Expeditious recruitment of circulating memory CD8 T cells to the liver facilitates control of malaria

Abstract

Circulating memory CD8 T cell trafficking and protective capacity during liver-stage malaria infection remains undefined. We find that effector memory CD8 T cells (Tem) infiltrate the liver within 6 hours after malarial or bacterial infections and mediate pathogen clearance. Tem recruitment coincides with rapid transcriptional upregulation of inflammatory genes in Plasmodium-infected livers. Recruitment requires CD8 T cell-intrinsic LFA-1 expression and the presence of liver phagocytes. Rapid Tem liver infiltration is distinct from recruitment to other non-lymphoid tissues in that it occurs both in the absence of liver tissue resident memory "sensing-and-alarm" function and ∼42 hours earlier than in lung infection by influenza virus. These data demonstrate relevance for Tem in protection against malaria and provide generalizable mechanistic insights germane to control of liver infections.

Keywords: CD8 T cells; liver-stage immunity; malaria.

Figure 1.. Accumulation of liver Tem after sporozoite challenge of RAS-vaccinated mice

(A) Experimental schematic for (B)–(F). CB6F1 mice received intravenous (i.v.) vaccinations with 10⁴ PbANKA (Pb) radiation attenuated sporozoites (RAS) 28 days apart. Mice were challenged with 2 × 10⁴ virulent Pb sporozoites 56 or 70 days after the first RAS vaccination. Blood, spleens, and livers were collected for T cell analysis via flow cytometry prior to or 12- and 24-h post-infection (hpi). (B) Numbers of CS₂₅₂-specific Tem cells in the blood (per mL) and spleen of RAS-vaccinated mice 1 month after 2^nd RAS dose. Tem cells are defined as live singlets that bind to the CS₂₅₂ tetramer and are CD8α⁺CD11a^hiCD62L⁻CD69⁻. (C) Representative flow cytometry (gated on live CD8α⁺CD11a^hi singlet events) depicting CS²⁵²-specific (left) Tem and Trm phenotype populations (right) in the liver of RAS-vaccinated mice 56 days after the first RAS vaccination. Tetramer staining identifies CS₂₅₂-specific cells. Numbers represent frequencies. (D) Pairwise comparison of CS₂₅₂-specific Trm and Tem phenotype cells recovered from the livers of vaccinated mice in B). Liver Trm cells are defined as CD8α⁺CD11a^hiCD62L⁻CD69⁺. (E) Representative flow cytometry (gated on live CD8α⁺CD11a^hi singlet events) depicting CS₂₅₂-specific (left) Tem and Trm phenotype populations (right) in the liver of mice 12 hpi with virulent Pb sporozoites. Numbers represent frequencies. (F) Frequencies and numbers of CS₂₅₂-specific Tem phenotype cells recovered from livers prior to or 12 and 24 hpi with Pb sporozoites. (G) Unvaccinated (infected control) and RAS-vaccinated (as in 1A) mice were challenged with 10⁴ virulent Pb spz. RNA was extracted from the liver hepatocyte fractions at 12, 24, and 44 hpi. Pb 18S ribosomal RNA transcripts were quantified via real-time qRT-PCR and normalized to mouse GAPDH transcripts. All data are combined from two independent experiments, each with at least 3 mice per group. Symbols represent individual mice and mean ± SEM are depicted. (D) Paired two-tailed t test was performed assuming similar standard deviation (SD). (F) One-way ANOVA (Tukey post hoc test) comparing the mean of each column to the mean of every other column was performed. (G) Unpaired two-tailed t tests assuming similar SD were performed between RAS- and un-vaccinated groups at each time point. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns, p > 0.05.

