Tissue-resident memory T (TRM) cells serve as vanguards of antimicrobial host defense in nonlymphoid tissues, particularly at barrier epithelia and in organs with nonrenewable cell types (e.g., brain). In this study, we asked whether an augmented ability to sense Ag complemented their role as early alarms of pathogen invasion. Using mouse polyomavirus, we show that brain-resident mouse polyomavirus–specific CD8 T cells, unlike memory cells in the spleen, progressively increase binding to MHC class I tetramers and CD8 coreceptor expression. Using the two-dimensional micropipette adhesion-frequency assay, we show that TRM cells in brain, as well as in kidney, express TCRs with up to 20-fold higher affinity than do splenic memory T cells, whereas effector cells express TCRs of similar high affinity in all organs. Together, these data demonstrate that TRM cells retain high TCR affinity, which endows them with the high Ag sensitivity needed for front-line defense against infectious agents.

Anatomic location shapes the phenotypic and functional heterogeneity that defines subsets of memory T cells. Central memory T cells reside in secondary lymphoid organs, whereas effector memory T cells travel through the vasculature and enter nonlymphoid tissues. The third and largest subset of memory T cells does not recirculate and remains in fixed position in nonlymphoid tissues (1). These tissue-resident memory T (TRM) cells inhabit cutaneous and mucosal epithelia, portals of pathogen invasion where in situ initiation of immune defenses may prove essential for limiting host morbidity and mortality (2). These cells also occupy nonbarrier sites (1, 3); the CNS may especially rely on TRM cells to protect the large populations of nonregenerative cells. Brain CD8 TRM cell responses have been characterized for acutely cleared CNS infections, such as vesicular stomatitis virus (VSV) and West Nile virus (46).

JC polyomavirus (JCV) is an opportunistic pathogen in the human virome that can cause the life-threatening, demyelinating CNS disease progressive multifocal leukoencephalopathy (PML) under conditions of immunocompromise. Elevated frequencies of JCV-specific CD8 T cells correlate with improved PML prognosis in HIV/AIDS patients (7). Using the two-dimensional micropipette adhesion-frequency assay, we identified high TCR affinity as a property of virus-specific CD8 T cells responding to persistent mouse polyomavirus (MPyV) infection in the brain. MPyV-specific TRM cells in the kidney, a major site of human polyomavirus persistence, also expressed high-affinity TCRs. High TCR affinity facilitates the ability of TRM cells to sense viral Ags during low-level persistent infections.

C57BL/6NCr female mice, purchased from the Frederick Cancer Research and Development Center of the National Cancer Institute (Frederick, MD), were housed in accordance with the guidelines of the Institutional Animal Care and Use Committee of The Pennsylvania State University College of Medicine. Anesthetized mice (7–12 wk old) were injected intracerebrally in the right frontal lobe with 2 × 106 PFU MPyV strain A2 in 30 μl DMEM 5% FBS. Heat-inactivated (70°C for 30 min) MPyV stock had no infectious virus by plaque assay (limit of detection 5 PFU/ml; data not shown).

TaqMan real-time PCR was performed with 10 μg template DNA purified from tissue. Primers and amplification parameters were described previously (8).

Mice were injected i.p. with 250 μg rat anti-CD8 (YTS169.4; Bio X Cell, West Lebanon, NH), rat anti-CD4 (GK1.5; Bio X Cell), or ChromPure whole rat IgG (Jackson ImmunoResearch, West Grove, PA) at 10 and 12 days postinfection (dpi) and then weekly until 60 dpi. Lack of staining of PBMCs by anti-CD4 (RM4-5) or anti-CD8β (53-5.8) confirmed depletion.

Brains, kidneys, and spleens were harvested from transcardially perfused mice. After collagenase digestion, brain and kidney cells were isolated on Percoll gradients and exposed to Fixable Viability Dye (eBioscience, San Diego, CA) and Fc Block (BioLegend, San Diego, CA) prior to staining with Db-LT359–368 tetramers (National Institutes of Health Tetramer Core Facility, Atlanta, GA) and Abs to the following molecules: CD8α (53-6.7), CD4 (RM4-5), CD44 (IM7), CD69 (H1.2F3), CD103 (M290), CD62L (MEL-14), IFN-γ (XMG1.2), and IL-2 (JES6-5H4) (all from BD Biosciences, San Diego, CA) and TNF-α (TN3-19.12; eBioscience). BrdU (Sigma) was injected i.p. (1 mg/24 h), and mice were euthanized 60 h later. BrdU uptake was detected using a BrdU Flow Kit (BD Biosciences). Ex vivo LT359 peptide stimulation and intracellular cytokine staining were done as previously described (8). Samples were acquired on an LSR II or LSRFortessa (BD Biosciences), and data were analyzed using FlowJo software (TreeStar, Ashland, OR).

