Abstract

   Programmed cell death-1 (PD-1) receptor signaling dampens the
   functionality of T cells faced with repetitive antigenic stimulation
   from chronic infections or tumors. Using intracerebral (i.c.)
   inoculation with mouse polyomavirus (MuPyV), we have shown that CD8 T
   cells establish a PD-1^hi, tissue-resident memory population in the
   brains (bT[RM]) of mice with a low-level persistent infection. In MuPyV
   encephalitis, PD-L1 was expressed on infiltrating myeloid cells,
   microglia and astrocytes, but not on oligodendrocytes. Engagement of
   PD-1 on anti-MuPyV CD8 T cells limited their effector activity.
   NanoString gene expression analysis showed that neuroinflammation was
   higher in PD-L1^−/− than wild type mice at day 8 post-infection, the
   peak of the MuPyV-specific CD8 response. During the persistent phase of
   infection, however, the absence of PD-1 signaling was found to be
   associated with a lower inflammatory response than in wild type mice.
   Genetic disruption and intracerebroventricular blockade of PD-1
   signaling resulted in an increase in number of MuPyV-specific CD8
   bT[RM]and the fraction of these cells expressing CD103, the αE integrin
   commonly used to define tissue-resident T cells. However, PD-L1^−/−
   mice persistently infected with MuPyV showed impaired virus control
   upon i.c. re-infection with MuPyV. Collectively, these data reveal a
   temporal duality in PD-1-mediated regulation of MuPyV-associated
   neuroinflammation. PD-1 signaling limited the severity of
   neuroinflammation during acute infection but sustained a level of
   inflammation during persistent infection for maintaining control of
   virus re-infection.

   Keywords: viral encephalitis, tissue-resident memory, CD8 T cells,
   PD-1, PD-L1, mouse polyomavirus, neuroinflammation

Introduction

   The inhibitory receptor PD-1 plays a dominant role in T cell
   exhaustion, a state of progressive loss of T cell function resulting
   from repetitive antigen stimulation such as in chronic viral disease or
   tumor development ([43]1). Extensive work using experimental models of
   chronic infection [lymphocytic choriomeningitis virus (LCMV)-clone 13,
   simian immunodeficiency virus (SIV)] as well as analysis of T cells
   from individuals infected with human immunodeficiency virus (HIV),
   hepatitis C virus, and hepatitis B virus, demonstrate that blockade of
   PD-1 signaling restores T cell functionality ([44]2). Engagement of
   PD-1 by its ligands PD-L1 (CD274/B7-H1) or PD-L2 (CD273/B7DC) results
   in recruitment of SHP-phosphatases proximal to the T cell receptor
   (TCR). These phosphatases inactivate kinase cascades induced by TCR
   signaling and thereby inhibit downstream pathways required for cytokine
   production, proliferation, and cytotoxicity ([45]3, [46]4). PD-L1 is
   expressed on a variety of cell types while the expression of PD-L2 is
   limited to antigen presenting cells ([47]5–[48]7). CNS infection with
   neurotropic coronavirus induced the expression of PD-L1 but not PD-L2
   on glia ([49]8). In addition, only PD-1:PD-L1 interactions are
   responsible for inhibiting CD8 T cell effector function in mouse
   cytomegalovirus (MCMV) CNS infection ([50]9). PD-1 mediated T cell
   exhaustion is characterized by increased expression of the
   transcription factors Eomesodermin (Eomes) and B lymphocyte-induced
   maturation protein-1 (Blimp-1), co-expression of inhibitory receptors
   PD-1, Tim3, and 2B4 and diminished effector function [Interferon
   (IFN)-γ and degranulation] ([51]2, [52]10).

   Accumulating evidence challenges the concept that PD-1 expression is
   solely synonymous with T cell dysfunction and senescence
   ([53]11–[54]13). PD-1 regulates T cell mobility in tissues ([55]14) and
   T cell survival ([56]10, [57]15). Additionally, in chronic high-viremic
   infections, T cell dysfunction is not absolute as shown by the
   emergence of CD8 T cell epitope-escape HIV late in infection ([58]16),
   and increased viral titers after depletion of CD8 T cells in chronic
   SIV infection ([59]17, [60]18). Recent reports support the concept that
   CD8 T cell exhaustion is a bona fide state of differentiation adapted
   by T cells, which enables them to survive and retain functionality
   during persistent infection ([61]19, [62]20). Reversal of T cell
   exhaustion by blockade of the PD-1:PD-L1 axis indicates that exhausted
   T cells span a spectrum of dysfunction with the least exhausted cells,
   characterized as Tim3^−PD-1^intTCF-1^hiCXCR5^hi cells in LMCV infection
   ([63]21, [64]22), being more susceptible to functional resurrection
   after checkpoint inhibitor blockade. PD-1 or PD-L1 antibody-mediated
   blockade has shown remarkable effectiveness for certain types of cancer
   ([65]23). Expression of PD-1 has recently been reported in human lung-
   and brain-resident CD8 T cells, with PD-1 signaling proposed to limit
   inadvertent deployment of effector mechanisms ([66]24). Tissue-resident
   memory CD8 T cells are an essential component of the first line of
   defense against re-infection in non-lymphoid tissues ([67]24–[68]26).
   Whether PD-1 regulates CD8 T cell activity and affects memory
   differentiation in non-lymphoid tissues is an open question. Here we
   asked, in CNS persistent infection, if PD-1 operates to dampen CD8 T
   cell effector activity and modulate neuroinflammation.

   Mouse polyomavirus (MuPyV), a non-enveloped virus with a covalently
   closed circular ~5-kb double-stranded DNA genome, is the founding
   member of the family Polyomaviridae. MuPyV establishes a systemic
   low-level persistent infection in mice; however, whether the virus
   persists as a smoldering infection or cycles between latency and
   reactivation has yet to be experimentally ascertained. Inoculation i.c.
   with MuPyV gives rise to a stable population of virus-specific PD-1^+
   tissue-resident memory CD8 T cells in the brain (bT[RM]) ([69]27,
   [70]28). Although the role of PD-1 in modulating T cell function has
   been extensively investigated for virus-specific lymphoid CD8 T cells
   in the setting of chronic viremia ([71]29), only a few studies have
   examined PD-1's impact on T[RM] responding to a persistent viral CNS
   infection ([72]8, [73]9, [74]30). In humans, JC polyomavirus (JCPyV)
   causes several aggressive CNS diseases, the most common being the
   frequently fatal demyelinating disease progressive multifocal
   leukoencephalopathy (PML) ([75]31). JCPγV-specific CD8 T cells in PML
   patients express PD-1 ([76]32). MCMV, Theiler's murine
   encephalomyelitis virus (TMEV), and JHM mouse hepatitis virus (JHMV)
   CNS infections demonstrate that excessive CD8 T cell effector activity
   is immunopathologic ([77]8, [78]9, [79]30, [80]33). Whether PD-1
   regulates neuroinflammation and formation of CD8 T[RM] in a persistent
   infection model is incompletely understood.