Figure 2.. Liver infection drives Tem infiltration of the liver

(A, E, and G) Experimental schematics. CS and/or OT-I TCR-retrogenic (Rtg) memory cells were enriched from the spleens of DC-Lm-vaccinated donor mice (see Figure S2) via a CD8α⁺ T cell enrichment and adoptively transferred (AT) i.v. (5 × 10⁵ cells of one or both types) into either RAS-vaccinated mice that received 1 dose of 10⁴ RAS 1–2 months prior to rechallenge or unvaccinated mice. Mice receiving Rtg cells remained uninfected or were challenged with Pb sporozoites. Livers were harvested at either 6 or 12 hpi. (B) Quantification of Rtg Tem cell numbers and frequencies in livers of RAS-vaccinated mice that were uninfected or 12 hpi with 3 × 3 10⁴ virulent Pb sporozoites. Rtg Tem cells are defined by an expression profile of CD8α⁺CD11a^hiCD45.1⁺ CD62L⁻. CS₂₅₂-Rtg (CS-R) cells expressing eGFP are detected in the FITC channel, whereas OT-I-Rtg (OT-I-R) cells expressing mCherry are detected in the PE-TxRed channel. (C and D) Quantification of liver-localized CS-R and OT-I Tem (C) and endogenous (D) CS₂₅₂-specific Tem cells at 6 hpi with Pb sporozoites in RAS-vaccinated mice. (E–I) Quantification of liver-localized CS-R and/or OT-I-R Tem cells in unvaccinated mice 6 h postinoculation with either Pb sporozoites (E and F), 10⁶ recombinant virulent Lm expressing secreted OVA peptide (G and H), or cecal ligation and puncture surgery (G and I). All data are combined from 2 independent experiments, each with 2–5 mice per group. Symbols represent individual mice and mean ± SEM are depicted. (B, C, and F) One-way ANOVA (Tukey post hoc test) comparing the mean of each column to the mean of every other column was performed. (D, H, and I) Unpaired two-tailed t tests assuming similar SD were performed. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns p > 0.05.

Figure 3.. Rapid recruitment of Tem is unique to the liver

(A) Experimental schematic. Mice 5 weeks post-recovery from influenza A (IAV) X31 (H3N2) infection were injected with memory CS-R and OT-I-R Tem cells and treated by intranasal (in) infusion of PBS or IAV PR8-OVA (H1N1). Mice were injected i.v. with labeled anti-CD45.2 antibody prior to tissue harvest to distinguish vascular from parenchymal T cells. Lungs were harvested for T cell analysis 6–48 hpi. (B) Representative flow cytometry showing how TCR-Rtg cells were categorized as IV⁺ (intravascular) or IV⁻ (extravascular). (C) Quantification of lung-localized extravascular (IV⁻) TCR-Rtg Tem cells from mice at various time points after PR8-OVA infection. (D) Quantification of lung-localized intravascular (IV⁺) TCR-Rtg Tem cells from mice at various time points after PR8-OVA infection. (E) Normalized comparison of Rtg Tem accumulation over time in the liver and lungs after infection with their respective pathogens. All data are combined from 2 independent experiments, each with 3–4 mice per group. Symbols represent individual mice and mean ± SEM is depicted. (C and D) One-way ANOVA (Tukey post hoc test) comparing the mean of each column to the mean of every other column was performed. (E) Unpaired two-tailed t tests assuming similar SD comparing uninfected and 6 hpi groups were performed. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns, p > 0.05.

Figure 4.. Liver-stage malaria infection rapidly triggers an inflammatory state in the liver

(A) Experimental schematic. Age matched female naive and RAS-vaccinated (one dose of 10⁴ RAS i.v. 28 days before sporozoite challenge) CB6F1 mice were unchallenged or received 3 × 10⁴ virulent Pb sporozoites. Livers were harvested at 6 hpi for RNA-seq. (B) Heatmap displaying the top 60 differentially expressed genes (defined by adjusted p value) across the groups. Genes are sorted based on hierarchical clustering. (C) Volcano plots comparing individual gene expression differences between various groups. Genes with a log2 fold change between −2 and 2 are excluded. Genes of particular interest are labeled. Dashed lines indicate thresholds of significance for log2 fold change (x axis, ±2) and adjusted p value (y axis, <0.05). (D–F) Enrichment plots and associated heatmaps for gene pathways previously defined by the Gene Ontology database. Data are from a single experiment with 3 biological replicates per group. Expression values were obtained via analysis with DESeq2, R, and edgeR. Thresholds for significance: log2 fold change >2 and <−2; normalized p value <0.05.

