Memory T cells persisting in the brain following MCMV infection induce long-term microglial activation via interferon-γ

Abstract

Murine cytomegalovirus (MCMV) brain infection stimulates microglial cell-driven proinflammatory chemokine production which precedes the presence of brain-infiltrating systemic immune cells. Here, we show that in response to MCMV brain infection, antigen-specific CD8(+) T cells migrated into the brain and persisted as long-lived memory cells. The role of these persistent T cells in the brain is unclear because most of our understanding of antimicrobial T cell responses comes from analyses of lymphoid tissue. Strikingly, memory T cells isolated from the brain exhibited an effector phenotype and produced IFN-γ upon restimulation with viral peptide. Furthermore, we observed time-dependent and long-term activation of resident microglia, indicated by chronic MHC class II up-regulation and TNF-α production. The immune response in this immunologically restricted site persisted in the absence of active viral replication. Lymphocyte infiltrates were detected until 30 days post-infection (p.i.), with CD8(+) and CD4(+) T cells present at a 3:1 ratio, respectively. We then investigated the role of IFN-γ in chronic microglial activation by using IFN-γ-knockout (GKO) mice. At 30 days p.i., GKO mice demonstrated a similar phenotypic brain infiltrate when compared to wild-type mice (Wt), however, MHC class II expression on microglia isolated from these GKO mice was significantly lower compared to Wt animals. When IFN-γ producing CD8(+) T cells were reconstituted in GKO mice, MHC class II up-regulation on microglial cells was restored. Taken together, these results suggest that MCMV brain infection results in long-term persistence of antigen-specific CD8(+) T cells which produce IFN-γ and drive chronic microglial cell activation. This response was found to be dependent on IFN-γ production by viral Ag-specific T cells during the chronic phase of disease.

Figure 1. Early leukocyte trafficking into the brain during MCMV encephalitis consists of predominantly macrophages and neutrophils

Age-matched Wt mice were infected with 1.5 × 10⁵ TCID50 of MCMV through the intracerebroventricular (i.c.v) route. Single cell suspensions of brain tissue obtained from MCMV-infected mice (2–4 animals) per time point were banded on a 70% Percoll cushion. Brain leukocytes at the 30–70% Percoll interface were collected, labeled with APC-conjugated Abs specific for CD45 and Cy7-labeled anti-CD11b Abs and analyzed using flow cytometry (Canto, BD, CA), with FlowJo software (TreeStar, Inc.). a. Dot plots shown are representative of 3 experiments at 5 and 30 d p.i.. The CD45(hi)CD11b(hi) population was significantly decreased at 30 d p.i. when compared to 5 d p.i. b. Histograms showing differential expression of Ly6-G and F4-80 among the CD45(hi)CD11b(hi); markers used to identify neutrophils and macrophages, respectively. Gray plot on the histograms denotes isotype Ab binding for each respective MAb, data expressed as mean (±SEM) percentage of cells infiltrating the brain were pooled from three independent experiments. c. Data showing absolute numbers of cells infiltrating the brain including CD45(int)CD11b(+), microglia at each indicated time point were pooled from three independent experiments.

Figure 2. Persistence of T lymphocytes in the MCMV-infected brain

Single cell suspensions of brain tissue obtained from MCMV-infected mice (2–4 animals) per time point were banded on a 70% Percoll cushion. Brain leukocytes at the 30–70% Percoll interface were collected, labeled with APC-conjugated Abs specific for CD45 and Cy7-APC-labeled anti-CD11b Abs, and analyzed using flow cytometry and FlowJo software. a. Dot plots from uninfected control and MCMV-infected animals are shown and are representative of 3–5 experiments at 30 d p.i.. Control mice showed minimal peripheral cell trafficking. b. Representative dot plots showing the percentages of CD4(+) and CD8(+) T lymphocytes in infected brains at 5, 14 and 30 d p.i.. c. Data showing the mean (±SEM) absolute number of cells infiltrating the brain were pooled from 3 independent experiments. PE-labeled anti-CD8(+) and FITC-labeled anti-CD4 Abs were used to determine the total number of CD4(+) and CD8(+) T lymphocytes within the infiltrating CD45(hi) population. Data presented show the mean absolute number of cells in each population, **p < 0.01 versus MCMV-infected Wt.

