Fig 1: Phenotype of Memory CD8 T Cells Generated following Infection Is Diverse in CC StrainsB6, BALB/c, and CC mice were infected with LCMV-Armstrong on d0. Phenotype of TM cells (CD11ahi/CD8alo) was determined on d75. (A) Summary graphs of percentage of CD8 TM cells expressing CD127, CD62L, CD27hi, KLRG1, or Cx3Cr1. (B) Summary graphs of percentage of CD8 TM cells displaying an effector memory (Tem) (Cx3Cr1hi/CD27lo; top), peripheral memory (Tpm) (Cx3Cr1int/CD27hi; middle), or central memory (Tcm) (Cx3Cr1lo/CD27hi; bottom) phenotype. (C) Percentage of CD8 TM cells (d75) expressing CD62L (x axis) relative to percentage expressing CD127, CD27hi, KLRG1, or Cx3Cr1 (y axis). (D) B6, NIH Swiss (SW), and CC mice were infected with LCMV-Armstrong on d0. Phenotype of TM cells (CD11ahi/CD8alo) was determined on d75+. Violin plots of the percentage of TM cells expressing CD127, CD62L, CD27hi, and KLRG1 for B6 or SW mice or CC strains are shown.Data from 1–3 individual experiments. n = 2–20 mice per group (see Table 1). Error bars for summary plots indicate standard error of the mean and dashed red lines at percentage seen in B6 mice. For linear correlations, red dots indicate B6 mice, blue dots indicate BALB/c mice, and black dots indicate CC strains. Statistical significance of R-squared values based on linear regression analysis. For violin plots, solid lines are at the 25th and 75th quartiles and dashed line indicates the median. See also Figures S5 and S6 and Table S4.
Fig 2: Hypoxia-inducible factor-1a (HIF1a) is dispensable for natural killer (NK) cell effector function.(A) Representative histograms (top) and quantification (bottom) of CD69 MFI on NK cells and frequency of KLRG1+ and IFN?+ NK cells at day 1.5 post-infection (pi) of 1aKO or FL control mice. Data are from 2–3 independent experiments with 4–6 mice per group. (B) Representative histograms (top) and quantification (bottom) of CD69 and Granzyme B MFI (GrzmB) of NK cells, and frequency of KLRG1+ NK cells at day 3 pi. Data are from two independent experiments with four mice per group. (C) 1aKO or FL control mice were injected with PBS or Poly (I:C) then 3 days later 20 × 106 splenocytes at 1:1 ratio of CFSE-labeled MHCI-sufficient (CFSEHigh) and -deficient (CFSELow) were intravenously transferred into mice. MHC-deficient splenocyte elimination was measured 3 hr post-transfer by flow cytometry. Data are from three independent experiments with 3–4 mice per group. Data depict mean ± SEM, with each data set containing data indicated number of mice per group from independent experiments. Unpaired t-test was performed on (A, B) or one-way ANOVA (C). Statistical significance indicated by n.s., no significant difference; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Fig 3: Maturation of ILC2s through lung-gut axis.a CCR2-RFP;CCR4-mNeonGreen mice were i.p. injected with IL-25 or IL-33. After 24 h, expression of mNeonGreen and red fluorescent protein (RFP) in lung or gut ILC2s was analyzed by flow cytometry. b Analysis of mNeonGreen and RPF expression of gut ILC2s from CCR2-RFP;CCR4-mNeonGreen mice after intraperitoneal administration of IL-33 for the indicated days. The cell frequency of indicated subgroups of gut ILC2s was calculated and shown as mean ± SD (lower panel). (n = 3 for each group). c Adoptive transfer of lung or gut ILC2s (5 × 104) from WT CD45.1 mice to NOD-PrkdcscidIL2rgtm1/Bcgen (B-NDG) mice by intravenous injection. Six days after of transfer, lung and gut ILC2s from the recipient mice were analyzed by flow cytometry. The cell frequency of ILC2s were calculated and shown as mean ± SD. (n = 3 for each group). d Adoptive transfer and development of ILC2Ps in the lung and gut. ILC2Ps (Lin-CD127+ST2+Sca1+KLRG1-) from BM of CD45.1 mice were isolated and intravenously injected into B-NDG mice. One week after transfer, the recipient mice were i.p. injected with 400 ng/mouse/day IL-33 for the indicated days. The cell frequency of ILC2s in the lung and gut were calculated and shown as mean ± SD. (n = 3 for each group). e Tracing of lung ILC2s. Solubilizing 