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. 2017 Mar 29:8:335.
doi: 10.3389/fimmu.2017.00335. eCollection 2017.

CD103+ CD8 T Cells in the Toxoplasma-Infected Brain Exhibit a Tissue-Resident Memory Transcriptional Profile

Tyler A Landrith  1 , Suhas Sureshchandra  1 , Andrea Rivera  1 , Jessica C Jang  1 , Maham Rais  1 , Meera G Nair  1 , Ilhem Messaoudi  1 , Emma H Wilson  1
Affiliations

CD103+ CD8 T Cells in the Toxoplasma-Infected Brain Exhibit a Tissue-Resident Memory Transcriptional Profile

Tyler A Landrith et al. Front Immunol. .

Abstract

During chronic infection, memory T cells acquire a unique phenotype and become dependent on different survival signals than those needed for memory T cells generated during an acute infection. The distinction between the role of effector and memory T cells in an environment of persistent antigen remains unclear. Here, in the context of chronic Toxoplasma gondii infection, we demonstrate that a population of CD8 T cells exhibiting a tissue-resident memory (TRM) phenotype accumulates within the brain. We show that this population is distributed throughout the brain in both parenchymal and extraparenchymal spaces. Furthermore, this population is transcriptionally distinct and exhibits a transcriptional signature consistent with the TRM observed in acute viral infections. Finally, we establish that the CD103+ TRM population has an intrinsic capacity to produce both IFN-γ and TNF-α, cytokines critical for parasite control within the central nervous system (CNS). The contribution of this population to pro-inflammatory cytokine production suggests an important role for TRM in protective and ongoing immune responses in the infected CNS. Accession number: GSE95105.

Keywords: CD103; CD8+ T cell memory; Toxoplasma gondii; chronic infection; neuroimmunology; tissue-resident memory cells.

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Figures

Figure 1
Figure 1
Kinetics and specificity of CD103+ CD8 T cells in the Toxoplasma-infected brain. Brain mononuclear cells harvested from Toxoplasma gondii-infected mice at indicated time points and gated on live CD3+ CD8+ cells. (A) Numbers indicate the proportion of CD69+ CD103+ and CD69+ CD103− CD8 T cells in the brain at the indicated time points. (B) Absolute cell counts for the data are shown in panel (A). (C) Percentage of dextramer+ CD8 T cells in the brain. (D) Percentage of SIINFEKL dextramer+ CD8 T cells expressing CD103. Data from week 12 postinfection. Numbers indicate average ± SEM of three biological replicates for each time point. Significance was determined using two-way ANOVA with multiple comparisons post test. ****p < 0.0001. Data are representative of two independent experiments with similar results.
Figure 2
Figure 2
Distribution of CD103+ CD8 T cells in the Toxoplasma gondii-infected brain. Immunofluorescent staining in situ for CD8 and CD103 in representative sagittal section-infected brain at 5 weeks postinfection. (A) E-cadherin staining in choroid plexus of lateral ventricle, 40× magnification, zoomed in. (B) Laminin staining for vasculature of frontal cortex, 40× magnification, zoomed in. (C) T. gondii staining in the frontal cortex, 25× magnification, zoomed in. All images are representative of n = 3 biological replicates at 5 weeks post infection. White arrows indicate examples of staining positive for both CD8 and CD103. Scale bar indicates 20 µm.
Figure 3
Figure 3
Transcriptional characteristics of brain CD103+ CD8 T cells. CD8 T cells were isolated from the brain and spleen of chronically infected mice and sorted according to CD103 expression. (A) Differentially expressed genes (DEGs) for the three indicated analyses. Numbers indicate combined upregulated and downregulated DEGs with fold change >2 and FDR <5%. (B) PCA plot for normalized read counts (reads per kilobase of transcript per million mapped reads) for all samples.
Figure 4
Figure 4
Distinct transcriptional profile of brain CD103+ CD8 T cells relative to brain CD103− CD8 T cells. (A) After conducting differential gene expression analysis between brain CD103+ and brain CD103− CD8 T cells, the genes with a fold change >2, FDR <5%, and mean RPKM >1 were input into MetaCore for functional enrichment. Of the 239 genes input, the following terms with FDR <5% were extracted from the categories “Disease by Biomarker” and “GO Process.” (B) Heatmap of genes under GO process term “Positive regulation of cell migration.” (C) Heatmap of genes under Disease term “RNA virus.” (D) Heatmap of genes under enrichment term “Arteriosclerosis.” Individual replicates in heatmap are pooled from n = 5 mice. Values in legend are scaled values representative of RPKM. Red indicates a highly expressed gene, and blue indicates a gene with a low expression value. (E) Table of fold changes and false discovery rate for genes relevant to tissue residence and memory phenotype. (F) Flow cytometry for differential expression of S1PR1 and KLRG1.
Figure 5
Figure 5
Comparison of tissue resident memory T cells (TRM) from acute vesicular stomatitis virus (VSV) to Toxoplasma gondii. Microarray data from the study by Wakim et al. were obtained and analyzed via GEO. (A) Venn diagram of DEGs in T. gondii and VSV (25) for the brain CD103+ CD8 T cells relative to brain CD103− CD8 T cells. (B) Comparison of fold changes for the 27 differentially expressed genes common to both models. RNA-Seq DEGs were determined according to the following criteria: fold change >2, FDR <5%, p < 0.05, and mean RPKM >1. Microarray DEGs were significant if the p value and adjusted p value were less than 5%. (C) Results of enrichment analysis for DEGs unique to microarray analysis of TRM in VSV. (D) Results of enrichment analysis for DEGs unique to RNA-Seq analysis of TRM in T. gondii infection.
Figure 6
Figure 6
Production of pro-inflammatory cytokines in CD103+ CD8 T cells. Brain mononuclear cells from chronically infected mice were restimulated with α-CD3/α-CD28 antibodies for 6 hours and intracellular cytokine staining was performed. Data were gated on live CD3+ CD8 T cells and then split into CD103+ and CD103– subsets for subsequent analysis. (A) Representative flow plot indicating percentage of restimulated CD103+ and CD103– CD8 T cells producing IFN-γ and TNF-α. Numbers indicate mean percentage ± SEM of n = 5 biological replicates. (B) Paired data plot of the data shown in (A). (C) Geometric MFI of IFN-γ production comparing CD103+ to CD103– CD8 T cells subsets. (D) Geometric MFI of TNF-α production for the same comparison. Significance was determined using a paired two-sample t-test. *p < 0.05, **p < 0.01; p values are one tailed. Data are representative of two independent experiments with similar results.
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