PD-1 expression has been proposed to constitute a facet of the T RM differentiation program to prevent inadvertent deployment of poised mRNAs for effector molecules. 1 In chronic LCMV infection, TCR signaling upregulates PD-1 expression at the effector stage of the splenic CD8 T cell response, with sustained PD-1 driving differentiation of exhausted T cells (T EX ) to prevent immunopathology. 2, 3 The state of PD-1 expression and its dependence on antigen by tissue-resident memory CD8 T cells during persistent viral infection remains to be defined. For example, CD8 bT RM cells from mice with acutely resolved vesicular stomatitis virus (VSV) encephalitis express PD-1 transcripts, but not PD-1 receptors, whereas bT RMs from mice persistently infected with mouse cytomegalovirus are PD-1 +. [4] [5] [6] This discrepancy in PD-1 expression by bT RM cells raised the question whether antigen and/or inflammation is involved in maintenance of PD-1 expression by bT RM cells during CNS infection. Tissue-intrinsic factors are also dominant determinants of the dependence on antigen for CD8 T RM cell generation and/or maintenance. Antigen is required for T RM cell formation and CD103 upregulation in the brain and dorsal root ganglion 5, 7, 8 but not in the skin, small intestine, female reproductive tract, and salivary glands 7, [9] [10] [11] [12] . The role of antigen in maintenance of expression of PD-1 and CD103 by CD8 T RM cells in the brain remains to be determined.
The PD-1 promoter of virus-specific CD8 T cells undergoes dynamic epigenetic reprogramming during development of memory T cells and T EX cells. 13 In acutely resolved LCMV-Armstrong infection, virus clearance was associated with remethylation of the Pdcd-1 promoter and loss of PD-1 expression; however, in the high-level chronic LCMV clone 13 infection model, the Pdcd-1 promoter remained unmethylated in T EX cells even after virus levels fell below detection 13, 14 . Notably, these epigenetic analyses were only performed on splenic LCMV-specific CD8 T cells in an infection where PD-1 is expressed by antiviral CD8 T cells in all nonlymphoid organs. 15 This led us to investigate the epigenetic programming of bT RM cells during persistent viral encephalitis.
MuPyV is a natural mouse pathogen which establishes a low-level persistent infection. CNS infection with MuPyV yields a stable population of virus-specific bT RM cells. 16 Here, we show that during persistent MuPyV infection, PD-1 is expressed by bT RM cells but not splenic memory anti-MuPyV CD8 T cells, despite virus loads being similar in both organs, suggesting dissociation between the viral load and PD-1 expression. We further show that maintenance of PD-1 expression by bT RM cells is independent of cognate viral antigen and inflammation. As seen for splenic virus-specific CD8 T cells in chronic LCMV infection, the pdcd-1 promoter of bT RM cells from MuPyV infected mice remains demethylated. However, the PD-1 locus in splenic anti-MuPyV CD8 T cells undergoes partial remethylation. Collectively, these findings indicate that PD-1 expression is part of the developmental program of bT RM cells to a persistent CNS viral infection.
