Organism-Level Analysis of Vaccination Reveals Networks of Protection across Tissues

Abstract

A fundamental challenge in immunology is to decipher the principles governing immune responses at the whole-organism scale. Here, using a comparative infection model, we observe immune signal propagation within and between organs to obtain a dynamic map of immune processes at the organism level. We uncover two inter-organ mechanisms of protective immunity mediated by soluble and cellular factors. First, analyzing ligand-receptor connectivity across tissues reveals that type I IFNs trigger a whole-body antiviral state, protecting the host within hours after skin vaccination. Second, combining parabiosis, single-cell analyses, and gene knockouts, we uncover a multi-organ web of tissue-resident memory T cells that functionally adapt to their environment to stop viral spread across the organism. These results have implications for manipulating tissue-resident memory T cells through vaccination and open up new lines of inquiry for the analysis of immune responses at the organism level.

Keywords: T cell memory; organismal immunology; single-cell analysis; systems biology; vaccines.

Figure 1.. Dynamics of viral spread at the whole-organism scale

(A) Viral genome alignment. Grey lines depict shared sequences. White boxes in the outer circle show genomic regions absent in the other strain. (B-C) Cohorts of mice used to track vaccinating (MVA subcutaneously; s.c.), lethal (WR intranasally; i.n.) and protective (MVA followed by WR) responses (B), and matching weight (C, left) and survival (C, right) measurements. Error bars, SEM (n = 5). (D) Schematic depicting the mouse tissues collected in this study (17 total including blood, not shown). (E) Organismal viral spread for indicated cohorts and times post-infection (top). Circle sizes, normalized expression for viral gene E3L (encoded by MVA and WR). (F-I) Whole-mount tissue imaging of skin (F), draining lymph node (inguinal; G), brain (H), and lung (I) at 2 or 3 day post-infection (d.p.i.) using indicated GFP-expressing virus strains and routes of infection. For skin (F) and dLN (G), insets indicate position of images on the right. For skin (F), MHC class II and nuclei (DAPI) were stained, and arrows indicate MVA-GFP⁺ cells. For brain (H) and lung (I), shown are tiled images (left), and representative sections (right). Autofluo., autofluorescence. See also Figure S1 and Table S1.

Figure 2.. Whole-tissue gene expression reveals local and systemic immune responses

(A-C) Principal component analysis (PCA) of whole-tissue mRNA profiles for 9 tissues (521 samples; A), skin (B), and liver (C). Colors, tissues (A) and cohorts (B-C); symbols, time after infection; axes, percentage of variance; lymph node, inguinal draining LN. (D) Heatmaps of differentially expressed genes (numbers in parentheses on top) from whole-tissue mRNA profiles ordered by hierarchical clustering (Pearson’s correlation) and tissue type. Shown are log2 fold-change values relative to matching, uninfected tissues (FDR-adjusted p-value < 0.05, absolute fold change > 2, n = 4). (E-G) Normalized read counts for indicated genes, cohorts and tissues. Error bars, SEM (n = 4). See also Figure S2 and Table S2.

Figure 3.. Analysis of ligand-receptor connectivity across tissues reveals a whole-body antiviral state

(A) Bar graphs showing numbers of differentially expressed (DE) genes (top) and RNA levels of Vaccinia virus gene E3L (qPCR; bottom). Error bars, SD. (B) Schematic overview of the analysis for ligand-receptor pair connectivity across tissues. Known ligand-receptor pairs were extracted from whole-tissue mRNA profiles and their potential links within and between tissues visualized using a circos plot. (C) Bar graph showing the numbers of ligand (L)-receptor (R) pairs emanating from indicated tissues upon MVA immunization (s.c., subcutaneous). (D) Inter-organ connectivity of ligand-receptor pairs at indicated times after MVA immunization. Line color, tissue source for ligands; line thickness, number of ligand-receptor pairs. (E) Bar graphs showing fold changes between MVA-infected and control tissues for Ifnb1 and two ISGs: Ifit3 and Ifitm3 (qPCR). Error bars, SD (n = 4). (F) Heatmap of all interferon-stimulated genes (ISGs) regulated across tissues upon skin MVA vaccination. Shown are log2 fold-change values relative to matching, uninfected tissues (FDR-adjusted p-value < 0.05 and absolute fold change > 2, n = 4). (G-H) Survival analysis of wild-type (WT), anti-IFNAR1 or isotype antibody-treated, and Ifnar1^−/− mice immunized subcutaneously with MVA at 1 (G) or 7 (H) days prior to intranasal WR challenge. Data are representative of three independent experiments. See also Figure S3 and Table S3.