4-OHT (2 µg/g mouse) were atomized and delivered into lung of Id2-Cre/ERT2;Rosa26-STOP-tdTomato mice by liquid aerosol devices as described in “Methods”. Expression of TdTomato in ILC2s were analyzed by flow cytometry two days after 4-OHT treatment (left panel). Two days after 4-OHT treatment, Id2-Cre/ERT2;Rosa26-STOP-tdTomato mice were i.p. injected with IL-33. The frequency of TdTomato+ ILC2s from the mice treated IL-33 with indicated days were calculated and shown as mean ± SD. The schematic diagrams in (c-e) were created with BioRender.com. f, g Depletion of ILC2s in the lung and gut. Ccr2-mNeonGreen-Cre;Rosa26-STOP-DTR mice were subjected to i.p. injection of 100 ng/mouse diphtheria (DT) every two days for six days. Cell frequency of ILC2s from the lung, gut and BM were analyzed by flow cytometry (f) and shown as mean ± SD (g). (n = 3 for each group). ***, P < 0.001 by Two-tailed unpaired Student’s t-test. NS, not significant (P > 0.05) by Two-tailed unpaired Student’s t-test. (P = 0.0003, 0.0007, 0.4971 for Lung, Gut, BM respectively by Two-tailed unpaired Student’s t-test). h The reconstitution of ILC2s in the lung and gut after ILC2 depletion. ILC2s were depleted as shown in (f) and the ILC2-depleted mice were subjected to the intraperitoneal injection of 400 ng/mouse IL-33 at day 0. After the indicated days, cell frequency of lung and gut ILC2s were analyzed by flow cytometry and shown as mean ± SD. (n = 3 for each group) Data are representative of at least three independent experiments. Source data are provided as a Source Data file.
Fig 4: The differential function of CCR2 and CCR4 in lung and gut ILC2s.a, b Deficiency of CCR2 abrogated the ILC2 counts in the lung (a) and gut (b). WT or Ccr2-/- mice were subjected to the i.p. injection of IL-25 or IL-33 followed by flow cytometry analysis. The cell frequency of indicated ILC2 subsets was calculated and shown as mean ± SD (right panel). (n = 3 for each group) (P = 0.0072, 0.0059, 0.0056 for a, P = 0.0040, 0.0066, 0.0012, 0.0196 for b). c CCR2 is required for the ILC2 relocalization from BM to the lung. ILC2Ps (Lin-CD127+Sca1+ST2+KLRG1-) and ILC2s (Lin-CD127+Sca1+ST2+KLRG1+) from BM of WT or Ccr2-/- mice were analyzed by flow cytometry. The indicated ILC2 subsets was calculated and shown as mean ± SD (right panel). (n = 3 for each group) (P = 0.0376, 0.0064, 0.0021). d Adoptive transfer of WT and Ccr2-/- ILC2Ps. ILC2Ps (Lin-CD127+Sca1+ST2+KLRG1-) were isolated from BM of WT and Ccr2-/- mice. WT and Ccr2-/- ILC2Ps were 1:1 mixed (5 × 104 for each) and i.v. injected into B-NDG mice. One week after transfer, the recipient mice were i.p. injected with 400 ng/mouse/day IL-33 for three days. The cell frequency of ILC2s in the lung and gut were calculated and shown as mean ± SD. (n = 3 for each group). (P = 0.0039, 0.0086, 0.0050 by Two-tailed unpaired Student’s t-test). The schematic diagram was created with BioRender.com. e Scatter plot comparing the gene expression pattern between WT ILC2s versus Ccr2-/- ILC2s from the lung and gut. ILC2s (Lin-CD45+CD127+KLRG1+) were isolated from WT or Ccr2-/- mice after treatment with IL-33 and subjected to bulk mRNA sequencing. Blue dots, upregulated genes in Ccr2-/- ILC2s; red dots, upregulated genes in WT ILC2s. Representative differentially expressed genes (DEGs) are depicted. f WT or Ccr4-/- mice were i.p. injected with IL-25 or IL-33 for three days. ILC2s from the lung and gut were analyzed by flow cytometry and cell frequency was shown as mean ± SD. (n = 3 for each group) (P = 0.0031 by Two-tailed unpaired Student’s t-test). g Comparison of gene expression in lung iILC2s from WT versus Ccr4-/- mice. iILC2s (Lin-CD45+CD127+ST2-KLRG1hi) were isolated from the lung of WT or Ccr4-/- mice after treatment with IL-25 followed by bulk mRNA sequencing. Blue dots, upregulated genes in Ccr4-/- ILC2s; red dots, upregulated genes in WT ILC2s. Representative DEGs are depicted. Data are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01 by Two-tailed unpaired Student’s t-test. Source data are provided as a Source Data file.
