Mice with FVB-derived sequence on chromosome 17 succumb to disseminated virus infection due to aberrant NK cell and T cell responses

Abstract

Zoonotic arenavirus infections can result in viral hemorrhagic disease, characterized by platelet loss, petechia, and multi-organ injury. The mechanisms governing these outcomes are likely impacted by virus strain and infection dose, as well as an individual's genetic background and immune constitution. To better understand the processes leading to severe pathogenesis, we compared two strains of inbred mice, C57BL/6J (B6) and FVB/NJ (FVB), that have diametrically opposed outcomes during disseminated lymphocytic choriomeningitis virus (LCMV) infection. Infection caused minimal pathogenesis in B6 mice, whereas FVB mice developed acute hepatitis and perished due, in part, to aberrant NK cell and T cell responses. Susceptible mice showed an outgrowth of cytolytic CD4⁺ T cells and loss of Treg cells. B6 congenic mice with the FVB allele at a 25Mb locus on chromosome 17 recapitulated FVB pathogenesis upon infection. A locus containing a limited number of variants in immune-related genes greatly impacts survival during infection.

Keywords: Cell biology; Components of the immune system; Genetics; Immunology; Virology.

Graphical abstract

Figure 1

FVB:Cl13 pathogenesis is heritable and affects multiple organs FVB, B6, and F1 (B6xFVB) mice were challenged with LCMV-Clone13 and monitored for physical signs of illness across time or assessed at day 6 post-infection for viral burden, liver histology, and blood Alanine Aminotransferase (ALT) activity. (A) The frequency of survival across time. (B) Body weight changes across time, including for mice surviving from day 8 onwards. (C) Body temperature changes based on rectal measurements, including for mice surviving past day 8. (D-I) Cohorts of infected mice were necropsied at day 6 post-infection. (D) Platelet counts as determined by complete blood cell count. Average levels in uninfected mice represented by horizontal gray line. (E) Viral burden in tissues based on plaque assay. Horizontal gray line marks the limit of detection. (F) Viral burden in sera. (G) H&E stains of spleen, liver, and lung sections. Black scale bar = 700 μm, white bar = 200 μm. (H) Whole spleen weight. (I) Alanine aminotransferase activity in sera. Results are combined from multiple experiments with 6–25 mix-sex mice per group per timepoint. Horizonal bars represent mean (D–F, H and I). Data are represented as mean ± SEM (B and C). Statistical analyses included Kaplan-Meier (A) and unpaired one-way ANOVA (B–F, H and I) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). The Grub’s outlier test was used to identify outliers in panel (H) and one data point was excluded (significant outlier, p < 0.05).

Figure 2

Spleen and liver damage in FVB mice begins early during Cl13 infection Mixed sex B6 and FVB mice and male F1 mice were infected with LCMV-Cl13 and evaluated 0, 2, 3, 5, and 6-day post infection for viral burden, blood ALT activity, spleen and liver histology and cleaved-caspase-3 immunohistochemistry. (A) Viral burden in spleen and liver at 2 and 3 dpi. Two to eight mice per group. (B) Daily alanine aminotransferase activity in sera. Three to twelve mice per strain per time point. (C) H&E liver histology at 0, 3, and 6 dpi. Black arrows highlight examples of fatty degeneration, with zoomed-in examples in black insets. White arrows represent examples of apoptotic bodies, with zoomed-in examples in white insets. (D) H&E spleen histology 0, 3, and 6 dpi. White circled area highlights early lesion formation. Examples of the varying cellular composition in zoomed-in insets. (E) Cleaved caspase-3 staining of livers at 0, 3, and 6 dpi. (F) Cleaved caspase-3 staining of spleens 0, 3, and 6dpi. Black outline marks concentrated areas of staining within marginal zone. All scale bars are 200 μm. Horizonal bars represent mean (A). Data are represented as mean ± SEM (B). Data are pooled from multiple independent experiments. Statistical analysis used unpaired one-way ANOVA (A and B) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 3

CD8⁺ T cell populations vary by tissue in Cl13-infected FVB mice B6, FVB, and F1 mice were challenged with LCMV-Cl13. At day 6 post-infection, spleen and liver cells were isolated and CD8⁺ T cells were analyzed by flow cytometry. (A) Representative flow plots and total cell count of CD8⁺CD44⁺ cells in spleen. (B) Representative flow plots and total cell count of CD8⁺CD44⁺ cells in liver. (C) Representative flow plots and total cell count of CD8⁺CD44⁺GrzB⁺ cells in spleen. (D) Representative flow plots and total cell count of CD8⁺CD44⁺GrzB⁺ cells in liver. (E and F) Single cell leukocyte preparations from the spleens and livers of B6 and FVB mice were stimulated ex vivo with anti-CD3/CD28, GP₃₃/NP₁₁₈ peptides, or were left unstimulated. Samples then stained for surface CD107a and CD107b and analyzed by flow cytometry. (E) Representative flow plots and total frequency of CD107a/b⁺CD8⁺ T cells from spleen. (F) Representative flow plots and total frequency of CD107a/b⁺CD8⁺ T cells from liver. Combined results of multiple independent experiments with 3–14 mice per group; all F1 mice were male, B6 and FVB mice were mixed sex. All flow cytometry data were gated on viability dye-negative, singlet cells. Horizontal bars represent mean. Statistical analysis used unpaired one-way ANOVA (A–D) and student’s t-tests (E and F) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 4