Naïve B6 mice received a "physiologic" number (200 cells/mouse) of Thy1.1-congenic TCR-I cells, and were inoculated i.c. the next day with MuPyV.LT206 virus. At day 9 postinfection (p.i.) the magnitude of the endogenous LT206-specific CD8 response in the brain, kidney, and spleen was similar to that of the donor TCR-I cells (Supplemental Fig. 1 ). Both the endogenous D b LT206-specific CD8 T cells and the TCR-I cells in acutely infected mice expressed PD-1 and CD69, with those in the brain having the highest level of expression (Fig. 1A) ; notably, virus levels in these organs were similar at day 9 and also at day 30 p.i. (Fig. 1B) . In addition to CD69, CD103 (α E integrin) is often used identify T RM cells. 17 At day 9 p.i., D b LT206-specific CD8 T cells in both lymphoid and nonlymphoid organs did not express CD103; however, by day 30 p.i., a fraction of CD103 + endogenous and donor CD8 T cells were seen in the brain, but not the kidney or spleen. Despite a decline in virus levels by day 30 p.i. (Fig. 1B) , PD-1 expression was sustained on both endogenous D b LT206-specific CD8 T cells and TCR-I cells in the brain and kidney, but not those in the spleen (Fig. 1A) , as was CD69; (Fig. 1A) . Analysis of PD-1 transcript levels in TCR-I cells from spleen at days 8 and 30 p.i. showed that splenic memory TCR-I cells expressed lower PD-1 mRNA levels than those from spleens of acutely infected mice, albeit this difference was not statistically significant (Fig. 1C) . Discordance between PD-1 mRNA levels and surface expression by CD8 T RM cells was similarly observed in the VSV acute encephalitis infection model. 4 To determine whether PD-1 functionally inhibited MuPyV-specific CD8 T cells, we assayed intracellular IFN-γ production by TCR-I cells after stimulation by LT206 peptide-pulsed PD-L1 + DC2.4 cells in the presence of a PD-L1 blocking mAb. As shown in Fig. 1C , PD-L1 blockade resulted in a significant increase in frequency of IFN-γ expressing TCR-I cells from brains, kidney and spleen of mice at day 9 p.i. At day 30 p.i., PD-1 blockade increased the frequency of IFN-γ + cells only of brain-resident TCR-I cells, which expressed the highest levels of PD-1 among T cells from all the organs examined.
We found that PD-1 hi expression by MuPyV-specific b TRMs cells was a stable phenotype and dissociated from virus infection levels (Fig. 1B) ; i.e. splenic anti-viral memory CD8 T cells were PD-1 − but spleen and brain virus loads were similar. In contrast, PD-1 expression by memory, TCR transgenic P14 CD8 T cells from LCMV clone13-infected mice declines following transfer to LCMV-immune mice or mice infected with a epitope-null mutant LCMV. 14 To directly ask whether cognate antigen and/or virus-associated inflammation was required for PD-1 expression by brain T RM cells, we sorted GFP + TCR-I cells from brains of Fig. 2A ) and by the absence of D b LT206 tetramer binding the endogenous CD8 T cells either in brain (Fig. 2B ) or in spleen (Supplemental Fig. 2B ) of uninfected recipient mice. Naïve TCR-I cells transferred into mice infected by MuPyV.A2 failed to proliferate and did not upregulate CD44, indicating that maintenance of PD-1 expression by TCR-I T RM cells in MuPyV.A2-infected recipients could not be attributed to cross-recognition of the D b LT359 epitope (Supplemental Fig. 2C ). These data suggest that TCR stimulation and the inflammatory environment of persistent MuPyV CNS infection are dispensable for maintenance of PD-1 expression by antiviral brain T RM cells.
As shown in Fig. 2D , donor TCR-I bT RM cells were predominantly CD103 + irrespective of the presence of antigen or inflammation in the recipient brains, suggesting preferential survival of the CD103 + population. Although fewer donor TCR-I bT RM cells were recovered from the brains of naïve and MuPyV.A2-infected recipients than from MuPyV.LT206infected mice at day 7 post-transfer (Fig. 2E) , these CD8 bT RM cells were found to persist in brains of naïve recipients for 25 days (Supplemental Fig. 1 ). To ask whether PD-1 expression was an intrinsic property of MuPyV-specific bT RM cells, TCR-I cells were isolated brains of mice at day 30 p.i. and transferred i.v. to either naïve or MuPyV.LT206 i.c. infected Thy1.2 congenic recipients. No donor cells, however, were detected at day 7 posttransfer in either cohort (data not shown), a finding mirroring that of Wakim et al. using the VSV acute encephalitis mouse model 5 and supporting the concept that CNS-specific factors are required for maintenance of bT RM cells.
To ask whether the brain environment per se induced PD-1, memory GFP + TCR-I cells from spleen were transferred i.c. into naïve or acutely i.c. infected (MuPyV.LT206) mice. We found that the donor splenic memory TCR-I cells (CD69 − PD-1 − , Fig. 1A ) 7 days posttransfer i.c. into naïve mice or mice given MuPyV.LT206 virus i.c. eight days earlier upregulated PD-1 and CD69 expression but not CD103 (Supplemental Fig. 1C ). Although splenic donor memory cells upregulated PD-1 expression in both naïve and acutely infected recipients, it merits noting that PD-1 expression is lower in the naïve than infected mice consistent with a role for antigen in driving PD-1 expression. Taken together, these data confirm the ability of the brain environment to upregulate PD-1 and CD69 on infiltrating MuPyV-specific CD8 T cells.
