The PD-1: PD-L1 pathway promotes development of brain-resident memory T cells following acute viral encephalitis
© The Author(s). 2017
Received: 4 February 2017
Accepted: 5 April 2017
Published: 13 April 2017
Previous work from our laboratory has demonstrated that during acute viral brain infection, glial cells modulate antiviral T cell effector responses through the PD-1: PD-L1 pathway, thereby limiting the deleterious consequences of unrestrained neuroinflammation. Here, we evaluated the PD-1: PD-L1 pathway in development of brain-resident memory T cells (bTRM) following murine cytomegalovirus (MCMV) infection.
Flow cytometric analysis of immune cells was performed at 7, 14, and 30 days post-infection (dpi) to assess the shift of brain-infiltrating CD8+ T cell populations from short-lived effector cells (SLEC) to memory precursor effector cells (MPEC), as well as generation of bTRMs.
In wild-type (WT) animals, we observed a switch in the phenotype of brain-infiltrating CD8+ T cell populations from KLRG1+ CD127− (SLEC) to KLRG1− CD127+ (MPEC) during transition from acute through chronic phases of infection. At 14 and 30 dpi, the majority of CD8+ T cells expressed CD127, a marker of memory cells. In contrast, fewer CD8+ T cells expressed CD127 within brains of infected, PD-L1 knockout (KO) animals. Notably, in WT mice, a large population of CD8+ T cells was phenotyped as CD103+ CD69+, markers of bTRM, and differences were observed in the numbers of these cells when compared to PD-L1 KOs. Immunohistochemical studies revealed that brain-resident CD103+ bTRM cells were localized to the parenchyma. Higher frequencies of CXCR3 were also observed among WT animals in contrast to PD-L1 KOs.
Taken together, our results indicate that bTRMs are present within the CNS following viral infection and the PD-1: PD-L1 pathway plays a role in the generation of this brain-resident population.
Infection of the central nervous system (CNS) presents unique challenges to effective pathogen control, as brain infection may rapidly progress causing substantial damage or even death. Neuroimmune responses are critical for antiviral defense, but extensive damage to this generally non-regenerating tissue must be avoided . It is well established that different immune mechanisms are very specifically tailored to control infections in particular organs. Recent studies have demonstrated that after clearance of many acute viral infections, CD8+ T lymphocytes generate a population of long-lived, non-recirculating tissue-resident memory cells (TRM) in non-lymphoid tissue; and it is becoming increasingly clear that these TRM cells play critical roles in controlling re-encountered infection and accelerating the process of pathogen clearance [2–5].
The CNS can be a target of acute viral infection, as well as a reservoir of latent and persistent virus. During acute viral infection, most pathogens are rapidly cleared through the generation of a large number of short-lived effector T cells (SLEC). Simultaneously, the T cell response is triggered to generate a subset identified as memory precursor effector cells (MPEC). These MPEC begin to develop into a tissue-resident memory (TRM) phenotype shortly after infection. Recent work by several groups provides evidence that there is a clear distinction between terminal effector and memory cells based on heterogeneity in expression of killer cell lectin-like receptor G1 (KLRG1) [6–8]. We have recently characterized brain-infiltrating T cells which persist within the tissue after acute murine cytomegalovirus (MCMV) infection. We showed that infiltrating CD8+ T cell populations shift from SLEC to clear infection to MPEC that protect against re-challenge. The shift of prominent SLEC populations to MPEC populations is concomitant with transition from acute through chronic phases of infection. In addition, these cells were found to selectively express the integrin CD103, a marker of brain TRM (bTRM) cells and persist long-term within the CNS .
Resolution of adaptive immune responses and generation of immunological memory is an essential process to confer long-term protective immunity particularly in immune-privileged tissue-like brain. Inflammation within different anatomical sites of brain dramatically increases the infiltration and migration of lymphocytes and effector molecules. We understand much about the infiltrating T cell mediated immune response and the penetration of T cells within the infected brain parenchyma . However, better understanding of the association between inflammation and the establishment of TRM will inform us about the protective effects of neuroimmune responses to re-infection or viral reactivation.