   In this study, we show that PD-1 acts to inhibit the effector functions
   of virus-specific CD8 bT[RM] during MuPyV encephalitis. NanoString
   inflammatory gene expression analysis of brains of wild type (WT) and
   PD-L1^−/− mice infected with MuPyV revealed that PD-1 signaling
   controls the inflammatory response during acute infection. In striking
   contrast, the absence of PD-1 signaling during persistent infection
   resulted in lower neuroinflammation than in WT mice. PD-1 was further
   found to affect the differentiation of virus-specific CD8 bT[RM].
   Together, these findings reveal a complex, dynamic impact of PD-1 on
   neuroinflammation and CD8 bT[RM] formation and activity during
   persistent viral encephalitis.

Materials and Methods

Mice and Virus Inoculation

   C57BL/6NCr (WT) female mice purchased from the Frederick Cancer
   Research and Development Center of the National Cancer Institute
   (Frederick, MD) and B7-H1^−/− (PD-L1^−/−) mice [generously provided by
   C.C. Bergmann (Lerner Research Institute, Cleveland, OH) with approval
   of L. Chen (Yale School of Medicine, New Haven, CT)] were housed in
   accordance with the guidelines of the Institutional Animal Care and Use
   Committees and the Department of Comparative Medicine at the
   Pennsylvania State University College of Medicine. The Pennsylvania
   State University College of Medicine Animal Resource Program is
   accredited by the Association for Assessment and Accreditation of
   Laboratory Animal Care International (AAALAC). The Pennsylvania State
   University College of Medicine has an Animal Welfare Assurance on file
   with the National Institutes of Health's Office of Laboratory Animal
   Welfare; the Assurance Number is A3045-01. TCR-I mice expressing a TCR
   transgene specific for the large T Ag (LT-Ag) 206–215 amino acids from
   the SV40 virus and the MuPyV mutant encoding this epitope have been
   previously described ([81]34, [82]35). At 7-12 weeks of age,
   anesthetized mice were i.c. inoculated by injecting the right frontal
   lobe with 30 μl of 2 × 10^7 plaque-forming units (PFU) of MuPyV strain
   A2 in DMEM 5% FBS, as previously described ([83]27, [84]33, [85]36).

Quantification of MuPyV Genomes and LT-Ag mRNA

   TaqMan real-time PCR was performed in an ABI StepOnePlus thermocycler
   (Applied Biosciences) with 10 μg of template DNA purified from tissues
   using the Maxwell 16 Research Instrument (Promega, Madison, WI)
   according to the manufacturer's instructions. Primers and amplification
   parameters are previously described ([86]37). To determine MuPyV LT-Ag
   mRNA copy numbers, total RNA from FACS-purified cells was insolated
   using TRIzol (Ambion, USA) and cDNA was prepared using RevertAid H
   minus reverse transcriptase (Thermo Scientific, Waltham, MA), as per
   manufacturer's instructions. Quantitative PCR (qPCR) was performed
   using FastStart Universal Probe Master (ROX) mix (Sigma-Aldrich) and an
   ABI PRISM 5700 sequence detection system (Applied Biosystems, Foster,
   CA), with protocol and primer set as previously described ([87]38).

Bone Marrow Dendritic Cell Culture

   Bone marrow was flushed from the femurs and tibias of WT and PD-L1^−/−
   mice using a 30-gauge needle and syringe loaded with DMEM 10% FBS. Red
   blood cells were lysed with ACK buffer. Bone marrow-derived cells were
   plated (5 × 10^6 cells/100 mm diameter Petri dish) and cultured in DMEM
   10% FBS with GM-CSF (20 ng/ml) at 37°C, with the media changed every 3
   days. After 10 days, differentiated bone marrow dendritic cells (BMDCs)
   were harvested by gentle trypsinization [trypsin-EDTA (0.25%)
   (ThermoFisher Scientific) for < 1 min] to release loosely adherent
   cells. BMDCs were re-plated (3 × 10^6 cells/100 mm diameter Petri dish
   in DMEM 10% FBS) with 100 U/ml IFN-γ (PeproTech US, Rocky Hill, NJ) to
   induce antigen presentation and PD-L1 expression and were incubated
   overnight at 37°C. BMDCs were transferred to a 96-well plate (3 × 10^5
   cells/well) and incubated with 10 μM LT359 peptide at 37°C for ~6 h
   prior to co-culture with T cells isolated from brain or spleen.

Cell Isolation, Intracellular Cytokine Staining, and Flow Cytometry

   For isolation of neural and mononuclear cells ([88]39), anesthetized
   mice were perfused transcardially with 30 ml heparinized PBS (100
   U/ml). Brains were minced and digested with Collagenase I (100 mg/100
   ml) for 15 min at 37°C. Single-step 37% Percoll centrifugation was used
   to remove myelin from brain homogenates. Cells were washed and
   incubated with antibodies against CD45 (1:200 dilution, 30-F11), CD11b
   (1:200 dilution, M1/70), GLAST (Miltenyi, Bergisch Gladbach, Germany,
   1:200, ACSA-1), and O4 (product code 130-095-891, Miltenyi, Bergisch
   Gladbach, Germany; 1:20 dilution). For all other experiments, post
   perfusion, finely minced brains were digested with collagenase I
   (Worthington Biochemical, Lakewood, NJ) (40 mg/100 ml) for 20 min at
   37°C, followed by a two-step 44%/66% Percoll gradient to remove myelin
   and cell debris. Spleen and brain cells were exposed to Fixable
   Viability Dye (eBioscience, San Diego, CA) and Fc Block (BioLegend, San
   Diego, CA) prior to staining with D^bLT359 tetramers (NIH Tetramer Core
   Facility, Atlanta, GA) and antibodies to the following molecules: CD8α
   (53–6.7), CD44 (IM7), CD11b (M1/70), CD45(30-F11), CD69 (H1.2F3), CD103
   (M290), IFN-γ (XMG1.2), and H-2D^b (KH95) purchased from BD Biosciences
   (San Diego, CA); and PD-1 (RMP1-30), PD-L1 (MIH5), Eomes (Dan11mag),
   MHC-II (M5/114.15.2), Tim3 (RMT3-23), 2B4 (eBio244F4), and Lag3
   (eBioC9B7W) purchased from eBioscience; and CD4 (RM4-5), CD45 (30-F11),
   CD11b (M1/70), FoxP3 (MF-14), CD25 (3C7), T-bet (4B10), PD-L2 (TY25),
   CD11c (N418), and CD127 (A7R34) from Biolegend (San Diego, CA). Brain
   and spleen cells were stimulated with 1 μM LT359-368 peptide
   (SAVKNY(Abu)SKL), no peptide or peptide-pulsed IFN-γ-treated BMDCs for
   5-6 h in the presence of brefeldin A, stained for viability and surface
   markers, fixed, and permeabilized with CytoFix/CytoPerm (BD
   Biosciences, San Diego, CA), then stained for intracellular IFN-γ.
   Anti-IFN-γ staining in the absence of peptide was <1% of CD8^+ CD44^hi
   gated cells (data not shown). Samples were acquired on an LSR II or
   LSRFortessa (BD Biosciences, San Diego, CA) and data analyzed using
   FlowJo software (Tree Star, Ashland, OR).