CD8 T RM cell populations defined by differential CD103 expression have been reported to vary in proliferative potential, transcriptome, antigen-dependence, and tissue distribution. 4, 5, 18, 19 CD103 expression by donor TCR-I cells, however, was higher than on the endogenous D b LT359 or D b LT206 CD8 T cells (Fig. 2C, D) , a difference that may be linked to the higher survival potential of CD103 + cells in the brain. 5 We thus asked whether MuPyV-specific CD103 − CD8 T cells infiltrating the brain express phenotypic and functional characteristics of T cell exhaustion. Both CD103 + and CD103 − D b LT359-specific CD8 T cells were maintained in the absence of circulating CD8 cells (Fig.  3A) , suggesting that each of these populations is comprised of T RM cells. CD103 + and CD103 − D b LT359-specific CD8 T cells also expressed similar levels of PD-1 and Tim3 (Fig.  3B ). Of note, the CD103 − subset expressed higher TCF-1 levels and had higher proportion of CXCR5 hi TCF-1 hi cells compared to the CD103 + cells. Elevated TCF-1 expression as well as the presence of a CXCR5 hi TCF-1 hi population by memory CD8 T cells in chronic infection models have been reported to exhibit improved functional capability. 20, 21 BLIMP-1 and Eomesodermin (EOMES) T-box transcription factors are highly expressed by exhausted CD8 T cells 22, 23 ; however, CD103 − D b LT359-specific CD8 T cells were found to express lower BLIMP-1 and higher EOMES levels than the CD103 + cells. These populations also showed similar Ki67 expression levels, indicating their comparable rates of proliferation during persistent MuPyV CNS infection (Fig. 3C) . Taken together, the pattern of expression of these molecules indicates that CD103 − cells lack a phenotype associated with T cell exhaustion. Moreover, CD103 − and CD103 + LT359 peptide-stimulated CD8 T cells from brains of persistent MuPyV-infected mice exhibited similar frequencies of intracellular IFN-γ production (Fig. 3D) . To assess IFN-γ production directly ex vivo by CD103 − and CD103 + MuPyV-specific CD8 T cells in brains of persistently infected mice, IFN-γ EYFP reporter mice were inoculated with MuPyV.A2 virus i.c. As shown in Fig. 3E , a higher fraction of CD103 + than CD103 − D b LT359-specific CD8 T cells were EYFP + , a pattern paralleled by expression of IRF4, a transcription factor upregulated by TCR stimulation. 24 The discrepancy between similar frequencies of IFN-γ producing CD103 + and CD103 − bTRM cells following LT359 peptide stimulation vs lower EYFP + and IRF4 + by the CD103-population may reflect regional differences between the CD103 + and CD103 − cells in engaging viral antigen-expressing cells and/or differences in TCR affinity. 16 Although the CD103 integrin tethers T RM cells to E-Cadherin and is important for their preferential localization at epithelial tissues, CD103 − CD8 T RM cells exist in particular organs, including the brain. 5, 25 CD103 expression by CD8 T RM cells may also be influenced by the nature of the viral infection, as lung CD8 T RM cells to influenza virus and respiratory syncytial virus express CD103 whereas those specific for human cytomegalovirus are CD103 −.26
Using the mouse model of chronic LCMV infection, it has been previously demonstrated that the Pdcd1 promoter is extensively demethylated at early stages of the immune response.
