Journal: bioRxiv

Article Title: Cell Programmed Nutrient Partitioning in the Tumor Microenvironment

doi: 10.1101/2020.08.10.238428

Figure Lengend Snippet: a-b , Representative OCR ( a ) and ECAR ( b ) tracings of MC38 tumor cell fractions with indicated injections of oligomycin (O), FCCP (F), and rotenone and antimycin A (R/AA). c-d Basal mitochondrial OCR ( c ) and cellular ECAR ( d ) of MC38 tumor fractions (n=5 mice). e , Unsupervised cluster analysis of differentially expressed metabolic mRNA transcripts of whole tumor, CD45-cancer cell, CD45+ CD11b+ F4/80hi TAM, CD45+ CD11b+ F4/80lo myeloid cell, CD45+ CD3+ CD8a+ T cell, and CD45+ CD3+ CD8a-(CD4+) T cell flow-sorted MC38 tumor populations (n=2 mice). f-g , Glucose transporter ( f ) and hexokinase ( g ) mRNA transcript levels of indicated MC38 tumor populations (n=2 mice). Dotted line approximates limit of detection. hi , Representative flow cytometry plots ( h ) and quantification ( i ) of pS6 levels in indicated MC38 tumor and spleen populations. j-k , Representative flow cytometry histograms ( j ) and quantification ( k ) of pS6 levels in cancer cells (CD45-CA9+), myeloid cells (CD45+ CD11b+ CD14+), T cells (CD45+ CD3+), and other immune cells (CD45+ CD3-CD14-) from patient ccRCC tumor and PBMC (n=4 patients). Each data point represents a biological replicate and error bars are SEM. a-d and f-g are representative of at least two independent experiments. P values were calculated using Welch’s 2-tailed t-test for (c-d) and Brown-Forsythe ANOVA test for (h, j). * p <0.05, ** p <0.01, *** p <0.001. ECAR: extracellular acidification rate; FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone; FMO: fluorescence minus one; MFI: median fluorescence intensity; OCR: oxygen consumption rate; PBMC: peripheral blood mononuclear cells; pS6: phosphorylated ribosomal protein S6 (Ser235/236).

Article Snippet: The anti-mouse and cross-reactive antibodies used were: CD45 BV510 (40-F11, Biolegend 103138), B220 e450 (RA3-6B2, ThermoFisher 48-0452-82), CD11b e450 (M1/70, ThermoFisher 48-0112-82), CD11b FITC (M1/70, Biolegend 101206), CD8a AF488 (53-6.7, Biolegend 100723), Ly6C FITC (HK1.4, Biolegend 128006), CD11c PE (N418, BioLegend 117308), FOXP3 PE (FJK-16s, ThermoFisher 12-5773-82), pS6 Ser235/236 PE (D57.2.2E, Cell Signaling 5316S), CD4 PerCP-Cy5.5 (RM4-5, BioLegend 100540), Ly6G PerCP-Cy5.5 (1A8, BioLegend 127616), F4/80 PE-Cy7 (BM8, BioLegend 123114), NKp46 PE-Cy7 (29A1.4, BioLegend 137618), CD3 PE-Cy7 (17A2, BioLegend 100220), CD3 FITC (17A2, BioLegend 100204), CD3 APC (17A2, BioLegend 100236), CD206 APC (C068C2, BioLegend 141708), GLUT1 AF647 (EPR3915, Abcam ab195020), EPCAM PE (G8.8, BioLegend 118206), Thy1.1 PerCP-Cy5.5 (HIS51, ThermoFisher 45-090082), CD45 PE (30-F11, ThermoFisher 12-0451-83), Ly6C BV570 (HK1.4, BioLegend 128030), CD68 BV605 (FA-11, BioLegend 137021).

Techniques: Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Cell Programmed Nutrient Partitioning in the Tumor Microenvironment

doi: 10.1101/2020.08.10.238428

Figure Lengend Snippet: a , Sorting gates of MC38 tumor cells used for mRNA transcript analyses. b , Expression of selected cell identity markers in flow sorted MC38 tumor cell population. Dotted line approximates limit of detection. c-d , GLUT1 levels determined by flow cytometry in MC38 ( c ) and CT26 ( d ) tumor populations. e, Quantification of pS6 levels determined by flow cytometry in indicated CT26 tumor and spleen populations. Each data point represents a biological replicate and error bars are SEM. c-e are representative of independent experiments performed at least twice. P values were calculated using the Brown-Forsythe ANOVA test. *** p <0.001.