Intracerebroventricular (i.c.v.) Cannulation

   Prior to surgery, ALZET osmotic pumps (Model 2002, DURECT Corporation,
   Cupertino, CA) were loaded with 6 mg/ml control rat IgG (Jackson
   ImmunoResearch, West Grove, PA) or PD-L1 rat IgG (Clone 10F.9G2;
   BioXCell, West Lebanon, NH), according to manufacturer's instructions,
   and connected to L-shaped cannulas (ALZET Brain Infusion Kit 3) with
   tubing trimmed to 2 cm. Pumps were incubated overnight at 37°C in
   autoclaved PBS to activate flow. Surgical procedures were performed
   similarly to those described ([89]40). Osmotic pumps were implanted
   subcutaneously via a scalp incision. After removal of soft tissue from
   the skull, the cannula was positioned into the left lateral ventricle
   (1.0 mm lateral to midline, 0.1 mm posterior to bregma, and 3.0 mm
   dorsoventral to the skull) and secured with ALZET Loctite adhesive. The
   scalp was sutured over the cannula. i.c.v. antibody administration
   lasted 14 days at a flow rate of 12 μl/day. Delivery of antibody to the
   lateral ventricle was confirmed by cutting the brain at the site of
   cannulation and measuring the volume of antibody remaining in the pump.

Micropipette Adhesion-Frequency Assay

   CD8 T cells from brains were isolated using magnetic bead-based
   positive selection columns (Miltenyi, Bergisch Gladbach, Germany).
   Coating of human RBCs with the peptide-MHC (p-MHC) D^bLT359 monomers,
   quantification of binding events, TCR surface densities, and TCR
   affinity calculations were performed as described earlier ([90]27,
   [91]41, [92]42). An adhesion frequency ≥ 0.1 between a T cell and a
   D^bLT359–coated RBC is scored as Ag-reactive.

Luxol Fast Blue (LFB)-Periodic Acid Schiff (PAS)-Hematoxylin Histology

   Mice were anesthetized with ketamine and xylazine, perfused
   transcardially with 10 mL of 10% heparin in PBS and 10 mL of 10%
   neutral buffered formalin (NBF). After perfusion, heads were removed
   and immersed in 10% NBF overnight at room temperature. The next day,
   brains were sectioned on a coronal brain cutting matrix. Formalin
   fixed-paraffin embedded (FFPE) samples were deparaffinized, then 10 μm
   sections stained with LFB-PAS, and counterstained with Harris-modified
   hematoxylin (Fisher), as described ([93]43). LFB-PAS stained sections
   were digitally imaged on a Keyence BZ-X710 all-in-one fluorescence
   microscope (4x magnification) and stitched together using ImageJ
   software (National Institutes of Health, Bethesda, MD). Analysis of
   myelination in the white matter tracts was performed as described
   ([94]44).

Immunofluorescence Microscopy

   Anesthetized mice were perfused transcardially with heparinized PBS
   followed by 10% NBF. Three-mm pieces were coronally using a cutting
   matrix and immersed overnight in 10% NBF and then embedded in paraffin.
   Ten μm FFPE brain sections were deparaffinized and rehydrated. Antigen
   retrieval was then performed using 10 mM sodium citrate buffer (pH
   6.0). Brain sections were stained with anti-NeuN (Clone A60; Millipore,
   Darmstadt, Germany) or anti-APC (Clone CC-1; Abcam, Cambridge, UK) for
   30 min at room temperature using the Mouse-On-Mouse Fluorescein Kit
   (Vector, Burlingame, CA), then stained overnight at 4°C with rabbit
   anti-VP1 [graciously provided by R. Garcea (University of Colorado,
   Boulder, CO)] followed by donkey anti-rabbit IgG conjugated to Alex
   Fluor 594 (Jackson ImmunoResearch, West Grove, PA). Astrocyte staining
   was performed using directly conjugated anti-GFAP (Clone GA5;
   eBioscience, San Diego, CA). Tissue sections were then mounted with
   ProLong Gold Anti-Fade Reagent with DAPI (Life Technologies, Carlsbad,
   CA). Images were acquired using a Leica DM4000 B LED microscope
   (Leica-Camera, Wetzlar, Germany).

NanoString Gene Expression Analysis

   RNA was isolated from a 2-mm thick brain section from the left
   hemisphere of the cerebrum using the Maxwell® 16 simplyRNA tissue kit
   with an in-solution DNAse digestion step. RNA from uninfected control
   groups (2 mice/pool) and infected groups (3 mice/pool) were subjected
   to NanoString gene expression analysis. 100 ng RNA was used to assess
   the expression of 254 mouse inflammatory genes provided in the
   nCounter® Inflammation mouse panels. Fold changes and p-value for the
   genes were calculated using nSolver software. Fold changes higher than
   1.5 fold and p ≤ 0.05 were considered significant. The gene list was
   imported into the Ingenuity Pathway Analysis (IPA) tool (Qiagen,
   Redwood City, CA) for enrichment analysis of the pathways and upstream
   regulators, using Ingenuity Knowledge Base (IKB) as reference data and
   the contextual analysis settings for mouse tissues ([95]Supplementary
   Table 1). The enrichment data and the p-values, calculated by Fisher's
   exact test, were exported and plotted using ggplot2 package in R
   software. Principal component analysis (PCA) was performed using START:
   Shiny Transcriptome Analysis Resource Tool hosted at
   [96]http://kcvi.shinyapps.io/START ([97]46).

Statistical Analysis

   p-values were determined by Mann Whitney, Wilcoxon matched-pairs signed
   rank test, or one-way or two-way ANOVA using GraphPad Prism software
   (La Jolla, CA). All p < 0.05 were considered significant.

Results

MuPyV-Infected Glial Cells and Infiltrating Monocytes Express High Levels of
PD-L1

   Using adoptively transferred transgenic CD8 T cells expressing a
   MuPyV-specific TCR, we previously showed that brain-resident, but not
   splenic, antiviral CD8 T cells were PD-1^hi ([98]28). Here, we examined
   the expression of PD-1 ligands by microglia, oligodendrocytes, and
   astrocytes, as well as by infiltrating monocytes in mice acutely
   infected with MuPyV ([99]Supplementary Figure 1). With the exception of
   oligodendrocytes, all of these cell types variably upregulated PD-L1
   after i.c. MuPyV inoculation, with the infiltrating monocytes having
   the highest frequency of PD-L1^+ cells ([100]Figure 1A). None of these
   cells showed expression of PD-L2 (data not shown). Although each of
   these cell populations was infected by MuPyV, microglia and
   infiltrating monocytes expressed at least a log higher LT-Ag
   transcripts than oligodendrocytes ([101]Figure 1B). The marginally
   higher expression of VP1 transcripts in astrocytes vs.
   oligodendrocytes, while not achieving statistical significance,
   reinforces previous studies showing that JCPyV more efficiently infects
   astrocytes than oligodendrocytes in brains of mice engrafted with human
   glial progenitor cells ([102]47). We further found that astrocytes, but
   not oligodendrocytes, express the viral capsid protein, VP1
   ([103]Figure 1C), a result in line with the human chimeric glial
   mouse-JCPyV infection model showing that astrocytes and not
   oligodendrocytes support productive infection ([104]47, [105]48). In an
   interesting observation, we found that PD-L1^+ astrocytes and microglia
   harbored a higher viral LT-Ag mRNA load as well ([106]Figure 1D). These
   data show that resident and infiltrating CNS cell types that express
   PD-L1 are also infected with MuPyV with a positive association between
   PD-L1 expression and virus infection.