This locus was remethylated in the acutely resolved LCMV Armstrong infection, but remained demethylated in chronic LCMV clone13 infection even after host control of infection, a result tying PD-1 to the exhaustion state of differentiation. 13, 14 Stability of the demethylated Pdcd1 promoter was further demonstrated in human EBV, CMV, and HIVspecific CD8 T cells. 13, 27 It is yet to be confirmed whether the epigenetic changes observed at peripheral exhausted T cells holds true for bT RM cell population as well. The marked difference in PD-1 expression by memory MuPyV-specific CD8 T cells in spleen and brain led us to ask whether cells in these organs differed in level and stability of Pdcd1 promoter demethylation. Pdcd1 methylation status was analyzed on Thy1.1 TCR-I CD8 cells sorted from brains and spleens of acute (day 8 p.i.) and persistent (day 30 p.i.) infected recipient mice (Fig. 4A) . As expected, the Pdcd1 promoter in naïve splenic TCR-I cells was almost entirely methylated (Fig. 4B,E) . At day 8 p.i., TCR-I cells in both spleen and brain exhibited extensive Pdcd1 promoter demethylation (Fig. 4C,E) . By day 30 p.i., however, TCR-I cells in the spleen showed increased methylation of the Pdcd1 promoter, while brain TCR-I cells retained a demethylated Pdcd1 promoter (Fig. 4D,F) . This intermediate level of methylation in TCR-I cells from day 30 infected mice was intermediate between cells from a naïve or day 8 infected mice. This observation is consistent with the lower trending PD-1 transcript levels seen at day 30 p.i. (Fig 1C) . The PD-1 hi phenotype of the brain TCR-I T RM cells further suggests that a minimum threshold of Pdcd1 promoter demethylation facilitates upregulation of PD-1 gene expression. Thus, these data show that the Pdcd1 promoter of CD8 bT RM cells remains more extensively demethylated than splenic memory CD8 T cells despite having similar persistent MuPyV infection levels.
Recent studies document expression of PD-1 by memory CD8 T cells in inflamed immuneprivileged organs, such as eye and brain. 6, 28 Expression of PD-1 on brain CD8 T cells has been shown to limit axonal bystander damage in a mouse coronavirus CNS infection model and mediate protection from experimental autoimmune encephalomyelitis. [29] [30] [31] [32] [33] Our finding that PD-1 blockade elevates effector function by anti-MuPyV CD8 bT RM cell is in line with the concept that PD-1 expression by tissue-resident T cells may serve to restrain immunopathology, which would prove especially deleterious in the infected CNS. The divergence in PD-1 expression between brain and spleen MuPyV-specific CD8 T cells during persistent infection, with bT RM cells maintaining PD-1 in the absence of cognate antigen and inflammation, supports the concept that the brain microenvironment favors PD-1 expression.
C57BL/6NCr female mice were purchased from the National Cancer Institute. B6.PL (Thy1.1) and C57BL/6-Tg (UBC-GFP) 30Scha/J mice ("GFP mice") and B6.129S4-Ifng tm3.1Lky /J, IFN-γ reporter "IFN-γ EYFP" mice were purchased from The Jackson Laboratory. TCR-I mice are transgenic for a TCR for the D b -restricted peptide corresponding to amino acids 206-215 of the SV40 Large T Ag ("LT206"). 22 The D brestricted LT359-368 epitope in MuPyV Large T Ag (strain A2; "MuPyV.A2") was replaced by the D b LT206 epitope ("MuPyV.LT206"). 22 6.5×10 4 PFU/mouse of MuPyV.A2 or MuPyV.LT206 were injected intracerebrally (i.c.). 16 Mice were bred and housed in accordance with the guidelines of the IACUC of The Penn State College of Medicine.
CD8 antibody (clone YTS 169.4; BioXCell) or rat IgG antibody (Jackson ImmunoResearch) was injected via intraperitoneal (i.p.) route at 250 μg antibody per mouse once per week starting from day 10 p.i. to day 30 p.i. Efficiency of depletion of CD8 + cells in blood was 90% (data not shown).
Naïve CD8 T cell (CD44 low CD62L high ) were purified from TCR-I mice using a negative selection isolation kit (Miltenyi Biotec), and unstained or CFSE-labeled cells were injected i.v. per tail vain in 100 μl PBS or by i.c. injection in 30 μl PBS.