CD8 T cells were purified by magnetic sorting of mononuclear cells isolated from brain, kidney, and spleen. Coating human RBCs with peptide-MHC (p-MHC), quantifying binding events and p-MHC and TCR surface densities, and calculating adhesion frequency and two-dimensional were as described (9, 10). A T cell that bound a Db-LT359–coated RBC with an adhesion frequency ≥ 0.1 was considered Ag reactive. No Ag-reactive binding events occurred with LCMV Db-NP396–coated RBCs (Supplemental Fig. 2B, 2C).

The p values were determined by an unpaired Student t test or one-way ANOVA using GraphPad Prism software (La Jolla, CA). All p values ≤ 0.05 were considered significant.

CD8 T cells likely check progression of JCV-PML disease (7) and are important for control of systemic MPyV infection (11). To investigate CD8 T cell responses to MPyV CNS infection, mice were inoculated intracerebrally, a route commonly used in neurotropic virus–infection models (12, 13). To determine whether the brain supports MPyV replication, mice received infectious or heat-inactivated virus, resulting in a 100-fold increase in viral genomes from days 1 to 4 postinfection or no increase, respectively (data not shown). Viral genome numbers in the brain contracted ∼100-fold from peak replication at 4 to 8 dpi, followed by a low-level persistent infection phase (Fig. 1A); similar kinetics were observed in the spinal cord, kidney, and spleen, as with i.p. and s.c. inoculation routes. Next, we asked whether antiviral T cell responses correlated with declining MPyV levels in the brain. Approximately half of brain-isolated cells at 8 d postinfection (dpi) were CD8 T cells, of which nearly 75% bound Db-LT359 tetramers (Fig. 1B), the immunodominant epitope (8). After peak infiltration at 8 dpi, both the magnitude of total CD8 T cells (Fig. 1C) and the frequency of Db-LT359–specific CD8 T cells in the brain remained stable into the persistent phase, unlike the response in the spleen, which contracted ∼30% (Fig. 1D). Most LT359-specific CD8 T cells in the brain produced IFN-γ at 30 dpi, with ∼25% coproducing TNF-α (Fig. 1E). The CD4 T cell response in the brain mirrored that of CD8 T cells, although it was ∼10-fold lower in magnitude throughout infection (Fig. 1C). These data show that brain-infiltrating CD8 T cells are predominantly directed to a single MPyV epitope, are functional, and are stably maintained in the setting of a nearly 3-log decrease in viral load.

FIGURE 1.

Characterization of MPyV-specific T cell responses in the brain. (A) Kinetic analysis by quantitative PCR of viral genome copy numbers in genomic DNA isolated from brain, spleen, kidney, and spinal cord. (B) Representative dot plot of T cell frequencies in brain-isolated mononuclear cells (left panel) and frequency of Db-LT359–tetramer+ cells of total CD8 T cells (right panel) at 8 dpi by flow cytometry. (C) Kinetic analysis of the number of total CD8 and CD4 T cell responses in brain. (D) Frequency of Db-LT359+ of CD44hi CD8+ T cells in brain and spleen over the course of infection. (E) Frequency of cytokine-producing CD44hi CD8 T cells in brain and spleen (with background subtracted) at 30 dpi following ex vivo stimulation with LT359 peptide. Data are cumulative from three independent experiments (n = 7–9 total mice/time point).

FIGURE 1.

Characterization of MPyV-specific T cell responses in the brain. (A) Kinetic analysis by quantitative PCR of viral genome copy numbers in genomic DNA isolated from brain, spleen, kidney, and spinal cord. (B) Representative dot plot of T cell frequencies in brain-isolated mononuclear cells (left panel) and frequency of Db-LT359–tetramer+ cells of total CD8 T cells (right panel) at 8 dpi by flow cytometry. (C) Kinetic analysis of the number of total CD8 and CD4 T cell responses in brain. (D) Frequency of Db-LT359+ of CD44hi CD8+ T cells in brain and spleen over the course of infection. (E) Frequency of cytokine-producing CD44hi CD8 T cells in brain and spleen (with background subtracted) at 30 dpi following ex vivo stimulation with LT359 peptide. Data are cumulative from three independent experiments (n = 7–9 total mice/time point).