Figure 1.

   [107]Figure 1
   [108]Open in a new tab

   Neural cells express PD-L1. (A) Representative contour plots with
   frequency of PD-L1^+ oligodendrocytes (CD11b^neg/CD45^neg/O4^+),
   astrocytes (CD11b^neg/CD45^neg/GLAST^+), microglia (CD11b^hi/CD45^int)
   and infiltrating monocytes (CD11b^hi/CD45^hi) from mock inoculated
   controls and MuPyV-infected mice at 8 dpi. The gates were drawn on the
   basis of the fluorescence minus one (FMO) controls. (B) LT-Ag mRNA copy
   number from FACS-purified astrocytes (Astro), microglia (Micro),
   infiltrating monocytes (Mono), and oligodendrocytes (Oligo). C[t]
   values were normalized to the amount of total RNA taken for cDNA
   synthesis. Each point represents data from a pool of 3 mice. (C)
   Fluorescence photomicrographs of FFPE brain tissue sections from mice
   euthanized at 4 dpi stained with antibodies specific for the indicated
   CNS cell markers (green) and for MuPyV capsid protein VP1 (red). Nuclei
   were counterstained with DAPI (blue). White arrows in merged images
   indicate VP1^+ cells (magnification 400X). (D) LT-Ag mRNA copy numbers
   from FACS-purified PD-L1^+ and PD-L1^− microglia and astrocytes. C[t]
   values were normalized with the C[t] value of TBP mRNA for each cell
   type between the PD-L1^+ and PD-L1^− samples. Each point connected by a
   line indicates cells from a pool of 3 mice. Data are cumulative from
   two independent experiments with 2–4 mice per group. Two-way ANOVA with
   Tukey multiple comparison test was performed. Values represent mean ±
   SD; *p ≤ 0.05.

Sustained PD-1 Expression by Antiviral CD8 T Cells During MuPyV Encephalitis

   We reasoned that higher TCR affinity by the CD8 bT[RM] would lead to
   augmented TCR signaling. Expression of the transcription factor IRF4 is
   reflective of TCR affinity and correlates with TCR signaling strength
   ([109]49, [110]50). In confirmation of this prediction, we found that
   the CD8 bT[RM] stained with tetramers for the dominant D^bLT359 MuPyV
   epitope exhibited higher levels of TCR-signaling, as reflected by the
   higher expression of IRF4 in the brains than in spleens of mice at 45
   days post-infection (dpi) ([111]Figure 2A).

Figure 2.

   [112]Figure 2
   [113]Open in a new tab

   bT[RM] express PD-1 during MuPyV infection. (A) Expression of IRF4 by
   D^bLT359-specific CD8 T cells from brains and spleens of WT mice at 45
   dpi in the indicated groups, represented as gMFI of the population. (B)
   Representative histograms of PD-1 expression by D^bLT359^+ CD8 T cells
   from brain (solid-line) or spleen (shaded) at the indicated dpi. (C)
   Representative contour plots with frequencies of Tim3/PD-1 and 2B4/PD-1
   expressing brain-infiltrating virus-specific CD8 T cells at 45 dpi. (D)
   Frequency and gMFI of IFN-γ^+ CD44^+ CD8 T cells from the brain and
   spleen at the indicated timepoints upon in vitro stimulation with the
   LT359 peptide-pulsed BMDC from WT and PD-L1^−/− mice. Each point
   connected by a line indicates cells from one mouse. Data are from two
   independent experiments with 4-5 mice per group. Mann Whitney tests (A)
   between WT and PD-L1^−/− groups and Wilcoxon matched-pairs signed rank
   test (D) were performed. Values represent mean ± SD; *p ≤ 0.05, **p ≤
   0.01, not significant (ns) p > 0.05.

   To understand if increased affinity of virus specific bT[RM] may
   reflect selective accumulation of TCR clonotypes in the brain, we
   performed TCR repertoire analysis using the the TCR-β ImmunoSEQ assay.
   FACS-purified D^bLT359 tetramer^+ CD8 T cells were isolated from the
   brains and spleens of mice 30 dpi. Notably, ~75 of the top 100 TCR
   clones from the spleen were absent in the brain. Productive entropy, a
   measure of the diversity of the TCR sequences, was also higher for the
   D^bLT359-specific memory CD8 T cells from the spleen than the D^bLT359
   specific bT[RM] ([114]Supplementary Figure 2A). The low diversity of
   TCR clones in the brain suggests that a subset of virus-specific
   peripheral CD8 T cells infiltrate or survive during the process of
   bT[RM] development.

   The dichotomy in PD-1 expression between brain and splenic
   MuPyV-specific TCR transgenic CD8 T cells was recapitulated by the
   endogenous D^bLT359-specific CD8 T cell response. This dichotomy in
   PD-1 expression was recapitulated by the endogenous D^bLT359-specific
   CD8 T cell response. D^bLT359 tetramer^+ CD8 T cells from the brain
   expressed PD-1 during the acute phase of infection (8 dpi), the
   expression peaked at 15 dpi, and was sustained into persistent
   infection ([115]Figure 2B). In contrast, D^bLT359-specific CD8 T cells
   in the spleen only transiently expressed a low level of PD-1 during
   acute infection ([116]Figure 2B). A sizable fraction of PD-1^hi
   MuPyV-specific CD8 T cells in the brains of persistently infected mice
   also expressed the inhibitory receptors Tim3 and 2B4 ([117]Figure 2C).

   Next, we asked whether engagement of PD-1 by its ligand, PD-L1,
   functionally inhibited MuPyV-specific CD8 bT[RM]. Bone marrow-derived
   dendritic cells (BMDCs) from WT and PD-L1^−/− mice were treated with
   IFN-γ to maximize MHC class I and PD-L1 expression ([118]Supplementary
   Figures 2B,C), then pulsed with LT359 peptide and used to stimulate T
   cells isolated from brains and spleens of MuPyV infected mice. LT359
   peptide-stimulated CD8 T cells from brains had a higher frequency of
   IFN-γ^+ cells and higher gMFI for IFN-γ at both 8 and 30 dpi when
   exposed to PD-L1^−/− BMDCs as compared to WT BMDCs. In contrast,
   D^bLT359-specific CD8 T cells from the spleen showed only a modest
   increase in IFN-γ production at 8 dpi when stimulated by PD-L1^−/−
   BMDCs, a time point coincident with PD-1 expression ([119]Figure 2D).
   Taken together, these data point toward PD-1-mediated inhibition of
   virus-specific CD8 T cell effector activity during MuPyV encephalitis.

