 ± 2.5

PBS + WNV A B C L W S 2.7 ± 0.4 2.7 ± 0.4 2.6 ± 0.4 2.8 ± 0.4 2.6 ± 0.4 13.4 ± 1.5

α-XCL1 + WNV A B C L W S 2.5 ± 0.5 2.5 ± 0.5 2.5 ± 0.5 2.6 ± 0.4 2.6 ± 0.4 12.7 ±1.9

Figures

Capilla,y Lumen

• XCL I

Layer

Perivas.cular CD8+TccD. Space interactions?

Glial Limitans

Astrocyte Endfeet WNV • N,urnn

Figure 1: Potential mediatory role of lymphotactin (XCL1) in DC recruitment and/or in stabilizing T-cellular interactions within the perivascular space during WNV encephalitis. Activated, virus-specific CD8+ T cells migrate to the perivascular spaces of the CNS in response to viral infection of the CNS with WNV. Within the perivascular space, infiltrating CD8+ T lymphocytes fail to be released from the epithelial layer until they achieve full reactivation which is mediated by CNS-localized DCs. Lymphotactin (XCL1) expression from activated and arrested T cells may attract DCs and/or may be instrumental in stabilizing DC-T cell interactions within this space.

Spleen

C: 20 .2 (/) (/) e! 15 C. >< w 10 Qj C: Qj (!) 5 32 0 LL 0 Un 2 4 6 8 10 Days Post Infection

Brain 20 *

0 Un 2 4 6 8 10 Days Post Infection

Figure 2: Lymphotactin levels during WNV encephalitis. 4-week-old mice were inoculated with 103 PFU of WNV by footpad injection. Spleen and brain tissues were collected at the indicated time points. mRNA levels of XCL1 were analyzed via qRT- PCR. Levels are shown as relative fold changes in gene expression compared to the housekeeping gene GAPDH. *, P < 0.05.

110 ...... Naive

--&- PBS 100 ~ eJl aXCLl =~ --- -=u 90 .... WNV ~ = -a- 80 PBS+WNV .... aXCLl 70 +WNV -1 0 1 2 3 4 5 Days post infection

Figure 3: Percent of weight change in mice from days -1 to 5 post West Nile infection. 4-week-old mice were either administered 150 µl lymphotactin neutralizing antibody (αXCL1 + WNV) or phosphate buffered saline (PBS + WNV) intracranially one day prior to WNV infection and then inoculated intracranially with 2.4 x 103 PFU of WNV. Mice were then weighed and monitored daily. Data reflects the mean and error bars are SEM. One-way ANOVA was used to determine statistical significance between WNV-infected groups and non-infected groups. ****, P < 0.0001

100 Naive ..... PBS -~ - .E aXCLl WNV =~ - ... 50 -&- PBS+WNV =~ - i: ~ ~ aXCLl +WNV ~ ** 0 0 2 4 6 8 10 Days post infection

Figure 4: Percent of survival to day 8 post West Nile Infection. 4-week-old mice were either treated with 150 µl lymphotactin neutralizing antibody (αXCL1 + WNV) or mock- treated (PBS + WNV) intracranially on day -1 post infection and then inoculated intracranially with 2.4 x 103 PFU of WNV and observed for 8 days. Mice found to be moribund were euthanized and considered to have died on that day. Survival differences were found to be significant between WNV-infected and non-infected groups. P = 0.0012

Brain

4x104 4x104 - Naive - PBS + WNV 3x104 aXCLI + WNV 3x104 t~ 8 2x104 ,-; - u~ ~ }x104

0 2 5 2 5

Days post infection

Figure 5: Protein levels of lymphotactin are lower in peripheral tissues following treatment with XCL1-neutralizing Ab. 4-week-old mice were either treated with 150 µl lymphotactin neutralizing antibody (αXCL1 + WNV) or mock-treated (PBS + WNV) intracranially on day -1 prior to infection and then inoculated intracranially with 2.4 x 103 PFU of WNV. Spleen and brain tissue were collected on days 2 and 5 post-infection and protein levels of lymphotactin were determined by ELISA. Data reflects the mean and error bars are SEM. Unpaired t test was used to determine significance between mock- treated (PBS + WNV) and Ab-treated (αXCL1 + WNV groups). *, P < 0.05.