TRM cells are characterized by their non-recirculating, resident nature in tissues. It is well reported that TRM cells often express αEβ7. αE, otherwise known as CD103, has been identified as a marker of particular types of TRM cells. High expression of CD103 and CD69 is a common feature of resident memory cells observed in epithelial tissue [11, 12]. Whereas, effector and resident memory cells in circulation appear to lack expression of both CD103 and CD69 [13, 14]. It has been shown that CD69 expression is required for the optimal formation of TRM following herpes simplex virus (HSV) infection in tissues such as the skin and dorsal root ganglia [2, 15]. In addition, experiments using the skin, lung, and gut show differential expression of CCR7, as well as CXCR3, which define the migration properties of T cells [16–18]. However, further insight into factors responsible for development of TRM is required. Given the importance of the formation of brain (bTRM) cells, there is surprisingly little known about how glial cells contribute to their formation.
The programmed death receptor-1 (PD-1): programmed death ligand-1 (PD-L1) pathway is central in controlling interactions between host defense and invading pathogens. Accumulating evidence suggests that during neuroinflammation, PD-L1 expression is increased on microglial cells, as well as astrocytes . These findings suggest that resident glial cells limit CNS pathology through suppression of proinflammatory cytokine production from brain-infiltrating T cells via activation of the PD-1: PD-L1 pathway . PD-L1 expression on glial cells has also been shown to limit immune-mediated tissue damage in models of multiple sclerosis, as well as during acute viral encephalitis [19, 21, 22].
We have previously investigated the role of PD-1: PD-L1 signaling in regulating immunopathology through functional inhibition of effector CD8+ T cells within the post-encephalitic brain following MCMV infection . In the present study, we investigated the involvement of PD-1: PD-L1 signaling in the retention of CD8+-gated CD103+CD69+ T cells and the development of bTRM. Using our murine model of MCMV infection, we performed phenotypic analysis of CD8+ lymphocytes residing within the chronically infected brain to characterize bTRM. We also compared their development in wild-type (WT) animals to that in PD1- and PD-L1 knockout mice.
This study was carried out in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (Protocol Number: 1402-31338A) of the University of Minnesota. All surgery was performed under ketamine/xylazine anesthesia and all efforts were made to minimize suffering.
Virus and animals
RM461, a MCMV expressing Escherichia coli β-galactosidase under the control of the human ie1/ie2 promoter/enhancer , was kindly provided by Edward S. Mocarski. The virus was maintained by passage in weanling female Balb/c mice. Salivary gland-passed virus was then grown in NIH 3T3 cells for two passages, which minimized any carry-over of salivary gland tissue. Infected 3T3 cultures were harvested at 80 to 100% cytopathic effect and subjected to three freeze–thaw cycles. Cellular debris was removed by centrifugation (1000×g) at 4 °C, and the virus was pelleted through a 35% sucrose cushion (in Tris-buffered saline [50 mM Tris–HCl, 150 mM NaCl, pH 7.4]) at 23,000×g for 2 h at 4 °C. The pellet was suspended in Tris-buffered saline containing 10% heat-inactivated fetal bovine serum (FBS). Viral stock titers were determined on 3T3 cells as 50% tissue culture infective doses (TCID50) per milliliter. Six to eight weeks old C57B/6 mice were obtained from Charles River Laboratories (Wilmington, MA), while PD-L1 KO and PD-1 KO animals were kindly provided by Arlene Sharpe (Harvard University) and Sing Sing Way (Cincinnati Children’s Hospital, Cincinnati, OH), respectively.