Cl13-infected FVB mice generate a robust granzyme-B+ CD4⁺ T population and lose Treg cells Leukocytes were isolated from the spleens and livers of Cl13-infected B6, FVB, and F1 mice at 6 dpi and analyzed for their expression of granzyme-B, capacity to degranulate, or their expression of Foxp3. (A–D) Representative flow plots and total cell count for:(A) CD4⁺CD44^hi T cells in spleen, (B) CD4⁺CD44^hi T cells in liver, (C) Granzyme-B⁺ (GrzB) cells among CD4⁺CD44⁺ cells in spleen, (D) GrzB⁺ cells among CD4⁺CD44⁺ cells in liver. (E and F) Single cell suspensions from spleen or liver of B6 or FVB mice were stimulated ex vivo with anti-CD3/CD28 or left unstimulated. Samples then stained for surface CD107a and CD107b and analyzed by flow cytometry. (E) Representative flow plots and total frequency of CD107a⁺CD107b⁺ CD4⁺ cells in spleen. (F) Representative flow plots and total frequency of CD107a⁺CD107b⁺ CD4⁺ cells in and liver. (G) Representative flow plots and total frequency of Foxp3⁺ CD4⁺ T cells in the spleens of uninfected B6 (n = 4) and FVB (n = 5) mice and day 6-infected B6 (n = 6) and FVB (n = 6) mice. (H) Representative flow plots and total frequency of Foxp3⁺ CD4⁺ T cells in livers of uninfected B6 (n = 2) and FVB (n = 2) mice and infected B6 (n = 6) and FVB (n = 6) mice. All F1 mice were male and B6 and FVB mice were mixed sex. Results are combined from multiple independent experiments with a total of 3–12 mice per strain. All flow cytometry data were gated on live, viability dye-negative cells. Horizontal bars represent mean. Statistical analyses included unpaired one-way ANOVA (A–D) and Student’s t test (E–H) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 5

T cells drive pathology and lethality in Cl13-infected FVB mice Cohorts of mixed-sex FVB mice were given depleting antibodies to remove CD4⁺ T cells or CD8⁺ T cells, or were given non-depleting isotype control antibody prior to LCMV-Cl13 challenge. The mice were assessed over time for physical signs of illness to criterion endpoints. Other mice were necropsied at day 6 and assessed for blood ALT activity and platelet counts, and spleen and liver histology. (A) FVB mice were depleted of CD4⁺ T cells (n = 9), CD8⁺ T cells (n = 9), or given isotype-control antibody (n = 10) prior to Cl13 challenge and were monitored for survival. (B) ALT in sera of CD4-depleted (n = 7), CD8-depleted (n = 4), or isotype-treated (n = 6) mice. (C) Platelet levels of CD4-depleted (n = 8), CD8-depleted (n = 4), or isotype-treated (n = 6) mice. (D) Representative histology of spleen and liver of mice with isotype or depleting antibody treatment. Each image is from a different mouse. White scale bar represents 200 μm. White boxed insets provide zoomed-in examples of varying cellular composition. All flow gated on live, viability dye-negative cells. Horizontal bars represent mean. Results from independent experiments were combined. Statistical analyses were Kaplan Meier (A) and unpaired one-way ANOVA (B and C) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 6

Pathogenesis in FVB mice is linked to a 25Mb locus on chromosome 17 Forward genetics was used to identify the FVB genetics driving pathogenesis after infection. At the N2 generation, mice were challenged and monitored for physical signs of illness. QTL mapping implicated Chr17. Other N2 mice with the FVB allele(s) at Chr17 were selected for further crosses to B6/J mice and then intercrossed to generate G6 mice that were homozygous at Chr17. (A) Illustration of the breeding scheme showing allele inheritance by generation. (B) Survival rate of Cl13-infected N2 mice (n = 63) compared to B6 (n = 12), FVB (n = 12), and F1 (n = 26) mice. (C) The frequency of the FVB allele at chromosome 17 (Chr17) in the 63 N2 mice that did or did not survive post infection. (D) Missense coding variants along a 25Mb locus at FVB Chr17. Below is an illustration of two lines of FVB-derived G6 mice with different recombination patterns within the original 25Mb Chr17-QTL. Each line is represented as either a light brown (G6_24) or dark reddish-brown bar (G6_20). Black portions of the bars represent sequence analogous to B6. (E) Survival of G6_20 (n = 19), G6_24 (n = 17), G6_25 (n = 9), FVB (n = 7), and B6 (n = 6) mice post Cl13 infection. (F) ALT at time of mandatory euthanasia (between 5 and 13 dpi) for G6_20 (n = 9), G6_24 (n = 10), and G6_25 (n = 7) compared to B6 (n = 15) and FVB (n = 9) mice. (G) Total platelet count of B6 (n = 5), FVB (n = 12), and G6_24 (n = 7) mice at day 6 dpi. (H) IFN-I levels in sera 24 h post-infection of B6 (n = 5), FVB (n = 7), and G6_24 (n = 7) mice. Horizontal bars represent mean. (F–H) Results were combined from multiple independent experiments and involved mixed sex mice. Statistical analysis used Kaplan Meier (B, E) and one-way ANOVA (F-H) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 7

