Normalizing the environment recapitulates adult human immune traits in laboratory mice

Abstract

Our current understanding of immunology was largely defined in laboratory mice, partly because they are inbred and genetically homogeneous, can be genetically manipulated, allow kinetic tissue analyses to be carried out from the onset of disease, and permit the use of tractable disease models. Comparably reductionist experiments are neither technically nor ethically possible in humans. However, there is growing concern that laboratory mice do not reflect relevant aspects of the human immune system, which may account for failures to translate disease treatments from bench to bedside. Laboratory mice live in abnormally hygienic specific pathogen free (SPF) barrier facilities. Here we show that standard laboratory mouse husbandry has profound effects on the immune system and that environmental changes produce mice with immune systems closer to those of adult humans. Laboratory mice--like newborn, but not adult, humans--lack effector-differentiated and mucosally distributed memory T cells. These cell populations were present in free-living barn populations of feral mice and pet store mice with diverse microbial experience, and were induced in laboratory mice after co-housing with pet store mice, suggesting that the environment is involved in the induction of these cells. Altering the living conditions of mice profoundly affected the cellular composition of the innate and adaptive immune systems, resulted in global changes in blood cell gene expression to patterns that more closely reflected the immune signatures of adult humans rather than neonates, altered resistance to infection, and influenced T-cell differentiation in response to a de novo viral infection. These data highlight the effects of environment on the basal immune state and response to infection and suggest that restoring physiological microbial exposure in laboratory mice could provide a relevant tool for modelling immunological events in free-living organisms, including humans.

Extended data figure 1

Frequency of CD8 T cell subsets in neonates vs. adult humans. CD8 T cell subsets were defined in adult PBMC (n=13) and cord blood PBMC (n=8) by fluorescence flow cytometry based on the following markers: Naïve=CD45RAhi CCR7hi, T_CM=CD45RAlo CCR7hi, T_EM=CD45RAlo CCR7lo and T_EMRA=CD45RAhi CCR7lo. Significance was determined using unpaired two-sided t-test. *** p<0.001, **** p<0.0001. Bars indicate mean ± S.E.M.

Extended data figure 2. Co-housing laboratory mice with petstore mice induces accumulation of T_RM-phenotype CD8 T cells and other innate cells in tissues of laboratory mice

a) CD8 T cell density within the indicated tissues of adult laboratory mice (n=5) and cohoused mice (n=7). Representative immunofluorescence staining, CD8β (red), DAPI (nuclei, blue), scale bars = 50 μm. b) Phenotype of CD8 T cells was compared between laboratory mice (n=9) and age-matched laboratory mice that were co-housed (n=9, representative flow cytometry plots shown). Samples gated on CD44hi cells isolated from the indicated tissue (vasculature populations were excluded, see methods). c) Enumeration of CD11b+ granulocytes and Ly6Chi inflammatory monocytes in spleens of laboratory (n=6) and cohoused (n=6) mouse. Significance was determined using unpaired two-sided Mann-Whitney U-test. ** p<0.01, bars indicate mean ± S.E.M.

Extended data figure 3

LEM metagene analysis. For each comparison, standard GSEA was performed using the ImmSigDB database of gene-sets. Genes in the top 150 enriched sets (FDR<0.001, ranked by P-value) were filtered to only leading edge genes and subsequently clustered into groups (metagenes) using an NMF algorithm. Hierarchical clustering of genes within individual metagenes was performed to obtain the final heatmap. Metagenes with qualitatively discernible ‘blocks’ of gene-set membership were annotated according to the identity of corresponding enriched gene-sets. Heatmaps for Adult vs. Neonatal, Petstore vs. Laboratory, Cohoused vs. Laboratory, Neonatal vs. Adult, Laboratory vs. Petstore, and Laboratory vs. Cohoused comparisons are shown. Individual genes within each metagene are listed in Supplemental table 1. Pairwise overlaps between metagenes from different comparisons are visualized in Figure 4c.

Extended data figure 4

Environment altered antimicrobial resistance and CD8 T cell differentiation. Laboratory mice were co-housed with petstore mice as described in figure 3. a) Bacterial load in the spleen 3 days post-challenge with 8.5 × 10⁴ CFU of Listeria monocytogenes (LM) among laboratory (n=8), LM-immune (n=9), cohoused (n=9) and petstore mice (n=9) among 2 independent experiments. b) Survival of laboratory mice (n=15), cohoused mice (n=19) and petstore mice (n=15) after challenge with 10⁶ Plasmodium berghei ANKA parasitized RBCs among 2 independent experiments. c) Laboratory (n=9) and cohoused (n=8) mice were infected with LCMV. Four weeks later, LCMV-specific CD8 T cells (identified with H-2D^b/gp33 MHC I tetramers) were evaluated for expression of the indicated markers. Top row, gated on live CD8α+ T cells. Bottom 3 rows, gated on live CD8α+ H-2D^b-gp33+ T cells. Significance was determined using Kruskal-wallis (ANOVA) test (in a) and log-rank (Mantel-Cox) test (in b). * p<0.05, *** p<0.001, **** p<0.0001. Bars indicate mean ± S.E.M.