PD-L1 Deficiency Results in a Heightened Inflammatory Environment

   Given that disruption of PD-1:PD-L1 signaling augmented IFN-γ
   production by anti-viral CD8 T cells in vitro, we hypothesized that
   PD-1 could be playing a role in controlling neuroinflammation in vivo.
   Upregulation of MHC class II expression on microglia is a commonly used
   indicator of neuroinflammation ([120]9, [121]51, [122]52). Comparing
   MHC class II expression on microglia from brains of MuPyV infected WT
   and PD-L1^−/− mice, we found significantly higher I-A^b (MHC class II)
   surface expression on microglia from PD-L1^−/− mice at 8 dpi
   ([123]Figure 3A); however, by 45 dpi MHC II expression levels on
   microglia of WT and PD-L1^−/− mice were comparable ([124]Figure 3B).
   These data point toward an acute-to-persistent infection phase
   remission of the heightened inflammatory environment in the PD-L1^−/−
   mice. Furthermore, we found that brain microglia from mice that were
   adoptively transferred with IFN-γ-sufficient MuPyV-specific TCR
   transgenic CD8 T cells upregulated I-A^b expression, but no I-A^b
   upregulation was seen in mice that received IFN-γ deficient-transgenic
   TCR CD8 T cells ([125]Figure 3C). These data support the likelihood
   that IFN-γ released by CNS-infiltrating, virus-specific CD8 T cells
   contributes to neuroinflammation during MuPyV encephalitis.

Figure 3.

   [126]Figure 3
   [127]Open in a new tab

   Lack of PD-1 signaling augments CD8 T cell IFN-γ production in vivo.
   (A) Representative histograms of MHC class II expression on microglia
   from WT and PD-L1^−/− mice at 8 dpi (left panel) with mean ± SD of MHC
   II gMFI (right panel). (B) MHC II gMFI (mean ± SD) on microglia at 45
   dpi. (C) MHC II gMFI on microglia from WT mice at 8 dpi after i.v.
   transfer of IFN-γ sufficient or IFN-γ^−/− naïve TCR-I cells at day−1.
   Combined data from 2 to 3 independent experiments with 2–4 mice per
   group; each value indicates cells isolated from an individual mouse.
   Two-way ANOVA with Tukey multiple comparison test (A) or Mann Whitney
   test (C) was performed. **p ≤ 0.01.

   To obtain a more comprehensive view of the role of PD-1 signaling in
   controlling neuroinflammation, NanoString gene expression analysis
   using a 254 gene mouse inflammation panel was performed on brains from
   acutely (8 dpi) and persistently (35 dpi) infected mice. At 8 dpi, a
   higher number of genes were upregulated in PD-L1^−/− (50 genes) vs. WT
   mice (25 genes) ([128]Figure 4A). However, in persistent infection, the
   inflammatory landscape profoundly changed, and did so differently
   between WT and PD-L1^−/− mice. For example, in WT mice, a number of
   genes were uniquely expressed in the 8 dpi and 35 dpi datasets. In
   contrast, the gene set upregulated in PD-L1^−/− mice at 35 dpi
   represented a subset of those same genes that were upregulated at 8 dpi
   ([129]Figures 4B,C). These differences were further reinforced by
   principal component analysis (PCA) which showed that the inflammatory
   gene expression profile in the brains of WT mice at 35 dpi were
   significantly different from both uninfected as well as 8 dpi WT mice
   ([130]Figure 4D). These observations suggest that the neuroinflammatory
   environment changes considerably over the course of MuPyV infection in
   the WT mice, while PD-L1^−/− mice show fewer differences between acute
   and persistent infection.

Figure 4.

   [131]Figure 4
   [132]Open in a new tab

   Brains of WT mice and PD-L1^−/− mice undergo dynamic and disparate
   changes in inflammatory transcriptomes from acute to persistent phases
   of infection. (A) Volcano plots representing differentially expressed
   genes (fold change ≥1.5 and p ≤ 0.05) of MuPyV infected WT brain
   samples vs. mock inoculated WT control brains and MuPyV infected
   PD-L1^−/− brains vs. mock inoculated PD-L1^−/− brains at 8 dpi and 35
   dpi. Combined data from a total of 3 pools of 3 mice per pool.
   Downregulated and upregulated genes are represented in blue and red
   dots, respectively. Similarly, numbers in blue and red represent the
   number of downregulated and upregulated genes, respectively. The y-axis
   represents –log(p-value) for each gene in the NanoString mouse
   inflammation panel. (B,C) Venn diagrams showing the overlap in the
   differentially regulated genes between each group as indicated. (D) PCA
   of each pooled sample was performed using normalized counts of all
   genes in the array. Uninf^1 and Uninf^2 are two independent sets of
   mock infected controls samples run along with the 8 dpi and 35 dpi
   MuPyV-infected samples, respectively.

   Ingenuity pathway analysis (IPA) revealed major differences in the
   neuroinflammation signaling pathways in MuPyV-infected WT and PD-L1^−/−
   mice. A heightened inflammatory state is indicated in PD-L1^−/− mice by
   engagement of pathways involved in interferon signaling, dendritic cell
   maturation and recognition by Pattern Recognition Receptors (PRRs) at 8
   dpi ([133]Figure 5A). Conversely, at 35 dpi the neuroinflammation
   signaling pathway, interferon signaling, role of PRRs in virus
   recognition and iNOS signaling exhibited higher enrichment scores and
   –log(p-value) in WT mice than their PD-L1^−/− counterparts ([134]Figure
   5B). These data provide further evidence for a sustained inflammatory
   response in WT mice but not in PD-L1^−/− mice. Using upstream regulator
   analysis of IPA, we found that TNF-α and IFN-γ, mediator of CD8 T cell
   effector function and other inflammatory mediators like IL-15, IL-21,
   NOS2, STAT1, and NF-κB had higher z-score and –log(p-value) at 35 dpi
   in PD-L1^−/− mice than WT mice ([135]Supplementary Figure 3). These
   data suggest that PD-1 protects against neuroinflammation during acute
   MuPyV infection, but paradoxically, ablation of this signaling pathways
   results in failure to sustain an inflammatory response during
   persistent infection.

Figure 5.

   [136]Figure 5
   [137]Open in a new tab

   PD-1 signaling alters inflammatory pathways in the brain during MuPyV
   infection. Pathway enrichment analysis of the differentially expressed
   genes between MuPyV-infected WT vs. mock-inoculated WT control (left
   panel), and MuPyV-infected PD-L1^−/− vs. mock-inoculated PD-L1^−/−
   control brain (right panel) at 8 dpi (A) and 35 dpi (B) using IPA
   software. The y-axis represents pathway and the x-axis represents
   enrichment ratio. Bubble size represents the z-score and color
   represents the -log(p-value) calculated by Fisher's exact test.