NK cells drive early disease in mice with the FVB-Chr17 QTL Mixed-sex cohorts of B6, FVB, G6_24 or G6_25 were challenged with LCMV-Cl13 and evaluated via flow cytometry at 0, 3, or 6-day post-infection (dpi) for the frequency of NK cells in spleen and liver. Other cohorts of infected G6 mice were treated with NK cell depleting antibody or isotype-control antibody and were assessed for blood ALT activity and platelet counts at day 3 dpi. (A) Representative flow plots, total frequency, and total cell count of CD3⁻DX5⁺ NK cells from spleen of B6 (n = 4), FVB (n = 4), and G6 (n = 4–5) mice. (B–D) Analysis of NK cells from Cl13-infected B6 (n = 3) and G6_25 (n = 3) mice 6 dpi. (B) Gating strategy for discriminating tissue resident NK cells (trNK) and conventional NK cells (cNK). Lymphocyte and doublet discrimination gates not shown. (C) Total cell count of trNK and cNK cells. (D) Representative flow plots of GrzB expression by trNK and cNK cells. (E) G6_24 mice were given either isotype (n = 5) or NK1.1-depleting antibody (n = 8) prior to infection and ALT was recorded in the sera 3 dpi (left). G6_24 mice were given either isotype (n = 3) or NK1.1-depleting antibody (n = 4) prior to infection and total blood platelet count was recorded 3 dpi (right). All flow gated on live, viability dye-negative, singlet cells. Horizontal bars represent mean. Statistical analyses used unpaired one-way ANOVA (A) and unpaired student’s t-tests (C–E) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 8

The FVB-Chr17 QTL drives increased cytotoxic T cell populations, pathogenesis, and lethality Cohorts of B6, G6_24, or G6_25 mice were challenged with LCMV-Cl13 and the frequency and total number of granzyme-B⁺ T cells in spleen and liver was assessed at day 6 post-infection. (A) Representative flow plots, total frequency, and total cell count of CD4⁺CD44⁺GrzB⁺ cells in spleen. (B) Representative flow plots, total frequency, and total cell count of CD4⁺CD44⁺GrzB⁺ cells in liver. (C–E) Groups of G6_24 mice were treated with isotype (n = 7), anti-CD4- (n = 5), or anti-CD8-depleting (n = 5) antibodies and then challenged with LCMV-Cl13. (C) Survival of G6_24 mice across time post-infection. (B) ALT at day 6 post-infection in G6 mice treated with isotype, CD4-, or CD8-depletion antibody. (C) Platelet counts at day 6 in blood of G6 mice treated with isotype, CD4-, or CD8-depletion antibody. All flow gated on viability dye-negative, singlet cells. Horizontal bars represent mean. Statistical analyses used Kaplan Meier (C), unpaired Student’s t test (A and B), or one-way ANOVA (D and E) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Figure 9

The FVB-Chr17 QTL drives loss and dysfunction of Tregs CD4⁺Foxp3⁺ Treg cells were quantified in the spleens and livers of mixed sex B6 and G6 mice before and 6-day post-infection (dpi). (A) Representative flow plots identify CD4⁺Foxp3⁺ cells in spleen; the graph shows their frequency among all CD4⁺ cells. (B) The number of CD4⁺Foxp3⁺ cells per spleen at 6 dpi. (C) Ratio of the number of splenic CD4⁺Foxp3⁺ cells to the total number of activated CD4⁺CD44⁺ cells. (D) Representative flow plots and frequency of CD4⁺Foxp3⁺ cells in liver. (E) Total number of CD4⁺Foxp3⁺ cells 6 dpi. (F) Ratio of CD4⁺Foxp3⁺ cells to all activated CD4⁺CD44⁺ cells in the liver. (G) CD4⁺ T cells from B6 (n = 3), FVB (n = 3), or G6_24 (n = 3) mice were purified and cultured under T regulatory (Treg) conditions with IL-2 and TGFβ for three days. Data combined from three independent experiments (n = 1 mouse/experiment). Representative flow plots depict Foxp3 expression by CD4⁺ T cells of each genotype (left). The frequency of Foxp3⁺ cells among live CD4⁺ cells (right). The FACS analyses were gated on viability dye-negative, singlet cells. Horizonal bars represent mean (A, B, D, E, and G). Data are represented as mean ± SEM (C, F). Results from multiple experiments are combined. Significance was determined by unpaired Student’s t test (A–F) or one-way ANOVA (G) (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).