Figure 5.. Mechanisms underpinning rapid Tem recruitment to the liver

(A) Experimental schematic. Naive CD45.2 mice received 5 × 10⁵ CD45.1⁺eGFP⁺ memory CS-R Tem cells via adoptive transfer, and some recipients were challenged with Pb sporozoites. Some mice also received various treatments including blocking antibodies/drugs (α-LFA-1 and α-IFNγ) and depleting antibodies/drugs (clodronate liposomes, α-NK1.1, α-CD41). Isotype control antibodies were delivered at the same time as the corresponding experimental antibody. (B–F) Quantification of CS-R Tem cells 12 (D) or 6 (B, C, E, and F) hpi with 3 × 10⁴ Pb sporozoites. Treatment doses, route of administration, and treatment administration time: α-LFA-1,300 μg i.v. 3 h prior; clodronate liposomes, 200 μL i.v. 1 day prior; α-IFNγ, 1 mg intraperitoneally (i.p.) 1 day prior; α-NK1.1, 300 μg and 100 μg i.v. 2 and 1 days prior, respectively; and α-CD41, 500 μg i.v. 3 h prior. Experiments were performed in previously naive CB6F1 mice, except for (D), in which CS-R cells were transferred into RAS-vaccinated mice. All data are combined from 2–3 independent experiments, each with 2–5 mice per group. Symbols represent individual mice and mean ± SEM is depicted. (B–F) One-way ANOVA (Tukey post hoc test) comparing the mean of each column to the mean of every other column was performed. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns, p > 0.05.

Figure 6.. Tem cells mediate immunity to liver infection in a recruitment-dependent fashion

(A, D, and K) Experimental schematics for protection against virulent Pb sporozoites. 10⁴ live sporozoites were injected i.v. into mice. Liver parasite burden was measured 44 hpi. (B) Quantification of liver localized CS₂₅₂-specific liver Trm and Tem cells in DC-Lm-CS₂₅₂-vaccinated mice 2 days after the 2^nd α-CXCR3 dose. (C) Normalized parasite burden showing degree of liver sporozoite infection 44 hpi in naive or isotype control and α-CXCR3-treated DC-Lm-CS₂₅₂-vaccinated mice. (E) Representative flow from separated parabiont mice demonstrating that antigen-specific circulating memory CD8 T cell populations equilibrate in the liver, whereas liver Trm compartments do not equilibrate. Top row: DC-Lm-CS₂₅₂-vaccinated parabiont; bottom row: DC-Lm-GAP50₄₁-vaccinated parabiont. (F) Mice that received either DC-Lm-CS₂₅₂ or DC-Lm-GAP50₄₁ vaccinations were joined via parabiosis. Mice were surgically separated 14 days after parabiosis surgery. 14 days post separation, tissues were harvested for quantification of liver localized CS₂₅₂-specific liver Trm and Tem cells. (G) Normalized parasite burden showing degree of liver sporozoite infection 44 hpi in mice that were DC-Lm-GAP50₄₁ immunized or CS_252⁻ and GAP50_41⁻-vaccinated parabionts. (H) Experimental schematic for protection against virulent Lm-OVA. 5 × 10⁵ OT-I Tem cells were isolated from the spleens of DC-Lm-OVA vaccinated C57BL/6 mice and adoptively transferred into naive recipient mice. (I and J) Quantification of virulent Lm-OVA colony-forming unit (CFU) recovered from the livers and spleens of infected mice 48 hpi. Horizontal dashed lines indicate the limit of detection. (L and M) Parasite burden showing degree of liver sporozoite infection 44 hpi in DC-Lm-CS₂₅₂ and DC-Lm-GAP50₄₁-vaccinated mice that were pre-treated with various antibodies. All data are combined from 2 independent experiments, each with 2–5 mice per group. Symbols representing individual mice and mean ± SEM are depicted. (C, G, I, J, and L) One-way ANOVA (Tukey post hoc test) comparing the mean of each column to the mean of every other column was performed. (B, F, and M) Unpaired two-tailed t tests assuming similar SD were performed. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns, p > 0.05.