Figure 1. Laboratory mice lack differentiated memory CD8 T cell subsets, which is more similar to neonates than adult humans

a,b) CD8 T cell density within adult laboratory mouse (n=5) and human cervix (n=3). Representative immunofluorescence staining of frozen sections (scale bars, 50 μm) is shown. CD8β (red), DAPI (nuclei, blue). c) CD8 T cell phenotype was compared between adult human blood (n=13), adult laboratory mouse blood (n=10), and human cord blood (n=8) among 2 independent experiments by fluorescent flow cytometry (representative plots shown). Top panels gated on CD8+/CD3+ cells. Bottom panels gated on antigen experienced CD8+/CD3+ cells, as defined by conventional lineage markers in each species (red and green quadrants). d) Enumeration of granzyme B+ CD8 T cell frequencies amongst antigen experienced subsets. Significance was determined using unpaired two-sided Mann-Whitney U-test (in b) or Kruskal-wallis (ANOVA) test (in d). * p<0.05, ** p<0.01, **** p<0.0001, bars indicate mean ± S.E.M.

Figure 2. CD8 T cell subsets vary between feral, petstore, and laboratory mice

a) CD8 T cell subsets were compared in PBMC between laboratory mice (n=9), feral mice that were trapped in the wild (n=10), and mice obtained from a pet store (n=6), among 2 independent experiments. b) Phenotype of CD44lo/CD62Lhi (naïve) and CD44hi (antigen-experienced) CD8+ PBMC was compared by fluorescent flow cytometry. c,d) CD8 T cell density in non-lymphoid tissues was compared between laboratory and pet store mice by quantitative immunofluorescence microscopy (QIM). Immunofluorescence staining of frozen sections of indicated tissues (n=8 animals per group, scale bars = 50 μm). CD8β (red), DAPI (nuclei, blue). Significance was determined using unpaired two-sided Mann-Whitney U-test. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, bars indicate mean ± S.E.M.

Figure 3. Co-housing with petstore mice changes the immune system of laboratory mice

a) Proportion of CD44hi (antigen-experienced) CD8+ PBMC in laboratory mice housed in either SPF conditions (blue, n=10 for each time point) or co-housed with pet store mice (green, n=9 for each time point) among 2 independent experiments. b) CD8 T cell phenotype in blood of laboratory mice after co-housing with pet store mice. Representative flow plots (n=15) are shown. c) Survival of laboratory mice after co-housing with pet store mice (n=65 mice sampled). d) Representative phenotypes of CD44hi CD8+ PBMC isolated from laboratory mice co-housed with pet store mice for 100 days (n=15) and non-cohoused age-matched controls (n=10) was compared by fluorescent flow cytometry. e) Enumeration of CD8 T cells in non-lymphoid tissues between laboratory and cohoused mice by QIM (n=8 animals per group). f) Ratio of indicated cell types isolated and enumerated by flow cytometry from cohoused (n=6) and laboratory mice (n=6) among 2 independent experiments. ILC = innate lymphoid cells, Th = CD4 T helper cells, Treg = Regulatory CD4 T cells, GC = Germinal center. Single positive staining for T-bet, Gata3, and Rorγt were used to determine Th1, Th2, and Th17 lineages, respectively. g) Serum antibody concentrations (n=7 per group). h) Principal component analysis of gene expression data from PBMC of laboratory (n=8), cohoused (n=7) and pet store mice (n=8). Significance was determined using unpaired two-sided Mann-Whitney U-test (in e) or one-way ANOVA with Bonferroni post-hoc analysis (in g). *p<0.05, ** p<0.01, ***p<0.001. Bars indicate mean ± S.E.M.

Figure 4. Microbial experience matures mouse immune transcriptome from neonatal to adult human and impacts immune system function

a) Experimental design and b) GSEA plots showing the enrichment of gene signatures among the indicated mouse group comparisons relative to human adult vs. neonatal comparison. Signatures consist of top 400 significantly differentially expressed genes of laboratory (n=8), cohoused (n=7) and petstore mice (n=8). c) Pairwise overlaps of metagenes identified through leading-edge metagene analysis and corresponding GO terms. d) Bacterial load in the liver 3 days post-challenge with 8.5 × 10⁴ CFU of Listeria monocytogenes (LM) among indicated groups (n=9 for all except laboratory mice where n=8). e) Parasitic load in peripheral blood 5 days post Plasmodium berghei ANKA parasitized RBC challenge among the indicated groups of laboratory (n=15), cohoused (n=19), and petstore mice (n=15). f) 28 days after LCMV Armstrong infection, the proportion of H-2D^b/gp33-specific CD8 T cell MPECs (KLRG1-, CD127+) and SLECs (KLRG1+, CD127−) in PBMC of cohoused (n=8) and laboratory (n=9). Data are cumulative of 2 independent experiments. d–f) Data points represent individual mice. Significance was determined using Kruskal-wallis (ANOVA) test (in d), one-way ANOVA test (in e) and unpaired two-sided t-test (in f) * p<0.05, ** p<0.01, **** p<0.0001. Bars indicate mean ± S.E.M.