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We asked whether CD4 T cell help or continuous infiltration by circulating CD8 T cells in response to ongoing infection contributed to stability of this population. We depleted circulating cells by administering anti-CD4, anti-CD8, or control rat IgG at 10 dpi, to allow peak infiltration of CD8 T cells into the brain, and then weekly until 60 dpi. Total numbers of brain-infiltrating CD4 T cells decreased in anti-CD4–treated mice, demonstrating their recruitment from the circulation during persistent infection (Fig. 2A). No change in numbers of total or MPyV-specific CD8 T cells in the brain were observed in CD4 or CD8 Ab-treated mice, indicating that neither CD4 help nor replenishment from the circulation is needed to maintain this population (Fig. 2A). This finding parallels parabiosis experiments in which endogenous memory cells in the brain do not equilibrate with donor parabiont cells, suggesting that effector-phase T cells seed the memory population (14). Next, we administered the thymidine analog BrdU to mice 60 h prior to sacrifice at 30 dpi. Approximately 15% of Db-LT359–specific CD8 T cells in both the brain and spleen incorporated BrdU (Fig. 2B), suggesting that in situ proliferation contributed to the long-term maintenance of brain-infiltrating memory CD8 T cells.

FIGURE 2.

MPyV-specific CD8 T cells survive long-term in the brain. (A) Mice were Ab depleted of circulating CD8 or CD4 T cells, and the numbers of T cells infiltrating the brain were determined by flow cytometry. Data are cumulative from two independent experiments (n = 7–8 total mice/group). (B) Mice were given BrdU i.p. for 60 h prior to sacrifice at 30 dpi. Shown are the mean frequencies (±SD) of BrdU+ Db-LT359+ CD8 T cells in brain and spleen. Data are cumulative from two independent experiments (n = 9 total mice). (C) Representative line graphs of expression of phenotypic markers of tissue-resident memory cells by brain-infiltrating (open graphs) or splenic (shaded graphs) Db-LT359+ CD8 T cells at the indicated time points postinfection (left panels). Mean (± SD) geometric (g)MFI of each marker (right panels). Data are cumulative from three independent experiments (n = 7–9 mice/time point). **p < 0.01.

FIGURE 2.

MPyV-specific CD8 T cells survive long-term in the brain. (A) Mice were Ab depleted of circulating CD8 or CD4 T cells, and the numbers of T cells infiltrating the brain were determined by flow cytometry. Data are cumulative from two independent experiments (n = 7–8 total mice/group). (B) Mice were given BrdU i.p. for 60 h prior to sacrifice at 30 dpi. Shown are the mean frequencies (±SD) of BrdU+ Db-LT359+ CD8 T cells in brain and spleen. Data are cumulative from two independent experiments (n = 9 total mice). (C) Representative line graphs of expression of phenotypic markers of tissue-resident memory cells by brain-infiltrating (open graphs) or splenic (shaded graphs) Db-LT359+ CD8 T cells at the indicated time points postinfection (left panels). Mean (± SD) geometric (g)MFI of each marker (right panels). Data are cumulative from three independent experiments (n = 7–9 mice/time point). **p < 0.01.

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We asked whether the canonical CD62L-selectinlo CD69hi CD103+ phenotype used to define TRM cells in acute infection models applied to brain-infiltrating virus-specific CD8 T cells during persistent MPyV infection. Over 90% of Db-LT359–specific CD8 T cells in the brain upregulated CD69 by 15 dpi, but those in the spleen remained CD69−/lo at all time points examined (Fig. 2C). All MPyV-specific CD8 T cells in the brain and spleen were CD62Llo, a phenotype expected in persistently infected hosts. Notably, most MPyV-specific CD8 T cells in the brain remained CD69hi, despite declining virus levels (Fig. 1A). Because CD69 antagonizes expression of S1P1 receptors, which must be downregulated for establishment of a TRM compartment (15), CD69hi appears to be an indispensable phenotype for TRM cells. Beginning at 15 dpi, we observed a gradual increase in CD103 expression by Db-LT359–specific CD8 T cells; however, even at 30 dpi, only one third of these cells were CD103+ (Fig. 2C), with no further increase in CD103 expression by 60 dpi (data not shown). This contrasts with acutely cleared VSV infection in the CNS, in which the CD103+ fraction increases from ∼60 to 80% of VSV-specific CD8 T cells from 20 to 40 dpi (4). Most MPyV-specific CD8 T cells in the brain were CD103, despite being stably maintained, indicating that these cells were functionally brain resident during persistent infection. Together, these data suggest that persistent Ag impedes CD103 upregulation, in line with other studies (16), but not establishment of a TRM population. Evidence that CD103 expression by CD69hi CD62Llo CD8 T cells varies among nonlymphoid organs (15) further supports the likelihood that CD103 is not a reliable TRM marker. The role of cytokines and Ag in development and retention of TRM cells responding to low-level persistent infections remains to be determined. The gradual decline in virus levels during persistent infection and stability of MPyV-specific CD8 T cells imply that Ag may be dispensable for retention of these TRM cells, as for skin-resident HSV-specific TRM cells (17).