Figure 4.. Protective memory responses to a respiratory viral challenge induce expression changes in lung, liver and spleen

(A-B) Cohorts of mice used to track memory protective responses using MVA immunization at skin (ear or flank) followed by intranasal WR challenge at day 21 (A), and matching weight and survival measurements (B). Error bars, SEM (n = 4). (C) Dot plots showing log2 fold-change in gene expression (Y axis) in tissues collected at 1.5 day post-WR challenge (on day 21 or 80 after MVA) relative to uninfected controls against log2 average expression (X axis) (n = 4). Red dots, genes with FDR < 0.05 and absolute log2 fold change > 1. (D-E) Heatmaps showing differentially expressed genes in lung, liver, and spleen at indicated days (d) after intranasal WR challenge of wild-type (D-E) and/or Tcra^−/− (E) mice immunized with MVA at flank skin 21 days earlier (FDR-adjusted p-value < 0.05 and absolute fold change > 2, n = 4). Shown are log2 fold-change values relative to matching, uninfected tissues (D), and normalized read counts scaled per row (4 replicates/condition; E). Day 0, mice immunized only. Interferon-stimulated genes (ISGs) are indicated in black on the right. See also Figure S4 and Table S4.

Figure 5.. Skin vaccination seeds tissue-resident memory CD8⁺ T cells in multiple distant tissues to confer host protection

(A-B) Relative viral DNA amount measured by qPCR in the brain (day 2 post-WR; A) and weight measurements (B) of wild-type (WT) and T-cell deficient (Tcra^−/−) mice immunized subcutaneously with MVA three weeks prior to intranasal WR challenge. Arbitrary units, a.u. (WT values set to 1) (A). Error bars, SD (n = 5) in A, and SEM (n = 7) in B. *; Student’s t-test p-value < 0.05. (C-E) Flow cytometry analysis of Vaccinia virus peptide B8R-specific (H2-K^b B8R_20–27) memory CD8α⁺ T cells (gated on live CD3ε⁺CD44⁺CD62L⁻ cells) from indicated tissues from mice vaccinated at skin with MVA 3 weeks (C), 3 months (E, left) or 15 months (E, right) earlier, and quantifications in percentage of parent gate (%) and absolute count per 10⁵ live cells (cells) (D). Error bars, SD (n = 5). (F) Flow cytometry analysis of vascular and parenchymal B8R-specific memory CD8α⁺ T cells (gated on live CD3ε⁺CD44⁺CD62L⁻ cells) by intravascular staining with CD45 (CD45 i.v.). Bar graphs show quantifications in percentage of parent gate (%) and absolute count per 10⁵ live cells (cells). Error bars, SD (n = 3). *; Student’s t-test p-value < 0.05. (G-H) Timed parabiosis experiments show the role of tissue-resident cells in host protection. One parabiont (CD45.2) was immunized at skin with MVA at indicated days before (−28, −14, −1) or after (+14) joining with the other parabiont (CD45.1) (G). For groups −28, −14 and −1, mice were joined for 2 weeks before splitting, whereas for group +14, mice were joined for 2 weeks before immunization of one parabiont and split after another 2 weeks. Weight measurements of immunized (dark grey) and naïve (light grey) parabionts after WR challenge are shown in panel H. Data are representative of two to three independent experiments. Error bars, SEM (n = 5). *; Student’s t-test p-value < 0.05. See also Figure S5.