Fig 5: Drp1 Controls the Metabolic Reprogramming of Activated T Cells(A) Mitochondria (TOM20) distribution in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated with anti-CD3-coated beads (referred to as B, labeled with anti-CD3 antibody, red) (n = 4).(B) Fluo3-AM-loaded +/+ cre+ control and fl/fl cre+ Drp1 KO T cells were incubated with the aCD3 antibody. After acquiring Fluo3-AM baseline fluorescence, a secondary antibody was added, and fluorescence was acquired up to 6 min. The fold increase in maximum (at 2 min) and residual (at 5 min) Fluo3-AM fluorescence relative to baseline is reported in the graph on the right (n = 5 ctrl, 4 KO).(C) Expression levels of the indicated (phospho)-protein in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells stimulated in vitro for the indicated time. Quantification of the KO:ctrl ratio for the indicated (phospho)-proteins is reported in the graph on the right (AMPK-mTOR, n = 5; cMyc, n = 4; S6, n = 3). cMyc levels are reported 48 hr post-stimulation (maximal upregulation), but similar results were also obtained at 5 hr.(D and E) Expression levels and relative quantifications of the indicated (phospho)-protein from +/+ cre+ control and fl/fl cre+ Drp1 KO T cells activated in vitro for 5 hr in the presence of the calcium chelators 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and EDTA (D, n = 3) or the AMPK inhibitor Compound-C (E, n = 3).(F and G) RNA sequencing (RNA-seq) analysis in 3-day in vitro-activated +/+ cre+ control and fl/fl cre+ Drp1 KO T cells.(F) Heatmap of cMyc-dependent metabolic genes in T cells (cMyc-MG) expression in +/+ cre+ control and fl/fl cre+ Drp1 KO T cells, with glycolytic genes highlighted.(G) The differential mRNA expressions (normalized association score) from enrichment gene set association analysis (GSAA) of the cMyc-MG group from (F) and additional metabolic pathways (whose heatmaps are reported in Figure S3F). TCA, tricarboxylic acid; PPP, pentose phosphate pathway; FAS, fatty acid synthesis; FAO, fatty acid oxidation. For each group, transcriptional enrichment in KO cells compared with controls is highlighted in red, downregulation in blue and no net difference in black (n = 3).(H–J) Seahorse analysis of extracellular acidification rate (ECAR) (H) and oxygen consumption rate (OCR) (I and J) rates in 6-day in vitro-activated +/+ cre+ control and fl/fl cre+ Drp1 KO CD8+ T cells (2-DG, 2-deoxyglucose; Rot/an, rotenone and antimycin). FA oxidation was measured with BSA-palmitate with or without etomoxir (J). The following parameters were quantified: glycolysis (Glyc), maximal glycolytic capacity (MGC); basal OXPHOS (basal OX), maximum respiratory capacity (MRC), and basal (basal) and maximal (max) FA oxidation (n = 3).(K) MFI for IL7Ra (n = 17), CD44 (n = 12), KLRG1 (n = 9), IFN? (n = 10), TNF-a (n = 6), IL-2 (n = 4), and IL-4 (n = 7) and for the Tbet:Eomes ratio (n = 8) in 6-day in vitro-activated CD8+ +/+ cre+ control and fl/fl cre+ Drp1 KO T cells under the indicated polarizing conditions.Data are represented as mean ± SEM. Scale bar, 10 µm in (A). Significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S3.
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