Day 2 Day 5

PBS

a-XCLI

WKV DAPI

=J 30 8 + ~ 20 ;.:. ~ 10

0 PBS +WNV aXCLl + WNV

Figure 6: Lymphotactin promotes viral control within the CNS during WNV encephalitis. 4-week-old mice were either administered 150 µl lymphotactin neutralizing antibody (αXCL1 + WNV) or phosphate buffered saline (PBS + WNV) intracranially on day -1 post infection and then inoculated intracranially with 2.4 x 103 PFU of WNV. Brain tissue was collected on day 2 and 5 post-infection, submersion fixed in 4% paraformaldehyde, cryopreserved, and then sectioned to 8 µm. Representative confocal microscopic images of WNV antigen (green) and DAPI for nuclear stain are shown. Inset image represents naïve brain tissue. Quantifications of WNV Ag+ confocal images are provided considering data from day 5 images. Cells within the image indicating positive staining were counted manually and the cell counts per image were averaged within each treatment groups. P = 0.28

Day 2 Day 5

PBS

a-XCLI

TUNEL DAPI Day2 Da)' 5

60 20

Figure 7: Lymphotactin plays a protective role against virus-associated neuronal cell death. 4-week-old mice were either treated with 150 µl lymphotactin neutralizing antibody (αXCL1 + WNV) or mock-treated (PBS + WNV) intracranially on day -1 post infection and then inoculated intracranially with 2.4 x 103 PFU of WNV. Brain tissue was collected on day 2 and 5 post-infection, submersion fixed in 4% paraformaldehyde, cryopreserved, and then sectioned to 8 µm. Confocal microscopic images of TUNEL staining (green) and DAPI for nuclear stain are shown. Inset image is representative of brain tissues collected from naïve controls. Quantifications of TUNEL+ cells within the confocal images are shown. Cells within the image indicating positive staining were counted manually and the cell counts per image were averaged within each treatment groups. Unpaired t test used to determine significance between antibody and naïve groups. *, P < 0.05.

Day 2 Day 5

PBS

a-XCLI

CD31 CD3 DAPI

CD3+ Cells

PBS+WNV uXCLl+WNV PBS +WNV aXCLl +WNV

Figure 8: Lymphotactin impacts the perivascular localization of T cells during WNV encephalitis. 4-week-old mice were either administered 150 µl lymphotactin neutralizing antibody (αXCL1 + WNV) or phosphate buffered saline (PBS + WNV) intracranially on day -1 post infection and then inoculated intracranially with 2400 PFU of WNV. Brain tissue was collected on day 5 post-infection, submersion fixed in paraformaldehyde, cryopreserved, and sectioned to 8 µm. Representative confocal microscopic images of CD3 (red), CD31 (green), and DAPI are shown. Image in inset is naïve collected on day 5. Quantifications of confocal images are provided. Cells within the image were totaled manually and the cell totals per image were averaged within each treatment groups. F test used to determine significance between PBS and αXCL1 groups in CD3+ cells. **, P < 0.01.

. Capilla,y Lumen • • XCRI XCLI • Laye, * Perivascular Space

Glial Limitans

Parenchyma

As.trocyte Endfeet • • Nemon

Figure 9: Lymphotactin-mediates DC-T cell interactions within the perivascular space during WNV encephalitis. Activated, virus-specific CD8+ T cells retained within the perivascular space produce lymphotactin to ensure and stabilize interactions with CNS- localized DCs. This stabilized interaction is required for efficient T cell reactivation and subsequent entry into the WNV-infected parenchyma to promote viral clearance and immunoprotection within the CNS.

DISCUSSION

In order to provide protection in the CNS, CD8+ T cells must be fully reactivated which requires interaction with CNS-localized DCs, yet it is unclear how DCs are recruited to the critical regions of the brain in order to achieve this critical interaction during viral infection. Previous studies have indicated that CD8+ T cells rapidly produce the chemokine lymphotactin in response to viral and tumor antigens (Lin et al, 2008).