Figure 6.. Tissue-resident memory CD8⁺ T cells are activated in lung and liver as virus spreads

(A) Experimental workflow: mice were immunized with MVA at skin 21 days prior to intranasal WR challenge (or PBS as control) for 0.5 day, and single lung and liver memory CD8⁺ T cells (live CD3ε⁺CD44⁺CD62L⁻ cells), including virus-specific cells were sorted by FACS prior to single-cell (sc) RNA-seq. (B) Heatmap of 1,476 single memory CD8⁺ T cells (columns) from lung and liver showing the top 100 differentially expressed genes (rows) between control (PBS only) and WR-challenged mice in each tissue type (FDR < 0.01, expression fold change > 3). (C) Heatmap of 20 single virus-specific (H2-K^b B8R_20–27) CD8⁺ T cells (columns) showing the top 25 differentially expressed genes (rows) between cells from lungs of mice challenged with B8R_20–27 peptide (20 μg) or saline as control (FDR < 0.01, expression fold change > 3). UMI, unique molecular identifiers. (D-E) Impact of WR challenge on single CD8⁺ memory T cell states. Visualization of single cells from lung (top) and liver (bottom) from control (MVA vaccination only) and WR-challenged mice using t-SNE (D). t-SNE plots from D are shown in E and colored based on the B8R activation score (scaled average expression of genes differentially expressed in both WR (B) and peptide B8R_20–27 (C) challenges). (F-G) Tissue-resident memory CD8⁺ T cells (T_RM) are activated in both lung and liver upon intranasal WR challenge. t-SNE plots from D are shown in F and colored based on the T_RM cell score (scaled average expression of genes associated with the T_RM phenotype). Blue dots indicate a T_RM score > mean T_RM score of all cells + 1 SD. In panel G are shown the distributions of B8R activation scores (E) in T_RM cells (F). (H) T_RM scores for indicated CD8⁺ T cell populations sorted from lung after intravascular (IV) immunostaining. Error bars, SD (n = 4). *; Student’s t-test p-value < 0.05. See also Figure S6 and Table S5.

Figure 7.. Local tissue environments shape the functional abilities of memory CD8⁺ T cells to bolster organ-specific responses

(A) Heatmap of 1,476 single memory CD8⁺ T cells (columns) showing the top 40 differentially expressed genes (rows) between lung and liver of control (MVA vaccination only) and WR-challenged mice (FDR < 0.01, expression fold change > 3). (B) Visualization of single cells from lung and liver in MVA-vaccinated mice (Control) using t-SNE. Vaccinia virus peptide B8R-specific (H2-K^b B8R_20–27) CD8⁺ T cells (Tetramer⁺) are labeled in the bottom plot for lung (25.4%, 94/370 cells) and liver (25.3%, 93/368 cells). (C) Expression levels of tissue-specific genes in single cells. t-SNE plot from B colored based on expression levels (UMI, unique molecular identifiers) of indicated genes. (D) Illustration of the impact of tissue-adapted memory CD8⁺ T cells on their respective tissue of residence. From left to right, vaccination at skin seeds memory CD8⁺ T cells in lung and liver, which secrete factors controlling tissue responses upon WR challenge. (E) Secreted factor induction in lung and liver at day 0.5 after WR challenge of MVA-vaccinated mice. Fold change values were calculated using single-cell RNA-seq profiles from WR-challenged versus vaccinated only mice as control. (F-G) Tissue-level expression changes for target genes of secreted factors produced in memory CD8⁺ T cells. Mice were immunized at skin with MVA and challenged 3 weeks later with WR intranasally for 1.5 days. Shown are log2 fold-change values of knockout (KO) relative to wild-type (WT) mice for selected (F) and all differentially expressed genes (G) (FDR-adjusted p-value < 0.05, absolute fold change > 2, n = 4). (H) Schematic depicting the inter-organ mechanisms of protection reported in this study. Upon skin MVA vaccination, type I IFNs produced locally trigger a whole-body antiviral state within hours (i; left), and tissue-resident memory CD8⁺ T cells (T_RM) seeded in distant tissues help block viral spread (ii; right). See also Figure S7 and Table S6.