Article Snippet: The anti-mouse and cross-reactive antibodies used were: CD45 BV510 (40-F11, Biolegend 103138), B220 e450 (RA3-6B2, ThermoFisher 48-0452-82), CD11b e450 (M1/70, ThermoFisher 48-0112-82), CD11b FITC (M1/70, Biolegend 101206), CD8a AF488 (53-6.7, Biolegend 100723), Ly6C FITC (HK1.4, Biolegend 128006), CD11c PE (N418, BioLegend 117308), FOXP3 PE (FJK-16s, ThermoFisher 12-5773-82), pS6 Ser235/236 PE (D57.2.2E, Cell Signaling 5316S), CD4 PerCP-Cy5.5 (RM4-5, BioLegend 100540), Ly6G PerCP-Cy5.5 (1A8, BioLegend 127616), F4/80 PE-Cy7 (BM8, BioLegend 123114), NKp46 PE-Cy7 (29A1.4, BioLegend 137618), CD3 PE-Cy7 (17A2, BioLegend 100220), CD3 FITC (17A2, BioLegend 100204), CD3 APC (17A2, BioLegend 100236), CD206 APC (C068C2, BioLegend 141708), GLUT1 AF647 (EPR3915, Abcam ab195020), EPCAM PE (G8.8, BioLegend 118206), Thy1.1 PerCP-Cy5.5 (HIS51, ThermoFisher 45-090082), CD45 PE (30-F11, ThermoFisher 12-0451-83), Ly6C BV570 (HK1.4, BioLegend 128030), CD68 BV605 (FA-11, BioLegend 137021).

Techniques: Expressing, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Heterozygous splice mutation in PIK3R1 causes human immunodeficiency with lymphoproliferation due to dominant activation of PI3K

doi: 10.1084/jem.20141759

Figure Lengend Snippet: PI3K signaling is constitutively active in patient immune cells. (A) Signaling diagram showing the major molecules downstream of the p110δ/p85α PI3K complex. PDK1: phosphoinositide-dependent kinase-1; mTORC: mechanistic target of rapamycin complex; GSK3: glycogen synthase kinase 3; S6K: ribosomal protein S6 kinase; 4EBP1: eukaryotic translation initiation factor 4E-binding protein 1; FOXO: forkhead box O. (B) Immunoblot for AKT phosphorylated at threonine 308, total AKT, and actin (top), and flow cytometric analysis of AKT phosphorylation at serine 473 (bottom) in T cell blasts from healthy relatives or control subjects (Ctrl) and the indicated patients. (C) Flow cytometric analysis of the phosphorylation of S6 at serine residues 235/236 and serine residues 240/244 in T cell blasts from healthy relatives or control subjects (Ctrl) and the indicated patients (stained in same experiment as in B, bottom). (D) Immunoblot for phosphorylation of FOXO protein threonines (T) and GSK3 protein serines (S), total protein, or β-tubulin loading control, as indicated. Four (B and C) or three (D) independent experiments on the two indicated patients and controls were performed.

Article Snippet: For phosflow analyses, anti-pAKT S473 Alexa Fluor 647 (D9E; Cell Signaling Technology), anti-pAKT T308 Alexa Fluor 488 (C31E5E; Cell Signaling Technology), anti-pS6 S235/236 PE (N7-548; BD), and/or anti-pS6 S240/244 Alexa Fluor 647 (D68F8; Cell Signaling Technology) were used.

Techniques: Binding Assay, Western Blot, Staining

Journal: The Journal of Experimental Medicine

Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal

doi: 10.1084/jem.20200866

Figure Lengend Snippet: Comparison of gene expression profile between Fr.2 and Fr.5 cells. (A) GSEA of Fr.2 and Fr.5 RNA-seq data . Selected gene sets are shown with normalized enrichment scores (NES) and false discovery rate (FDR) q-values (q-val). (B) Left: Flow cytometry analysis of intracellular (ic) expression of pS6, c-Myc, and pRb in Fr.2, Fr.5, and naive B cells. Right: Cumulative data of geometric mean fluorescence intensity (gMFI). n = 4 (pS6, pRb), n = 3 (c-Myc), representative of two independent experiments. (C) GSEA of Fr.2 and Fr.5 RNA-seq data. All Hallmark gene sets enriched in Fr.2 or Fr.5 (FDR q-value < 0.25) were listed with NES and nominal (NOM) P value (p-val). *, P < 0.05; **, P < 0.01; unpaired Student’s t test.

Article Snippet: PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.