MPyV-specific CD8 T cells in the brain showed a progressive increase in mean fluorescence intensity (MFI) of Db-LT359 tetramer, whereas in the spleen, tetramer MFI stayed uniformly lower during persistent infection (Fig. 3A). We also observed that brain-infiltrating cells expressed significantly higher levels of CD8 coreceptors than did those in the spleen as early as 8 dpi, and they showed a progressive increase in MFI (Fig. 3B). The cooperative trimolecular interactions among the TCR, CD8, and p-MHC complex obviate determination of affinity of a given TCR for its cognate p-MHC ligands (18). Therefore, to determine whether MPyV-specific memory T cells in the spleen and brain differed in TCR affinity, we used the micropipette adhesion-frequency assay with Db-LT359 monomers, in which the native α3 domain is replaced with that of HLA-A2, which cannot bind mouse CD8 (9). This assay allows interrogation of the affinity of a single T cell for p-MHC in a physiologically relevant membrane-anchored context, and it can detect Ag-specific populations with affinities below detection by tetramers (10, 19, 20). Reactivity to Db-LT359 was assessed for 56 CD8 T cells isolated from the brain, and 44 of which (78.6%) were determined to be Ag specific (Fig. 4A). In contrast, of 68 CD8 T cells isolated from the spleen, only 14 (20.6%) were found to be Db-LT359 specific. The micropipette adhesion-frequency data confirmed the observations made with tetramers that Db-LT359–specific CD8 T cells predominantly make up the brain TRM pool.

FIGURE 3.

Increase in tetramer binding and CD8 expression by MPyV-specific CD8 T cells in the brain. CD8 T cells from brain and spleen were analyzed for geometric (g)MFI of staining by Db-LT359 tetramers (A) and anti-CD8α (B), shown as representative line graphs (left panels) and bar graphs (mean ± SD) (right panels). Data are cumulative from three independent experiments (n = 7–9 mice/time point). ****p < 0.0001.

FIGURE 3.

Increase in tetramer binding and CD8 expression by MPyV-specific CD8 T cells in the brain. CD8 T cells from brain and spleen were analyzed for geometric (g)MFI of staining by Db-LT359 tetramers (A) and anti-CD8α (B), shown as representative line graphs (left panels) and bar graphs (mean ± SD) (right panels). Data are cumulative from three independent experiments (n = 7–9 mice/time point). ****p < 0.0001.

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FIGURE 4.

Comparison of TCR affinities of MPyV-specific CD8 T cells in brain, kidney, and spleen. CD8 T cells pooled from each organ during persistent (AC; 26–32 dpi) or acute (D; 8 dpi) infection were analyzed by micropipette adhesion-frequency assay. (A) Adhesion frequencies of CD8 T cells from the brain, spleen, and kidney using RBCs coated with different p-MHC surface densities. (B and D) Geometric mean (± 95% confidence interval) of two-dimensional binding affinity for Db-LT359; each mark represents an individual CD8 T cell. (C) Frequency distributions of the log of affinities for each organ examined. Gaussian curves were fitted to the data with r2 values as follows: spleen = +0.97; kidney = +0.71; brain = +0.64. Data are cumulative from two independent experiments (n = with 5–7 mice/pool). *p < 0.01, ****p < 0.0001. ns, not significant.

FIGURE 4.