However, the role of lymphotactin in mediating cytotoxic T lymphocyte response within the CNS during viral infection is unknown. In these studies, we begin to further clarify the role of lymphotactin during viral encephalitis. We show that lymphotactin is produced in response to WNV infection, peaking on day 4 post-infection in the peripheral tissues and on day 6 post-infection in the CNS. In order to gain a greater understanding of the neuroprotective role of lymphotactin during WNV encephalitis, we depleted lymphotactin intracranially via an anti-XCL1 neutralizing antibody. The neutralization of lymphotactin was found to be more effective in the peripheral tissues than in the CNS following WNV infection illustrating that neutralization of lymphotactin was more effective in the peripheral tissues compared to CNS tissues at early time points of WNV encephalitis. Following anti-XCL1 Ab treatment, WNV-infected neuronal cells were found to be increased as well as the overall number of neuronal cells undergoing apoptosis compared to mock-treated mice. These results suggest that lymphotactin has a role in controlling viral replication and proliferation, and protecting the CNS from viral - mediated neuronal cell death. Lymphotactin neutralization via XCL1-neutralizing Ab also resulted in a higher ratio of T cells in association with the CNS microvasculature compared to parenchymal T cells, suggesting that lymphotactin has a modulatory role in

the localization of T cells within the CNS during WNV encephalitis. Since it is known

that anti-WNV CD8+ T cells are crucial for viral control within the CNS as well as

neuronal protection during WNV infection, our results suggest that lymphotactin plays a

vital role in the full reactivation and thus the parenchymal migration of effector T cells

within the CNS during WNV encephalitis. Overall, these findings begin to illuminate the

immunoprotective role of lymphotactin during WNV encephalitis.

Lymphotactin expressed by T cells exclusively chemoattracts CD8+ DCs (Dorner

et al, 2009) and has been shown to increase early CD8+ T cell responses to Listeria

monocytogenes infection (Crozat et al, 2010). Previous studies have considered the effect

of lymphotactin on CD8+ T cell localization within lung lesions during infection with

Mycobacterium tuberculosis (Ordway et al, 2007). Lymphotactin has also been found to

inhibit a broad spectrum of HIV-1 isolates through direct interaction with the HIV-1

envelope which blocks viral attachment and entry into host cells at an early stage of infection (Guzzo et al, 2013). In parasite-resistant mice infected with Leishmania major, lymphotactin was found to be elevated in the draining lymph nodes, but not in parasite- suseptible mice (Vester et al, 1999). Lymphotactin expression is also upregulated in the cornea and trigeminal ganglia of mice infected with herpes simplex virus type 1 (Araki-

Sasaki et al, 2006). The elevation of lymphotactin has also been detected in several autoimmune diseases including rheumatoid arthritis, Chron’s disease, and insulin- dependent diabetes mellitus (Lei and Takahama, 2011). Previous studies pertaining to lymphotactin provide strong evidence that it is upregulated during infectious disease and that it is necessary for an early CD8+ T cell response against infection. These studies are similar to our current study in that they both consider the immunoprotective role of

lymphotactin during infectious disease and the effect of lymphotactin on the localization

of CD8+ T cells (Ordway et al, 2007). However, as far as we know, our studies are the

first to interrogate the role of lymphotactin within the CNS during an infectious disease.

All mice that had been inoculated with WNV exhibited accelerated and uniform

mortality by day 5 with no statistical difference between the lymphotactin neutralized and

PBS control groups (Fig. 4). This outcome was most likely due to the viral inoculum that may have disrupted the effectiveness of anti-XCL1 to effectively neutralize lymphotactin during WNV encephalitis. In fact, the amount of lymphotactin secreted has been shown to be directly correlated with the strength of the antigenic signal (Dorner et al, 2009).