PD-L1-Deficiency Affects the T[RM] Phenotype

   PD-1 signaling has been postulated to regulate CD8 T cell memory
   formation both in the context of peripheral as well as resident memory
   ([138]10, [139]53, [140]54). In MCMV infection, PD-1 signaling promoted
   bT[RM] formation, but had no effect on virus control ([141]53).
   However, unlike in MCMV acute infection, PD-L1^−/− mice showed only a
   modest, albeit significant, increase in the number of the
   D^bLT359-tetramer^+ CD8 T cells; this increase was not seen in the
   spleen ([142]Figure 6A). Unexpectedly, the frequency as well as number
   of D^bLT359-specific CD8 T cells expressing CD103 were modestly higher
   in persistently infected PD-L1^−/− mice than WT mice ([143]Figure 6B).
   We independently confirmed this apparent inverse relationship between
   PD-1 signaling and CD103 expression on MuPyV-specific CD8 T cells by
   continuous two-week delivery of anti-PD-1 vs. control IgG in
   persistently infected WT mice ([144]Figure 6C). Interestingly, viral
   load was unaffected upon either Pdcd1 ablation or antibody-mediated
   blockade of the PD-1 signaling ([145]Figure 6C and [146]Supplementary
   Figure 5C). TGF-β produced by CD25^+ FoxP3^+ CD4^+ T cells induces
   CD103 ([147]55). During the acute phase of infection, brain
   infiltrating CD25^+ FoxP3^+ CD4^+ T cells were found to express PD-1.
   Also, PD-L1^−/− mice had moderately higher numbers of CD25^+ FoxP3^+
   CD4 T cells compared to the WT mice ([148]Supplementary Figures 4A,B).
   This increase coincides with higher TGF-β transcripts in the PD-L1^−/−
   mice ([149]Supplementary Figure 4C). These data suggest that the
   expression of PD-1 on brain infiltrating CD4 T cells could be
   regulating the observed CD8 T cell response and phenotype in PD-L1^−/−
   mice.

Figure 6.

   [150]Figure 6
   [151]Open in a new tab

   Lack of PD-1 signaling is associated with an increased frequency of
   CD103^+ bT[RM] (A) Number of D^bLT359 specific CD8 T cells in WT and
   PD-L1^−/− mice. (B) Frequency and number of brain CD103^+ D^bLT359^+
   CD8 T cells in WT and PD-L1^−/− mice. (C) Frequency of
   CD103^+D^bLT359^+CD69^+ CD8 T cells and viral DNA load at 30 dpi in
   brains of WT mice with control IgG or PD-L1 antibody delivered by
   i.c.v. cannulae. (D) gMFI of IRF4 expression by D^bLT359^+ T cells at 9
   and 45 dpi. (E) TCR affinities of D^bLT359 monomer-binding CD8 T cells
   determined by 2D-micropipette adhesion assay. Data are from a pool of 5
   mice. (F) gMFI of PD-1, Tim3, and 2B4 (left panel), and Blimp-1, EOMES,
   and TCF-1 (right panel). (G) Frequency of TCF-1^+CXCR5^+ D^bLT359^+ CD8
   T cells from the brains of mice at 45 dpi. Data are from two to three
   independent experiments with 3–8 mice per group. Mann Whitney test
   between WT and PD-L1^−/− groups was performed in panels B, C, and D;
   two-way ANOVA with Tukey multiple comparison test in panel A was
   performed. Values represent mean ± SD; *p ≤ 0.05, **p ≤ 0.01, not
   significant (ns) p>0.05.

   Subcortical demyelination and enlargement of the ventricles are some of
   the hallmarks of JCPyV-associated-CNS-syndromes ([152]31). To detect
   demyelination MuPyV encephalitis, LFB-PAS-hematoxylin staining was
   performed on vehicle i.c. injected and MuPyV infected brains. No change
   was seen at 4 dpi between control and infected WT mice
   ([153]Supplementary Figure 5A). Small foci of demyelination and
   vacuolation were present in the cingulum bundle and the external
   capsule at 9 dpi, with enlarged ventricles and edema in the white
   matter tracts that became progressively worse by 30 dpi
   ([154]Supplementary Figure 5A). Given the heightened neuroinflammation
   in the PD-L1^−/− mouse brains, we were surprised to see comparable
   levels of demyelination in persistently infected WT and PD-L1^−/− mice
   [19 dpi ([155]Supplementary Figure 5B) and 30 dpi (data not shown)].

   Because MuPyV-specific bT[RM] express high affinity TCRs and are nearly
   all PD-1^hi, we next investigated the possibility that PD-1 might
   influence the generation of these cells. At 8 dpi, there was no
   difference in expression of IRF4 by D^bLT359-tetramer^+ CD8 T cells
   between WT and PD-L1^−/− mice, but at 45 dpi the bT[RM] from PD-L1^−/−
   mice showed lower IRF4 expression ([156]Figure 6D). Decreased IRF4
   expression was not only restricted to the immunodominant D^bLT359
   tetramer^+ CD8 T cells, but was also seen in the D^bLT359 tetramer^−
   CD8 T cells ([157]Supplementary Figure 6A). To see if the difference in
   IRF4 expression stemmed from differences in TCR affinity, we performed
   2D micropipette cell adhesion assays. Interestingly, no difference in
   TCR affinities between the D^bLT359-specific CD8 T cells from WT and
   PD-L1^−/− mice was detected ([158]Figure 6E). Expression of LT-Ag
   transcript in the brain was also found to be similar between WT and
   PD-L1^−/− mice, negating the possibility that differences in the
   expression of IRF4 could be ascribed to the differences in antigen
   expression ([159]Supplementary Figure 6B). Thus, a mechanism other than
   modulation of TCR affinity comes into play to dampen TCR signaling
   strength by virus-specific CD8 T cells in the absence of PD-1
   signaling.

   In LCMV clone 13 chronic infection, circulating CD8 T cells undergo
   severe exhaustion in the absence of PD-1:PD-L1 signaling ([160]10).
   Thus, we asked if MuPyV-specific CD8 bT[RM] developing in the absence
   of PD-1 signaling undergo terminal exhaustion as an alternative
   explanation for lower TCR signal strength. Genetic deficiency of PD-L1,
   did not change the expression of PD-1, 2B4, or Tim3 by MuPyV-specific
   CD8 T cells ([161]Figure 6F). Lack of PD-1 signaling also did not
   affect the expression of the transcription factors Eomes, Blimp-1 and
   TCF-1 (all known to be associated with exhaustion) by D^bLT359-specific
   CD8 T cells ([162]Figure 6F). TCF1^hiCXCR5^hi CD8 T cells have been
   recently defined as the PD-1^+ memory subset in LCMV clone 13
   infections that exhibited less exhaustion and improved functionality
   upon PD-1 blockade ([163]21, [164]22). In analogous fashion, we found
   that D^bLT359-specific CD8 bT[RM] in PD-L1^−/− mice had higher numbers
   of TCF1^hiCXCR5^hi cells ([165]Figure 6G). Taken together, these
   findings suggest that loss of PD-1 signaling during MuPyV-encephalitis
   led to increased CD103 expression and did not guide CD8 bT[RM] toward
   terminal exhaustion.