Intracerebroventricular infection of mice
Infection of mice with MCMV was performed as previously described . Briefly, female mice (6–8 weeks old) were anesthetized using a combination of ketamine and xylazine (100 mg and 10 mg/kg body weight, respectively) and immobilized on a small animal stereotactic instrument equipped with a Cunningham mouse adapter (Stoelting Co., Wood Dale, IL). The skin and underlying connective tissue were reflected to expose reference sutures (sagittal and coronal) on the skull. The sagittal plane was adjusted such that the bregma and lambda were positioned at the same coordinates on the vertical plane. Virulent, salivary gland-passaged MCMV RM461 (1 × 10  TCID50 units in 10 μL), was injected into the right lateral ventricle at 0.9 mm lateral, 0.5 mm caudal, and 3.0 mm ventral to the bregma using a Hamilton syringe (10 μL) fitted to a 27 G needle. The injection was delivered over a period of 3–5 min. The opening in the skull was sealed with bone wax and the skin was closed using 4–0 silk sutures with a FS-2 needle (Ethicon, Somerville NJ).
Brain leukocyte isolation and flow cytometry analysis
Leukocytes were isolated from the brains of MCMV-infected C57B/6 WT, PD-L1 KO, and PD-1 KO mice, using a previously described procedure with minor modifications [25–28]. In brief, whole brain tissues were harvested (n = 3–4 animals/group/experiment) and minced finely using a scalpel in RPMI 1640 (2 g/L D-glucose and 10 mM HEPES) and digested in 0.0625% trypsin (in Ca/Mg-free HBSS) at room temperature for 20 min. Single-cell preparations of infected brains were resuspended in 30% Percoll (Sigma-Aldrich) and banded on a 70% Percoll cushion at 900 × g for 30 min at 15 °C. Brain leukocytes obtained from the 30–70% Percoll interface were collected.
Following preparation of single-cell suspensions, cells were treated with Fc block (anti-CD32/CD16 in the form of 2.4G2 hybridoma culture supernatant with 2% normal rat and 2% normal mouse serum) to inhibit non-specific Ab binding. Cells were then counted using the trypan blue dye exclusion method, and 1 × 10  cells were subsequently stained with anti-mouse immune cell surface markers for 15–20 min at 4 °C (anti-CD45-PE-Cy5, anti-KLRG1-PE-Cy7, anti-CD11b-AF700, anti-CD103-PE, anti-CD127-APC, anti-CD69-e-F 450, anti-CXCR3-FITC, CCR7-PE-Cy7, anti-PD1-FITC (eBioscience, San Diego CA), and anti-CD8-BV-510 from (Biolegend)). For intracellular staining of Ki67 and Bcl-2, anti-Ki67FITC was obtained from eBioscience whereas PE-conjugated anti-Bcl-2(3F11) and PE-conjugated anti-TNP (isotype-matched control antibody for staining with anti-Bcl-2 A19-3) were from BD Pharmingen. Control isotype Abs were used for all fluorochrome combinations to assess non-specific Ab binding. Live leukocytes were gated using forward scatter and side scatter parameters on a BD FACSCanto flow cytometer and LSRII H4760 (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo software (FlowJo, Ashland, OR).
The brains were harvested from infected mice that were perfused with serial washes of phosphate-buffered saline (PBS), 2% sodium nitrate to remove contaminating blood cells, and 4% paraformaldehyde. The murine brains were subsequently submerged in 4% paraformaldehyde for 24 h and transferred to 25% sucrose solution for 2 days prior to sectioning. After blocking (1× PBS, 10% normal goat serum, and 0.3% Triton X-100) for 1 h at room temperature, brain sections (25 μm) were incubated overnight at 4 °C with the following primary antibodies: Rat anti-mouse CD8 (10 μg/mL; eBioscience) and Armenian Hamster anti-mouse CD103 (10 μg/mL; eBioscience). Brain sections were washed three times with PBS. After washing, secondary antibody (Goat anti-Rat FITC conjugate and Goat anti-Aremenian Hamster-conjugate Cy3) was added for 1 h at RT followed by nuclear labeling with Hoechst 33342 (1 μg/mL; Chemicon, Temecula, CA) and viewing under a fluorescent microscope.