Comparison of TCR affinities of MPyV-specific CD8 T cells in brain, kidney, and spleen. CD8 T cells pooled from each organ during persistent (AC; 26–32 dpi) or acute (D; 8 dpi) infection were analyzed by micropipette adhesion-frequency assay. (A) Adhesion frequencies of CD8 T cells from the brain, spleen, and kidney using RBCs coated with different p-MHC surface densities. (B and D) Geometric mean (± 95% confidence interval) of two-dimensional binding affinity for Db-LT359; each mark represents an individual CD8 T cell. (C) Frequency distributions of the log of affinities for each organ examined. Gaussian curves were fitted to the data with r2 values as follows: spleen = +0.97; kidney = +0.71; brain = +0.64. Data are cumulative from two independent experiments (n = with 5–7 mice/pool). *p < 0.01, ****p < 0.0001. ns, not significant.

Close modal

Using the adhesion-frequency values, together with TCR and p-MHC surface densities, we calculated that brain TRM cells have a 20-fold higher mean affinity for Db-LT359 than do cells in the spleen (Fig. 4B). To address whether increased TCR affinity was unique to TRM cells in the brain, we assayed 69 kidney-infiltrating memory CD8 T cells and found that 55.1% were Db-LT359 specific, with a mean TCR affinity that was significantly higher than that measured in the spleen (p = 0.0029). Cells in the brain and kidney occupied a 3-log range skewed toward higher affinities, in contrast to the spleen where affinities spanned 1-log (Fig. 4C). Notably, cells in the kidney did not differ significantly in affinity from those in the brain (Fig. 4B) and had higher MFIs for Db-LT359 tetramer and CD8 Ab staining than did those in the spleen (Supplemental Fig. 1A). Approximately 70% of CD8 T cells in the kidney expressed CD69, suggesting that most are TRM cells (Supplemental Fig. 1B). Elevated CD8 levels on T cells in both brain and kidney may constitute an additional phenotypic marker for TRM cells. Together, these data show that MPyV-specific CD8 TRM cells express higher-affinity TCRs, with cooperative binding by higher levels of CD8, than do memory cells in the spleen.

A number of studies documented that effector, but not memory, CD8 T cells are competent to enter nonlymphoid tissues (4, 21, 22). Effector CD8 T cells generated in vitro express higher effective TCR affinity than do their naive precursors (9). Together, these findings raise the possibility that effector T cells in both lymphoid and nonlymphoid tissues may express high-affinity TCRs during acute infection. We compared the TCR affinities of virus-specific CD8 T cells in acutely infected mice. From the brain, TCR affinities for Db-LT359 were determined for 40 of 53 cells assayed, a frequency of 75.4% (Supplemental Fig. 2A). In both the spleen and the kidney, the frequency of Db-LT359–specific CD8 T cells was lower: 40 of 86 cells (46.5%) cells in the spleen and 15 of 33 (45.5%) cells from the kidney (Supplemental Fig. 2A). Unlike memory cells, effector T cells in all three organs expressed TCRs of similarly high affinity (geometric mean affinities: 7.72 × 10−4, 5.20 × 10−4, and 7.48 × 10−4 μm4 for the brain, kidney, and spleen, respectively) (Fig. 4D). For both acute and persistent infection time points, splenic Db-LT359–specific CD8 T cells had significantly higher TCR surface density than did those from the brain (Supplemental Fig. 2D, 2E), and this difference is taken into account when calculating TCR affinity (10). Similar to memory cells in the brain and kidney, CD8 T cells from all organs isolated during acute infection spanned a 3-log range of affinities.

These findings suggest that high-affinity TRM cells originate from high-affinity effector cells that enter nonlymphoid tissues during acute infection; further, these data raise the possibility that the nonlymphoid microenvironment may be conducive for retaining T cells having high-affinity TCRs. High TCR affinity would improve the ability of TRM cells to detect infected cells expressing low levels of Ag, not only during persistent polyomavirus infection, but also during early reinfection when rapid control may be critical in limiting injury to organs with a large population of essential, nonrenewable cells (e.g., brain).

This work was supported by the PML Consortium, LLC (to A.E.L.) and by the National Institutes of Health (R01 NS088367 and R01 AI102543 to A.E.L., T32 AI007610 and F31 NS083336 to E.L.F., R01 AI096879 and NS071518 to B.D.E., and T32 AI007610 and F31 NS081828 to A.E.K.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

dpi

day postinfection

JCV

JC polyomavirus

MFI

mean fluorescence intensity

MPyV

mouse polyomavirus

p-MHC

peptide-MHC

PML

progressive multifocal leukoencephalopathy

TRM

tissue-resident memory T

VSV

vesicular stomatitis virus.

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The authors have no financial conflicts of interest.

Supplementary data