Quantitative TaqMan-based PCR was performed using 10ng template DNA purified from tissues, as described. 22 Quantitative real-time PCR for transcript analysis FACS-purified GFP + TCR-I cells were lysed in TRIzol (Ambion) and total RNA was isolated per manufacturer's instruction. cDNA was prepared using random primers and RevertAid H Minus Reverse Transcriptase enzyme (ThermoFisher Scientific). Taqman primer probe set for Pdcd1 and Tbp from IDT Technologies was used to perform qPCR using the ABI StepOnePlus Real-Time PCR System. Sequences of the primer-probe sets are as follows: PD-1, 5′-ATTTGCTCCCTCTGACACTG-3′; 5′-GTACCCTGGTCATTCACTTGG-3′; 5′-/56-FAM/TCCCTCACC/ZEN/ TTCTACCCAGCCT/3IANKFQ/-3′ AND TBP: 5′-TGTATCTACCGTGAATCTTGGC-3′, 5′-CCAGAACTGAAAATCAACGCAG-3′, 5′-/56-FAM/ACTTGACCT/ZEN/ AAAGACCATTGCACTTCGT/3IABkFQ/-3′. Relative fold change over naïve control was determined using the threshold cycle (2 −ΔΔ Ct ) method.
Mononuclear cells from brain and kidney were isolated from transcardially perfused mice by collagenase-DNase digestion and percoll gradient centrifugation, then treated with Fixable Viability Dye (eBioscience) and stained with APC-D b LT359 or BV421-D b LT206 tetramers (NIH Tetramer Core Facility) and antibodies, as described. 16 Samples were acquired on a BD LSRII or BD LSRFortessa, and analyzed using FlowJo software. GFP + cells stained with anti-CD8α and DAPI were sorted using a BD FACSAria SORP Cell Sorter.
DC2.4 cells were exposed to IFN-γ to upregulate MHC I and PD-L1 (data not shown), then pulsed with 10 μM LT206 peptide. Cells were added in presence of 50 μg/ml control rat IgG or PD-L1 blocking Ab (10F.9G2; BioXCell). After 5 h incubation with BD GolgiPlug TM , cells were stained with Fixable Viability Dye, anti-CD8α and anti-Thy1.1, then stained for intracellular IFN-γ.
Thy1.1 TCR-I cells isolated from spleens or brains of naïve mice or mice days 8, 30, or 45 p.i. were FACS-sorted to > 95% purity. Genomic DNA was isolated from the purified cells and subjected to bisulfite treatment. Bisulfite modified DNA was PCR-amplified using PD-1 promoter specific primers, cloned, and sequences were analyzed using BISMA software, as described. 13 Statistical analysis p values were determined by an unpaired Student t test with Mann-Whitney posttest or oneway ANOVA with Kruskal-Wallis posttest using GraphPad Prism software (La Jolla, CA). p values ≤ 0.05 were considered significant.
Refer to Web version on PubMed Central for supplementary material. Real-time PCR assay of MuPyV genome copy numbers from total genomic DNA isolated from brain, spleen and kidney at days 9 and 30 p.i. Oneway ANOVA, Kruskal-Willis test with Dunn's multiple comparisons test was used to determine statistical significance. (C) Real-time PCR analysis for expression of PD-1 on cDNA prepared from FACS-purified GFP + TCR-I cells from spleens of naïve, day 8-and day 30-infected mice. (D) Mononuclear cells from brain, kidney, and spleen of TCR-I recipient mice at days 9 and 30 p.i. were stimulated with LT206 peptide-pulsed DC2.4 cells in presence of control rat IgG ( ) or PD-L1 blocking Ab ( ). A nonparametric-paired Student's t-test with Wilcoxon matched-pairs signed rank test was used to determine statistical significance between control rat IgG and PD-L1 blocking Ab-treated samples for each organ at a single time point. Each data point represents each mouse in that group. Data from two independent experiments with 4-5 mice each group. Values are mean + SD; * p ≤0.05, ** p≤0.01, ns p>0.05. with Mann-Whitney test was used to determine statistical significance. Values are mean + SD; * p≤0.05, ** p≤0.01, not significant (ns) p>0.05 