Total DNA was extracted from murine brain tissues using the QIAamp DNA mini kit (Qiagen, Valencia, CA). Total RNA was extracted from murine brain tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA), treated with DNase and reverse transcribed to cDNA with oligo (dT)12–18, random hexmer, dNTPs (Gene Link, Hawthorne, NY), RNase inhibitor, and SuperScript™ III reverse transcriptase (Invitrogen). Mixtures of DNA or diluted cDNA, primers, and SYBR® Advantage® qPCR premix (ClonTech, Mountain View, CA) were subjected to real-time PCR (Stratagene, now Agilent Technologies, La Jolla, CA) according to the manufacturer’s protocol. Primer sequences were sense 5′- ATCTGAAACAGCCGTATATCATCTTG-3′ and antisense 5′- TCAGCCATCAACTCTGCTACCAAC-3′ for MCMV IE1 (100 bp), and sense 5′- TGCTCGAGATGTCATGAAGG-3′ and antisense 5′- AATCCAGCAGGTCAGCAAAG-3′ for HPRT (hypoxanthine phosphoribosyltransferase, 95 bp). The PCR conditions for the Mx3000P QPCR System were: 1 denaturation cycle at 95 °C for 10 s; 40 amplification cycles of 95 °C for 10 s, 60 °C annealing for 10 s, and elongation at 72 °C for 10 s; followed by 1 dissociation cycle. The relative product levels were quantified using the 2−∆∆Ct method  and were normalized to the housekeeping gene HPRT.
For comparing groups, two-tailed unpaired Student’s T test for samples was applied; p values ≤0.05 were considered significant.
Reduced numbers of bTRM cells in PD-L1 KO animals
PD-L1 supports development of long-lived memory cells following MCMV infection
bTRM cells are also reduced in PD-1 KO animals
Loss of PD-L1 or PD-1 resulted in fewer CD8+ T cells which co-express CD69 and CD103
PD-1 expression on bTRM cells following MCMV infection
Expression of CXCR3 on bTRM following MCMV infection
Bcl-2 expression on CD103+ and CD69+ CD8 T cells within the infected brain
The most significant finding presented in this study is that the PD-1: PD-L1 pathway contributes to development of bTRM cells within the MCMV-infected brains. Upon resolution of acute viral infection, the greatly expanded effector CD8+ T cell population rapidly contracts, leaving behind a small number of cells that survive to form long-lived memory cells [31, 35]. Some of these memory T lymphocytes persist long term in non-lymphoid tissues as TRM cells, which defend against re-infection [3, 14, 36]. We and others have previously shown that effector CD8+ T cell populations exhibit heterogeneity in expression of KLRG1 during activation and expansion [2, 3, 7, 9]. Through study of both acute and long-term CNS viral infection using WT, PD-L1 KO, and PD-1 KO animals, we report here that brain-infiltrating CD8+ T cells display distinct phenotypes of SLEC and MPEC populations from acute to chronic infection. In accordance with other studies where it has been reported that CD127 and KLRG1 are inversely expressed on SLEC and MPEC, our results show that during acute MCMV infection, KLRG1+ CD127− (i.e., the SLEC population) cells dominate. In contrast, later time points correlate with development of KLRG1− CD127+ cells in WT animals [9, 37, 38]. Importantly, CD127 expression was significantly reduced in PD-L1 KO and PD-1 KO animals. Taken together, these data demonstrate that the PD-1: PD-L1 pathway within the CNS promotes development of a bTRM cell population following viral infection.