PD-L1 Deficiency Results in Impaired Virus Control Upon Re-infection

   Lack of PD-1 signaling due to genetic ablation of PD-L1 resulted in an
   increased fraction of CD103^+ MuPyV-specific CD8 bT[RM] but did not
   affect virus levels. CD103 expression has been associated with improved
   functionality in CD8 T[RM] cells ([166]56). To test bT[RM] recall
   responsiveness, we re-inoculated >45 dpi WT and PD-L1^−/− mice i.c.
   with MuPyV and compared their ability to control this homologous virus
   challenge. Five days after re-inoculation, persistently infected WT and
   PD-L1^−/− mice showed similar increases in numbers of total CD8 T
   cells, but significantly more D^bLT359-specific CD8 T cells were seen
   only in the brains of the re-infected WT mice ([167]Figure 7A). The
   difference between total and virus-specific CD8 T cell numbers in the
   re-infected WT mice suggested that this increase was predominantly due
   to recall expansion of anti-MuPyV CD8 bT[RM] rather than recruitment.
   Importantly, re-infected PD-L1^−/− mice had an ~87-fold increase in
   virus load in the brain, whereas WT mice showed roughly a 6-fold
   increase in virus levels ([168]Figure 7B). D^bLT359-specific CD8 T
   cells from the brains of WT and PD-L1^−/− mice at day 5 post-challenge
   also exhibited similar abilities to produce IFN-γ and degranulate
   (e.g., CD107a/b^+) upon LT359 peptide stimulation ([169]Figure 7C). The
   recall response upon re-infection was mainly due to the activation of
   CD103^+ cells, as only CD103^+ cells showed high proliferative
   potential as indicated by their Ki67 expression. We also observed a
   moderate decrease in Ki67^+ cells in PD-L1^−/− mice ([170]Figure 7D).
   These observations are in line with data in [171]Figure 5B and
   [172]Supplementary Figure 3, showing PD-1 signaling deficiency results
   in failure to sustain an inflammatory environment, resulting in poorer
   virus control. Moreover, these data support the likelihood that
   neuroinflammatory factors induced by PD-1 signaling are required to
   maintain immunologic defense against resurgence of a persistent viral
   encephalitis.

Figure 7.

   [173]Figure 7
   [174]Open in a new tab

   Lack of PD-1 signaling results in impaired viral control. WT or
   PD-L1^−/− mice were re-infected i.c. with MuPyV or mock inoculated at
   35 dpi, then euthanized 5 days later. Total CD8 T cells and D^bLT359^+
   CD8 T cells from brains were analyzed by flow cytometry. (A) Number of
   total CD8 and D^bLT359^+ CD8 T cells per brain, (B) Brain viral DNA
   genome copies by qPCR at 5 days after re-infection. (C) Number of
   IFN-γ^+ (left panel) and IFN-γ^+ CD107a/b^+ (right panel) CD8 T cells
   upon in vitro stimulation with LT359 peptide. (D) Frequency of Ki67^+
   LT359^+ CD103^+/− CD8 T cells upon reinfection. Data from two
   independent experiments with 3–5 mice per group. Two-way ANOVA with
   Tukey multiple comparison test was performed. Values represent mean ±
   SD; *p ≤ 0.05, **p ≤ 0.01. (D) Frequency of Ki67^+ LT359^+ CD103^+/−
   CD8 T cells upon reinfection.

Discussion

   Accumulating evidence indicates that expression of PD-1 and CD103 by
   CD8 bT[RM] is dependent on virus context. For example, VSV and
   LCMV-Armstrong generate PD-1^− CD103^+ CD8 bT[RM], but CD8 bT[RM] in
   TMEV and MCMV encephalitis are PD-1^+ CD103^+ ([175]53,
   [176]57–[177]59). In contrast, during persistent MuPyV encephalitis,
   virus-specific CD8 T cells are uniformly PD-1^+, but only ~40% express
   CD103 ([178]28). In addition, irrespective of CD103 status,
   MuPyV-specific CD8 bT[RM] are maintained stably independent of resupply
   from the circulation; and CD103^+ and CD103^− cells have highly
   overlapping transcriptomes ([179]28, [180]60). However, the CD103^+
   subset preferentially produces IFN-γ ([181]28, [182]60) and
   proliferates upon i.c. MuPyV re-infection ([183]Figure 7D). Moreover,
   PD-L1^−/− mice have diminished ability to control virus levels upon
   homologous virus re-infection, despite having a higher proportion of
   CD103^+ MuPyV-specific CD8 bT[RM] than WT mice. Although CD103
   expression has been linked to CD8 bT[RM] formation in response to VSV
   CNS infection ([184]61), other studies show that CD8 T cell motility in
   the small intestine mucosa and brain are not dependent on CD103
   ([185]58, [186]62). In addition, intestinal pathogen-specific CD8 T[RM]
   to oral Yersinia pseudotuberculosis infection are comprised of CD103^+
   and CD103^− populations, with the latter preferentially localizing to
   infectious foci ([187]63). In sum, the potential contributions of CD103
   to anatomical localization and function of CD8 T cells in different
   tissues and in the setting of different pathogen infections remain to
   be determined.

   CD4 T cell-derived cytokines may be involved in upregulating CD103 on
   CD8 T[RM]. Nearly all MuPyV-specific CD8 T cells in the brains of CD4 T
   cell-deficient mice are CD103^− ([188]60). TGF-β induces CD103 on T[RM]
   ([189]64–[190]66); more TGF-β1 transcripts are detected in brains of
   PD-L1^−/− than WT mice persistently infected with MuPyV
   ([191]Supplementary Figure 4C). In this connection, all CD25^+ CD4 T
   cells isolated from the brains of acutely MuPyV infected mice are
   PD-1^+ and a fraction are FoxP3^+ as well ([192]Supplementary Figures
   4A,B). Together, these data raise the possibility that, in the absence
   of inhibitory PD-1 signaling, CD4 T cells in the brain produce more
   cytokines that induce CD103 expression on CD8 bT[RM].