Thus, the high viral titer in the brain may have led to a higher amount of lymphotactin secretion; overcoming the ability of the antibody to fully neutralize lymphotactin. Indeed, higher levels of lymphotactin were found in the brain when compared to the naïve mice

(Fig. 5). Another possibility for the high lymphotactin levels in the brain (Fig. 5) is due to

T cell overcompensation. As the initial secretion of lymphotactin was neutralized via the anti-XCL1 antibody, the T cells may have responded by secreting more lymphotactin and thus diluting the effectiveness of the lymphotactin antibody within the CNS. This may also be correlated with the high viral titers seen on day 5 post infection (Fig. 6), since an increase in viral titer would lead to an increase in the number of activated T cells that secrete lymphotactin. The viral titer that we used for our WNV encephalitis model was in the magnitude of 103 PFU of WNV-Kunjin; other studies have had successful infections

with dosages ranging from 102 – 104 PFU (Bingham et al, 2014) and thus suggests a

possibility of initiating a successful infection utilizing a lower viral titer. In order to

expand and confirm the neuroprotective role of lymphotactin in the restriction of WNV

33 infection or in the protection against WNV neuroinvasive disease, additional studies should assess clinical disease development and survival with lymphotactin neutralization at varying viral titers.

Secondary lymphoid organs are a site where mature lymphocytes colonize and interact with antigenic stimuli to execute an antigen-specific immune response. DCs, which have taken up antigen within peripheral tissues, migrate to secondary lymph tissues such as the draining lymph nodes, in order to present antigen to T cells for their activation (Dorner et al, 2009). After activation, CD8+ T cells migrate to the site of infection and perform viral clearance by killing virus-infected cells. For protection in the

CNS, CD8+ T cells must be fully reactivated through the interaction with DCs for complete effector function (Brewitz et al, 2017). Recent scientific evidences have led to the understanding of the CNS as a virtual secondary lymphoid organ (Anandasabapathy et al, 2011). In fact, T cell immune surveillance of the CNS is restricted to the perivascular spaces and DCs have been shown to be found in the brain, particularly within these spaces (Anandasabapathy et al, 2011), which suggests they have an immunomodulaory role within the CNS. Infiltrating T cells that have been localized within the perivascular space require interaction with DCs for their full activation which allows these cells to leave this space and migrate into the parenchyma where they provide their immunoprotective role. Based on these studies, we suggest that antigen-specific T cells localized within the perivascular space produce lymphotactin in order to ensure a stable interaction with DCs for full activation, similar to the DC-T cell interactions that occur within secondary lymphoid organs (Figure 9).

Previous studies have shown that lymphotactin has a mediatory role in DC-T cell interactions to ensure the full activation and proliferation (Crozat et al, 2010). During

WNV encephalitis, it is known that CNS infiltrating T cells also need to be fully activated and that their full reactivation is mediated by intracerebral DCs (Durrant, 2013). Our studies begin to clarify the role of lymphotactin in mediating DC-T cell interactions within the WNV-infected CNS and show that lymphotactin impacts the localization of T cells within the parenchyma where they are needed for viral clearance. In order to further clarify the specific role of lymphotactin in mediating DC-T cell interactions, further studies should assess the perivascular localization of DCs in relation to the T cells throughout the course of neuroinvasive disease with WNV. In addition, future studies should address the activation status of infiltrating T lymphocytes in the presence or absence of XCL1. This information would provide insight into how DCs are recruited to the brain to achieve interaction with the infiltrating T cells and whether this interaction is effectively establishing T lymphocyte reactivation.

WNV is an emerging neurotropic virus that continues to spread and cause increased outbreaks throughout many regions of the world. Due to the burden WNV, a mosquito-borne viral infection, has on public health as well as the lack of effective treatments to eliminate or prevent infection, further understanding of WNV infection and immunity is crucial. Although much work has defined some of the cell-specific processes involved in immune control, the governing process that regulates CNS infection and immune protection within the CNS needs to be further explored. Our study begins to identify the immunomodulatory role of lymphotactin within the CNS which is crucial for effector T cell reactivation and virologic control within the CNS during WNV

35 encephalitis. This further aids in our understanding of antiviral immunity within the CNS, which may provide insights into the development of novel therapeutics and strategies for enhancing neuroprotective immunity during WNV infection.

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