Studies of HIV-1 infection have reported expansion of CD8+CD127− effector-like T cells as a consequence of heightened immune responses . Experiments using acute LCMV and Listeria infections in mice have demonstrated emergence of CD127-expressing CD8+ T cells that arise during the effector phase and acquire phenotypical and functional properties of memory T cells [37, 40]. The down-regulation of CD127 during these chronic viral infections has been attributed to ongoing repetitive TCR stimulation, whereas elevated expression of CD127 on HCV-, HBV-, and RSV-specific memory CD8+ T cells has been explained by a lack of persisting antigen [38, 41]. Thus, the frequency of CD127 expression on bTRM cells in WT animals despite persistence of the latent viral genome may suggest an absence of ongoing TCR triggering within the MCMV-infected brain. In contrast, significantly reduced expression of CD127 on bTRM cells indicates prolonged, effector-like T cell responses in PD-1 KO and PD-L1 KO animals.
Phenotypic signatures indicative of bTRM, consisting of CD103, CD69, and CD127 expression, were observed at higher levels among WT animals than among PD-L1 KO and PD-1 KO mice during chronic infection. Similar to findings reported for other non-lymphoid organs, as well as from the brain with vesicular stomatitis virus [42, 43], we found 87.3 ± 5.6% of the CD8+ T cells persisting within the MCMV-infected brain express CD69. In contrast to brain infection with LCMV, which showed that CD103 was expressed only on a portion of bTRM , we observed that the vast majority of CD8+ T cells co-expressed CD103 and CD69 in WT mice during long-term infection. Expression kinetic studies show early induction of CD69 on brain-infiltrating T cells, as shown by Mutnal et al. , and CD69+CD103− cells appear to show effector function early after brain infection. It has been previously reported that expression of CD69 is required for efficient effector T cell retention in the skin and subsequent formation of TRM cells [11, 15, 45, 46]. This is because CD69 expression by TRM cells downregulates cell surface expression of S1P1, thereby blocking T cell movement out of tissues supporting their stationary state [3, 47, 48]. In this study, PD-1 KO and PD-L1 KO animals show a dominating population of CD69+CD103− at 30 dpi, a time point at which these mice have significantly fewer co-expressing CD69+ CD103+ cells when compared to WT. Accumulating evidence also indicates a contribution of cytokines like TGFβ, IL-15, IL-7, and IFN-α/β in the induction CD103 and CD69 [2, 49–51].
Development of TRM cells in a particular tissue clearly involves various factors such as T cell migration, entry into the tissue, retention, and survival. These factors are likely regulated or induced by locally derived signals. Therefore, effector T cell populations during acute infection and the retention of TRM within non-lymphoid tissue under specific environment conditions, such as the infected brain, are critical to understand. The chemokines CXCL9 and CXCL10 have been shown to facilitate entry of T cells into epithelium during infection of mucosal surfaces with HSV-2 . Similarly, CXCR3, the receptor for CXCL9 and CXC10, is required for the appropriate localization of effector T cells and for subsequent formation of TRM . Expression of CXCR3 on circulating T cells or its chemokine ligands, CXCL9 and CXCL10, in tumor tissues has been reported to be associated with elevated intratumoral T cell infiltration in melanoma and colorectal cancer patients [52–54]. Interestingly, previous studies from our laboratory have shown that microglial cells produce high levels of CXCL9 and CXCL10 in response to MCMV brain infection . Additionally, reports using a skin model suggest that CCR7 is responsible for exit of T cells out of the tissue, whereas CCR7− T cells remained in the skin as TRM cells [3, 11]. Likewise, results presented here show negligible expression of CCR7 at 7 and 30 dpi in all groups of animals, which are in line with other studies [3, 17]. Furthermore, differential expression of CXCR3+ on CD103+CD8+ T cells was observed among WT, PD-1 KO, and PD-L1 KO animals. A significantly higher level expression of CXCR3 on CD103+CD8+ T cells from the brains of WT mice in comparison to PD-1 KO and PD-L1 KO mice at 30 dpi was observed. Moreover, the PD-1: PD-L1 pathway has been reported to negatively regulate chemokine expression in various contexts. For example, increased expression of the chemokine CXCL9 and its receptor is associated with blocking of PD-L1 in dry eye disease mice . Here, differential expression profiles of CXCR3 on bTRM between WT, PD-1 KO, and PD-L1 KO mice were observed. This finding reveals a role for PD-1: PD-L1 in regulating the expression of CXCR3, which in-turn may regulate the retention of bTRM following MCMV infection.