Techniques: Comparison, Gene Expression, RNA Sequencing, Flow Cytometry, Expressing, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal

doi: 10.1084/jem.20200866

Figure Lengend Snippet: Cellular survival of pro-memory B cells. (A) Flow cytometry analysis of intracellular (ic) expression of pS6 and c-Myc in Fr. 3, Fr.5, and naive B cells (related to ). Representative of three independent experiments. (B) Left: Flow cytometry analysis of donor NP + IgG1 + CD73 + memory B cells. Congenically marked Bcl2l11 +/+ and Bcl2l11 f/+ ERT2cre B1-8 ge naive B cells were cotransferred as a 1:1 mixture into wild-type mice, which were immunized with NP-CGG/alum, administered with tamoxifen on day 8, and analyzed on day 21 (see also ). Right: Cumulative data of Bcl2l11 f/+ : Bcl2l11 +/+ ratio. n = 4, representative of two independent experiments. (C) Top: Flow cytometry analysis of aCasp3 expression in donor IgG1 + LZ B cells (related to ). Bottom: Cumulative data of aCasp3 + ratio. n = 4 (vehicle), n = 7 (tamoxifen), pooled from two independent experiments. (D) Left: Protocol of the control experiment related to . Congenically marked B1-8 ge ERT2cre and Prdm1 f/+ B1-8 ge ERT2cre naive B cells were mixed at a 1:1 ratio and cotransferred into wild-type CD45.1/1 recipient mice. After immunization with NP-CGG/alum, tamoxifen was administered on day 10, and spleens were analyzed on day 12. Right: Flow cytometry analysis of aCasp3 expression in donor NP + IgG1 + CD38 + Efnb1 + (Fr.5/6) B cells. Representative of three independent experiments. **, P < 0.01; unpaired Student’s t test (B) and *, P < 0.05; paired Student’s t test (C). FSC, forward scatter.

Article Snippet: PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.

Techniques: Flow Cytometry, Expressing, Control

Journal: The Journal of Experimental Medicine

Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal

doi: 10.1084/jem.20200866

Figure Lengend Snippet: Hyper mTORC1 activity in Bach2/Blimp1 double-deficient GC B cells. (A) Experimental design of inducible Bach2 and Blimp1 double knockout (dKO) in the adoptive transfer experiment. Bach2 +/+ Prdm1 +/+ , Bach2 +/+ Prdm1 f/f , or Bach2 f/f Prdm1 f/f ERT2cre B1-8 hi naive B cells were independently transferred into wild-type CD45.1 + mice. Mice were administered with tamoxifen, immunized with NP-CGG/alum, and analyzed on day 12. (B) Left: Flow cytometry analysis of donor NP + GC, donor NP + IgG1 + LZ, and donor NP + IgG1 + CD38 + cells. Right: Cumulative data of DZ:LZ ratio, and Fr.2, Fr.5, and Fr.7 cell numbers. Bach2 +/+ Prdm1 +/+ and Bach2 f/f Prdm1 f/f donor-derived NP + IgG1 + GC B cell numbers are 4,206 ± 1,813 and 1,243 ± 374 cells per 10 6 splenocytes, constituted 48 ± 1.3% and 31 ± 3.3% among total recipient GC B cells, respectively. n = 4 ( Bach2 +/+ Prdm1 +/+ ), n = 7 ( Bach2 +/+ Prdm1 f/f ), n = 8 ( Bach2 f/f Prdm1 f/f ) for DZ:LZ ratio, pooled from four independent experiments. n = 5 ( Bach2 +/+ Prdm1 +/+ ), n = 4 ( Bach2 +/+ Prdm1 f/f , Bach2 f/f Prdm1 f/f ) for cell number. Representative of two independent experiments. (C) GSEA of RNA-seq data from LZ B cells. Bach2 +/+ Prdm1 f/f , or Bach2 f/f Prdm1 f/f ERT2cre B1-8 hi naive B cells were independently transferred into wild-type CD45.1 + mice. Mice were administered with tamoxifen and immunized with NP-CGG/alum. NP-specific donor GC LZ and DZ B cells were sorted from five pooled recipients on day 10 for one RNA-seq sample. See also for heatmap of the top 50 differentially expressed genes. n = 2 for each population. Gene sets with false discovery rate (FDR) q-value (q-val) < 0.25 are shown in the table. (D) Left: Flow cytometry analysis of intracellular (ic) expression of c-Myc, pRb, and pS6 in control (Cont; Bach2 +/+ Prdm1 +/+ or Bach2 +/+ Prdm1 f/f ) LZ, dKO ( Bach2 f/f Prdm1 f/f ) LZ, and wild-type naive B cells. Right: Cumulative data of geometric mean fluorescence intensity (gMFI). n = 3, representative of two independent experiments. (E) Top: Flow cytometry analysis of pulse-labeled EdU incorporation in control ( Bach2 +/+ Prdm1 +/+ or Bach2 +/+ Prdm1 f/f ) and dKO ( Bach2 f/f Prdm1 f/f ) GC B cells. Bottom: Cumulative data of EdU + ratio. n = 3, representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired Student’s t test.

