Fig 2: ELID is mirrored by a decline of Rp Macs and increase of CD9 Macs(A and B) Mice were infected with S.Tm-GFP (n = 3 per time point). At the indicated time points, spleens were harvested and analyzed by flow cytometry using antibodies to the indicated populations or the GFP signal of the bacteria (A). Bar graphs (left y axis) indicates mean ratio from the population of F4/80, and dots (right y axis) indicate cells containing intracellular bacteria (presented as ratio from F4/80+ GFP+ population) (B).(C) Mice were infected as in (A), 24 hpi single GFP+ CD9 Macs and iNOS Macs were sorted, and scCFU was measured (n = 494, n = 553 respectively). Presented are the proportions of cells according to number of bacteria per cell.(D and E) Mice were infected as in (A). Twenty-four hpi spleens were harvested, fixed, stained, and imaged with CD9 and F4/80 antibodies, DAPI, and GFP for intracellular bacteria (D). An example of CD9 Macs containing multiple bacteria. Number of intracellular bacteria were quantified across F4/80+CD9+ or F4/80+CD9– cells (n = 94 cells from 3 mice) (E).Results are representative of four (A and B) or three (C–E) independent experiments. See also Figure S4.

Fig 3: Functional analysis reveals distinct inflammatory programs of iNOS Macs and reducing oxidized lipids programs of CD9 Macs(A) Monocytes from Ms4a3cre-RosaTdT chimera were transferred to naive mice and 16 h (top panel) or 4 days (bottom panel) post-transfer mice were infected with S.Tm or PBS (n = 5, per group per time point). Twenty-four hpi spleens were harvested and analyzed by FACS. Presented are calculated proportions of tdTomato+ of the mononuclear populations.(B) Mice were infected with S.Tm and 24 hpi spleens were harvested and analyzed by flow cytometry using CD11b, F4/80, CD9, and Ly6C antibodies. Presented are contour plots of iNOS Macs and CD9 Macs from the gated Lin–CD11b+F4/80+ population.(C–E) Mice were infected as in (B), and CM or NCM from naive mice (n = 5) or bystander or infected CD9 Macs and iNOS Macs from S.Tm challenged mice (n = 4) were sorted by FACS and analyzed by bulk RNA-seq. Presented are PCA plots (C) and a heatmap (D) of the significant differentially expressed genes between all groups and log2 fold expression changes of selected genes (E).Results are representative of two (A and C–E) or four (B) independent experiments. See also Figure S3 and Tables S1 and S2.

Fig 4: CD9 Macs provide a niche for intracellular S.Tm replication during the second phase of ELID(A and B) Spleens of E2+/+ (WT; n = 3) and E2−/− (n = 4) were harvested and analyzed by flow cytometry using antibodies to mark the indicated populations (A) and presented as the ratio of the population from WT mice (B).(C and D) WT (n = 5) and E2−/− mice (n = 5) were infected with S.Tm. Twenty-four hpi spleens were harvested and analyzed by flow cytometry using antibodies to mark the indicated populations (C) and presented as the ratio of the population from WT mice (D).(E) WT and E2−/− mice were infected with 1,000 CFU of S.Tm. Three days post-infection, spleen were harvested and plated for CFU (n = 5 per condition).(F and G) WT and E2−/− mice were infected with 1,000 CFU of S.Tm (n = 7 per condition). Mice were weighed (F) and monitored for survival for 5 days, presented on a Kaplan-Meier plot (G).Results are representative of three independent experiments in all panels. (B and D) Error bars indicate standard deviation. (F) Error bars indicate standard error. (B) ∗p < 0.05 Student’s t test. (D)∗p < 0.05, Mann-Whitney U test. (E) p < 0.01, Mann-Whitney U test. (F) ∗∗p > 0.01 two-way analysis of variance. (G) ∗∗p < 0.01 log-rank test.

Fig 5: Phenotype and cytokine production capacity of F5 memory cells is conserved after homologous or heterologous recall(A) Naive F5 x CD45.1 cells (2.105) were i.v. transferred in B6 mice 1-day prior immunization with VV-NP68 (i.n., 2.105 pfu) or EL4-NP68 cells (s.c., 2,5.106 cells). At 26 dpi, mice received a second immunization with VV-NP68 or EL4-NP68.(B) Thirty-one days post-recall, the number of F5 cells was measured in the spleen.(C) The expression of CD9, CD43, CD49a, and CD49d was measured on F5 memory cells 31 days after recall by flow cytometry.(D and E) Splenocytes were stimulated with NP68 (10 nM) for 4h in the presence (D) or absence (E) of GolgiStop.(D) The production of IFNγ, TFNα, and IL-2 was measured by flow cytometry.(E) The expression of PD-1 and TIM3 on F5 memory cells was determined in the spleen. The statistical significance of differences was determined with 1-way (B and C) or 2-way (D and E) ANOVA test (∗p > 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001). Data are represented as mean ± SD (n = 5 mice per group) and are representative of 2 independent experiments.