Figure 3. Antigen-specific CD8(+) T lymphocytes are a major source of IFN-γ at 30 d p.i

Single cell suspensions of brain tissue obtained from MCMV-infected mice (2–4 animals) per time point were banded on a 70% Percoll cushion. Brain leukocytes at the 30–70% Percoll interface were collected. For intracellular IFN-γ staining, brain infiltrating leukocytes (2 × 10⁶ cells/ml) were pulsed with either CD3/CD28 antibodies or with a MCMV-specific IE1 peptide for 5 h at 37°C and cells were treated with Brefeldin A. After incubation, cells were washed in FACS buffer and stained for the surface molecules CD45, CD11b, CD4, CD8 and for intracellular IFN-γ using a Cytofix/Cytoperm kit (BD Pharmingen), before analysis by flow cytometry. a. Representative dot plots showing the percentage of CD8(+) and CD4(+) T lymphocytes producing IFN-γ at 5 and 30 d p.i. in response to αCD3/CD28 and MCMV-IE1 peptide treatment b. Absolute numbers of CD8(+) cells producing IFN-γ were determined within the infiltrating CD45(hi)CD3(+) population. Data showing the mean (±SEM) absolute number of cells infiltrating the brain were pooled from three to five experiments. **p < 0.01 versus MCMV infected Wt mice at 5 and 30 d p.i.. Total RNA extracted from the brain of three to five mice/time point was reverse transcribed and examined for the expression of IFN-γ (c) and MCMV IE1 (d) mRNA by real-time PCR. Mean RNA transcript levels normalized to HPRT expression and presented as fold increase over mock-infected controls from three to five animals per time point.

Figure 4. Persistent, brain infiltrating T lymphocytes express an effector memory phenotype

Single cell suspensions of brain tissue obtained from MCMV-infected mice (2–4 animals) per time point were banded on a 70% Percoll cushion. Cells were stained using MAbs against CD45-APC, CD11b-APC-cy7, CD3-PE-Cy7, CD44-PE, and CD62L-FITC or with CD69-FITC, and the percentages of these respective markers were determined among the CD45(hi)CD11b(int/hi)CD3(+) cells, at indicated time points. Histogram overlays from isotype control (grey, filled) and MCMV-infected mice (solid) are shown. Pooled data indicating percentages of the various cell populations, based on a leukocyte gate, are expressed as mean ± SEM.

Figure 5. Up-regulation of MHC class II expression on resident microglial cells in response to MCMV brain infection

CD45(int)CD11b(+), microglia in single cell suspensions of brain tissue obtained from MCMV-infected mice at the indicated time points were stained with PE-labeled anti-MHC class II Abs and analyzed for expression using flow cytometry. Pooled data indicating the percentage of cells expressing MHC class II, based on a CD45(int)CD11b(+) gate, are presented as mean ± SEM. Histogram overlays from isotype control (grey, filled) and MCMV-infected mice (solid) are shown.

Figure 6. Chronic TNF-α production by activated microglia

CD45(int)CD11b(+), microglia in single cell suspensions of brain tissue obtained from MCMV-infected mice at the indicated time points were stained with FITC-labeled anti-TNF-α Abs and analyzed for expression using flow cytometry. a. Representative dot plots showing the percentage of TNF-α producing CD45(int)CD11b(+), microglial cells at the indicated time points. b. TNF-α expression from total brain homogenate obtained at 5 and 30 d p.i. is shown.

Figure 7. Up-regulation of MHC class II expression on resident microglial cells is IFN-γ-dependent

CD45(int)CD11b(+) microglia in single cell suspensions of brain tissue obtained from MCMV-infected Wt and GKO mice at 30 d p.i. were stained with PE-labeled anti-MHC class II Abs and analyzed for expression using flow cytometry. Pooled data indicating percentages of the MHC class II expressing cells, based on a CD45(int)CD11b(+) gate, are expressed as mean ± SEM. **p < 0.01 versus MCMV infected Wt at 30 d p.i..

Figure 8. Microglial cell activation is restored following adoptive transfer of Wt CD8(+) T-cells in GKO mice

a. MCMV-primed splenocytes and lymph node cells from β-actin-luciferase transgenic BALB/c mice were enriched for CD8(+) T-cells by using negative selection kit as described in the methods. The primed CD8(+) T-cells were adoptively transferred via tail vein injection into MHC-matched recipients, 1 d prior to the infection with MCMV. Dorsal bioluminescence images of recipient mice are shown at 2 h post-transfer (p.t.), 3 d p.i./4 d p.t., 7d p.i./8d p.t., 11 d p.i./12 d p.t., and 17 d p.i./18 d p.t.. b. Signal intensity of the luciferase expression, indicative of the number of immune cells present, was quantified in the brain as photons/sec/cm² at each time point. c. Histogram overlays with pooled data from Wt (Wt - MCMV), GKO (GKO – MCMV), and GKO mice that received CD8(+) T-cells (GKO Ad.-MCMV) are shown for MHC class II up-regulation on CD45(int)CD11b(+), microglial cells at 30 d p.i.. Grey line (filled) represents isotype control and red line (solid) for MHC class II expression.