   PD-1 expression by CD8 T cells may not inevitably result in T cell
   exhaustion. PD-1 has been shown to be dispensable for CD8 T cell
   exhaustion in chronic LCMV infection ([193]10). Severity and duration
   of chronic viral infections are associated with expression of multiple
   inhibitory receptors by CD8 T cells in addition to PD-1 ([194]67). In
   infected WT and PD-L1^−/− mice, however, PD-1^+ D^bLT359 specific CD8
   bT[RM] cells expressed equivalent levels of Tim3 and 2B4. Expression of
   the Blimp1, Eomes and TCF-1 transcription factors was also unaffected
   by the absence of PD-1 signaling. LT359 peptide-stimulated bT[RM] from
   WT and PD-L1^−/− mice were similarly capable of IFN-γ production and
   degranulation, indicating retention of function independent of PD-1
   signaling. MuPyV-specific CD8 bT[RM] from WT and PD-L1^−/− mice also
   expressed similar levels of PD-1 during persistent infection. We
   recently reported that PD-1 downregulation on splenic MuPyV-specific
   CD8 T cells is followed by Pdcd1 promoter loci remethylation ([195]28).
   PD-1 is stably expressed on anti-MuPyV bT[RM] in PD-L1^−/− mice. These
   data support the interpretation that PD-1 signaling does not regulate
   the methylation status of the Pdcd1 gene promotor. In the brains of
   PD-L1^−/− mice a number of upstream regulators were enriched during
   acute infection but reduced or below threshold during persistent
   infection (e.g., IFN-γ, IL-15, NOS, NFATC2, and IL12-A), suggesting
   dynamic epigenetic changes in these genes in the absence of PD-1
   signaling.

   Microglia, astrocytes and oligodendrocytes express PD-L1, under various
   inflammatory conditions, such as those induced by MCMV and TMEV CNS
   infections ([196]8, [197]33, [198]68, [199]69). Yet, PD-L1 in mouse
   coronavirus encephalomyelitis is only transiently and poorly expressed
   on microglia, although oligodendrocytes exhibit high and sustained
   expression ([200]8). In MCMV encephalitis, microglia and astrocytes
   express PD-L1 ([201]9). In MuPyV infection, PD-L1 was expressed on the
   infiltrating monocytes, microglia and astrocytes, but not on
   oligodendrocytes. IFN-γ is a potent inducer of PD-L1 ([202]9). The
   positive correlation between viral LT-Ag mRNA and PD-L1 expression in
   MuPyV-infected brain cells could be a result of contact of the infected
   cells with IFN-γ-secreting CD8 T cells. A similar mechanism termed
   “adaptive resistance” dampens anti-tumor CD8 effector activity
   ([203]70, [204]71). The alternate possibility that MuPyV-infected cells
   upregulate PD-L1 as a virus-immune evasion strategy is less likely,
   given that oligodendrocytes, which are also infected, fail to express
   PD-L1.

   Using a NanoString mouse inflammatory gene expression array, we
   uncovered a PD-L1-dependent difference in the inflammation
   transcription landscape over the course of MuPyV infection. Compared to
   WT mice, PD-L1 deficiency during acute infection involved upregulated
   expression of more genes and enrichment of neuroinflammatory pathways
   ([205]Figures 4, [206]5 and [207]Supplementary Figure 3). Of these,
   brains of PD-L1^−/− mice showed exclusive enrichment of NF-κB and
   apoptotic pathways, as well as IL-9, IL-12, and IL-3 signaling pathways
   ([208]Figure 5 and [209]Supplementary Figure 3). Although the
   inflammation transcriptome changed considerably as infection
   transitioned from acute to persistent infection in WT mice, it failed
   to do so in PD-L1^−/− mice ([210]Figure 4D). Despite this difference,
   WT and PD-L1^−/− mice shared the IL-15 signaling pathway as an upstream
   regulator. IL-15 is of particular interest, because it is expressed by
   CNS parenchymal cells as well as by infiltrating immune cells in
   response to an ongoing inflammatory insult ([211]72). In addition,
   IL-15 limits apoptosis in neuronal cells and suppresses nitric oxide
   production in neurons ([212]72). The importance of IL-15 in memory CD8
   T cell differentiation and establishing CD8 T[RM] in barrier tissues
   are well documented ([213]73–[214]76). An intriguing possibility is
   that a deficiency in IL-15 in PD-L1 deficient mice underlies the
   compromised ability of CD8 T cells to control virus levels upon
   re-infection and promotes neural cell death during persistent
   infection.

   The interferon signaling pathway also showed pronounced differences
   between WT and PD-L1^−/− mice. Genes in this pathway were found to be
   significantly enriched in persistently, but not acutely infected, WT
   mice with the reverse seen in PD-L1^−/− mice ([215]Figure 5). Type I
   and II IFNs have been shown to inhibit JCPyV replication in glial cells
   in vitro ([216]77, [217]78). In a clinical trial to evaluate IFN-γ as a
   prophylactic therapy for opportunistic infections in HIV^+ patients,
   none receiving IFN-γ developed PML while 10% of those in the placebo
   group did ([218]79). Type I IFNs have been shown to enhance the
   cytolytic activity of airway-resident memory CD8 T cells a secondary
   respiratory virus challenge ([219]80). We reported that Type I and Type
   II IFNs inhibit MuPyV replication in vitro and in peripherally
   inoculated mice, that MuPyV infection in vitro and in vivo induces
   IFN-β mRNA, and that Type I IFN regulates MuPyV-specific CD8 T cell
   memory differentiation and function ([220]81, [221]82). JCPyV, as well
   as BKPyV and SV40, infections induce a STAT1-dependent upregulation of
   interferon-stimulated genes and production of IFN-β ([222]83, [223]84).
   Thus, differences in IFN signaling pathways in WT and PD-L1^−/− mice
   may reflect dynamic changes over the course of MuPyV encephalitis in
   Type I IFN-mediated innate immunity and/or Type II IFN production by
   CNS-infiltrating virus-specific T cells. It merits emphasizing,
   however, that the NanoString data do not establish a temporal link
   between PD-1 signaling and CD8 bT[RM] development.

   In summary, PD-1 signaling plays a critical role in regulating the
   immune response against MuPyV infection of the CNS. Defects in the
   PD-1:PD-L1 signaling pathway may lead to increased neuroinflammation at
   the peak of the CD8 bT[RM] response, but neuroinflammation was not
   maintained during the persistent phase of this viral encephalitis.
   Thus, the inhibitory functions of PD-1 are necessary for the generation
   of a controlled, sustained inflammatory response in the CNS. These
   findings offer insights into the role of PD-1 in modulating an immune
   response of a persistent viral infection in the CNS beyond its
   documented function as a CD8 T cell inhibitory receptor.

Ethics Statement

   Institutional Animal Care and Use Committees and the Department of
   Comparative Medicine at the Pennsylvania State University College of
   Medicine. The Pennsylvania State University College of Medicine Animal
   Resource Program is accredited by the Association for Assessment and
   Accreditation of Laboratory Animal Care International (AAALAC). The
   Pennsylvania State University College of Medicine has an Animal Welfare
   Assurance on file with the National Institutes of Health's Office of
   Laboratory Animal Welfare; the Assurance Number is A3045-01.

Author Contributions

   Shwetank, EF, and AL contributed to the conception and design of the
   study and wrote the manuscript. Shwetank, EF, TM, HR, MT, ML, CN-W, GJ,
   and JC performed experiments, analyzed data, and performed the
   statistical analysis. All authors contributed to manuscript revision,
   read and approved the submitted version.

Conflict of Interest Statement

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Acknowledgments