Article Snippet: PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.

Techniques: Activity Assay, Double Knockout, Adoptive Transfer Assay, Flow Cytometry, Derivative Assay, RNA Sequencing, Expressing, Control, Fluorescence, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal

doi: 10.1084/jem.20200866

Figure Lengend Snippet: Partial rescue of memory B cell generation by rapamycin treatment in Bach2/Blimp1 double-deficient GC B cells. (A) Experimental design of B cell–specific rapamycin treatment using Bach2 and Blimp1 double-deficient B cells. Control ( Bach2 +/+ Prdm1 +/+ ) or double knockout (dKO; Bach2 f/f Prdm1 f/f ) ERT2cre B1-8 hi CD45.1 + naive B cells were independently transferred into wild-type mice. Mice were administered with tamoxifen, immunized with NP-CGG/alum, injected with rapamycin daily during days 4–11, and analyzed on day 12. (B) Flow cytometry analysis of intracellular (ic) expression of pS6 and c-Myc in control LZ, dKO LZ, and wild-type naive B cells (left), and pulse-labeled EdU incorporation in control and dKO GC B cells (right). Representative of three independent experiments. (C) Left: Flow cytometry analysis of donor NP + and donor NP + GC B cells. Right: Cumulative data of GC, IgG1 + memory, and IgG1 + CD73 + memory B cell number, and DZ:LZ ratio. n = 6–8 for GC and IgG1 + memory B cell number and DZ:LZ ratio, n = 3–5 for IgG1 + CD73 + memory B cell number. Pooled from two independent experiments. (D) Left: Flow cytometry analysis of donor NP + IgG1 + LZ and donor NP + IgG1 + CD38 + B cells. Right: Cumulative data for the Fr.5:Fr.2 ratio. n = 4–6, representative of two independent experiments. *, P < 0.05; **, P < 0.01; unpaired Student’s t test.

Article Snippet: PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.

Techniques: Control, Double Knockout, Injection, Flow Cytometry, Expressing, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal

doi: 10.1084/jem.20200866

Figure Lengend Snippet: Lower mTORC1 activity in GC B cells favors the memory fate. (A) Left: Experimental design of competitive cotransfer of rapamycin-sensitive and -resistant B1-8 ge B cells. Congenically marked Mtor +/+ and Mtor F2108L/F2108L B1-8 ge naive B cells were cotransferred as a 1:1 mixture into Mtor F2108L/F2108L hosts, which were immunized with NP-CGG/alum, administered with rapamycin daily during day 4–13, and analyzed on day 14. Right: Flow cytometry analysis of intracellular (ic) expression of pS6 in Mtor +/+ or Mtor F2108L/F2108L LZ B cells with or without rapamycin treatment. Representative of three independent experiments. (B) Flow cytometry analysis and cumulative data of donor NP + GC (left) and donor NP + memory B cells (right). n = 4, representative of two independent experiments. (C) Experimental design of competitive cotransfer of rapamycin-sensitive and -resistant B1-8 ge B cells with EdU labeling. Mice were prepared as in A, and proliferative GC-derived cells were labeled with EdU (injected i.p. on day 10 and then in the drinking water during days 10–13). (D) Left: Flow cytometry of donor NP + cells prepared as in C. Right: Cumulative data of EdU + Fr.7 memory B cell number and EdU + Fr.7:GC ratio. n = 4, representative of two independent experiments. (E) Left: Flow cytometry of Fr.2 and Fr.5 cells prepared as in C. Right: Cumulative data of Fr.5:Fr.2 ratio. n = 4, representative of two independent experiments. (F) Recall potential of IgG1 + memory B cells generated after rapamycin treatment. B1-8 ge B cells were transferred into Mtor F2108L recipient mice. After immunization with NP-CGG/alum, mice were treated with or without rapamycin as in C, and NP + IgG1 + memory B cells were sorted on day 14. Adoptive transfer with activated CD4 + T cells was performed as in . On day 6 after a boost with NP-CGG, donor NP + splenocytes were analyzed by flow cytometry (top), and donor NP + B cells and donor NP + plasma cells were quantified (bottom). n = 3 (vehicle), n = 4 (rapamycin), pooled from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; paired Student’s t test.

Article Snippet: PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.

Techniques: Activity Assay, Flow Cytometry, Expressing, Labeling, Derivative Assay, Injection, Generated, Adoptive Transfer Assay, Clinical Proteomics