The PD-1: PD-L1 pathway is well known to limit immune-mediated tissue damage caused by over-reactive T cells, particularly in immune-privileged sites like the brain. Previous reports which show upregulation of PD-L1 in inflamed brain suggest a role for this pathway in regulating T cell activation, as well as controlling immunopathological damage [19, 56]. Additionally, PD-1 was first regarded as an inhibitory marker and found to be upregulated on exhausted T cells, as defined by reduced ability to proliferate and produce cytokines . It has previously been proposed that increased expression of inhibitory markers, such as PD-1 and CTLA-4, on brain TRM cells may serve as a means to prevent this population from unintentional activation and unnecessarily self-attack . Similarly, our flow cytometry analysis reveals upregulation of PD-1 on CD103+CD8+ T cells, along with negligible expression on CD103−CD8+ cells in WT animals. Interestingly, inverse expression of PD-1 on the CD103+ and CD103− population was observed among the PD-L1 KO animals. It appears that expression of PD-1 by bTRM cells is not only a mechanism by which the immune system exerts brakes on unnecessary T cell stimulation and proliferation, but it also may itself promote longevity. Furthermore, the decreased expression of PD-1 on TRM cells in PD-L1 KO animals indicates a dysregulation of bTRM cells in the absence of the PD-1: PD-L1 pathway.
Evaluation of Bcl-2 expression in memory cells during chronic infection showed significant higher levels of this pro-survival factor in CD103+ and CD69+ CD8 T cells among WT when compared to PD-L1 KO animals. However, an evaluation of the proliferation potential of memory cells using Ki67 staining revealed no major difference among the two groups of animals. These data indicate increased survival of memory cells without any change in proliferation with an intact PD-1: PD-L1 pathway. Our finding was similar to Grayson et al. who reported surviving memory cells contain higher levels of Bcl-2 than naïve cells. These elevated levels of Bcl-2 may lead to diminished death phase after secondary infection, resulting in a net increase in memory cells .
Data indicate that TRM cells residing in a variety of tissues accelerate and improve clearance of pathogens upon re-challenge. However, the driving mechanisms still remain the subject of intense investigation [31, 59]. It has recently been reported that TRM cells respond to viral reactivation by the production of inflammatory cytokines, such as IFN-γ, along with immune cells being rapidly recruited from the circulation . The function of TRM cells in the brain may largely depend upon rapid IFN-γ production in combination with release of cytotoxic granules and perforin because IFN-γ-deficient bTRM fails to provide sufficient non-cytolytic antiviral function . The positioning of bTRM within the brain parenchyma could be critical to rapidly eliminate infected cells in response to reinfection or reactivation of latent CNS infections.
Taken together, our results indicate that bTRMs are present within the CNS following viral infection and the PD-1: PD-L1 pathway plays a role in the generation of this brain-resident population.
B cell lymphoma 2
- bTRM :
Brain-resident memory T cells
Central nervous system
Killer cell lectin-like receptor G1
Memory precursor effector cells
Short-lived effector cells
This work was supported by awards MH-066703 from the National Institute of Mental Health and NS-038836 from the National Institute of Neurological Disorders and Stroke.
Availability of data and materials
Data supporting the conclusions of this article are presented in the manuscript.
SP, SH, and JL conceived and designed the experiments. SP, SH, WS, and PC performed the experiments. SP, SH, AS, and JL analyzed the data. SP and JL wrote the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
This study was carried out in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (Protocol Number: 1402-31338A) of the University of Minnesota.
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