Journal: Communications Biology

Article Title: T cell-specific deletion of Pgam1 reveals a critical role for glycolysis in T cell responses

doi: 10.1038/s42003-020-01122-w

Figure Lengend Snippet: a, b WT and Pgam1 KO naive CD8 T cells were stimulated for 24 h, and then a the extracellular acidification rate (ECAR) and b oxygen consumption rate (OCR) were determined ( n = 3, technical replicate). Error bars represent the standard deviation. The results of the analyses are representative of at least three independent experiments with similar results. c WT and Pgam1 KO naïve CD8 T cells were stimulated with anti-TCR-β and anti-CD28 mAbs for 6 or 24 h, and the intracellular amounts of glycolytic intermediates were determined by metabolic profiling ( n = 3–4, biological replicates). d The intracellular amount of ATP of the cells in c . e The phospho-tyrosine and β-actin levels in WT and Pgam1 KO CD8 T cells stimulated for indicated periods were determined by an immunoblot analysis. The protein amount of β-actin was used as a loading control. The numbers below the bands indicate the densitometry ratio with WT stimulated for 24 h. f The results of the immunoblot analysis of phospho-Zap70 (Tyr319), phosphor-Plcγ1 (Tyr783), and β-actin (control) in cells in e are shown. The numbers below the bands indicate the densitometry ratio with WT stimulated for 24 h. The results of the immunoblot analyses are representative of at least three independent experiments with similar results and are presented as cropped images. The full-length blots are presented in Supplementary Fig. . * P < 0.05, ** P < 0.01 (Student’s t test).

Article Snippet: Naive CD8 T (CD44 low CD62L high ) cells and naive CD4 T (CD44 low CD62L high CD25 negative ) cells were prepared using a Naive CD8 + T-cell Isolation kit (cat# 130-096-543; Miltenyi Biotec, San Diego, CA, USA) and a Naive CD4 + T-cell Isolation kit (cat# 130-093-227; Miltenyi Biotec), respectively.

Techniques: Standard Deviation, Western Blot, Control

Journal: Communications Biology

Article Title: T cell-specific deletion of Pgam1 reveals a critical role for glycolysis in T cell responses

doi: 10.1038/s42003-020-01122-w

Figure Lengend Snippet: a Representative staining profile of CD25, CD44, and CD69 in WT and Pgam1 KO CD8 T cells stimulated with anti-TCR-β plus anti-CD28 mAbs for 36 h. b Representative staining profiles of phospho-STAT5 (Tyr694) in activated CD8 T cells. WT and Pgam1 KO naïve CD8 T cells were stimulated with anti-TCR-β mAb plus anti-CD28 mAb for 48 h, and then the cells were cultured with or without IL-2 for 24 h. c WT and Pgam1 KO naive CD8 T cells were labeled with eFluor670 and stimulated with anti-TCR-β mAb plus anti-CD28 mAb. The cell division was detected by flow cytometry at the indicated number of hours after the initial stimulation. d The immune response of antigen-specific CD8 T cells after Lm -OVA infection was analyzed by staining with an OVA-specific tetramer (Tet). A representative staining profile of Tet/CD8 gated on the CD8-positive cells in the spleen at 7 days post infection (upper). The percentages of cells are indicated in the circle. The absolute number of Tet + CD8 T cells in the spleen is shown (lower). Each point represents an individual mouse. e Representative results of the intracellular FACS analysis of IFN-γ/IL-2 in the WT and Pgam1 KO CD8 T cells cultured under IL-2 conditions on day 5. The percentages of cells are indicated in each quadrant. f The results of an ELISA for IL-2, IFN-γ, and TNF-α in the supernatants of the cells in e ( n = 3, biological replicate). The results of the analyses are representative of at least three independent experiments with similar results. The results are indicated with the standard deviation. * P < 0.05, ** P < 0.01 (Student’s t -test).

Article Snippet: Naive CD8 T (CD44 low CD62L high ) cells and naive CD4 T (CD44 low CD62L high CD25 negative ) cells were prepared using a Naive CD8 + T-cell Isolation kit (cat# 130-096-543; Miltenyi Biotec, San Diego, CA, USA) and a Naive CD4 + T-cell Isolation kit (cat# 130-093-227; Miltenyi Biotec), respectively.

Techniques: Staining, Cell Culture, Labeling, Flow Cytometry, Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Communications Biology

Article Title: T cell-specific deletion of Pgam1 reveals a critical role for glycolysis in T cell responses

doi: 10.1038/s42003-020-01122-w

Figure Lengend Snippet: a Representative results of the intracellular FACS analysis of phospho-ribosomal S6 (S6; Ser235/236) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1; Thr37/46) in WT and Pgam1 KO CD8 T cells stimulated with anti-TCR-β mAb plus anti-CD28 mAb for the indicated number of hours. The results of the FACS analyses are representative of at least three independent experiments with similar results. b The results of the immunoblot analysis of c-myc, Hif1α, SREBP1 and β-actin in WT and Pgam1 KO CD8 T cells stimulated with anti-TCR-β mAb plus anti-CD28 mAb for 24 h. The protein amount of β-actin was used as a loading control. The numbers below the bands indicate the densitometry ratio with WT. The results of the immunoblot analyses are representative of at least three independent experiments with similar results and are presented as cropped images. The full-length blots are presented in Supplementary Figure . c – e WT and Pgam1 KO CD8 T cells were stimulated with anti-TCR-β mAb plus anti-CD28 mAb for the indicated number of hours, and the mRNA expression of the enzymes for glycolysis ( c ), glutaminolysis ( d ), and lipid synthesis ( e ) were determined by qRT-PCR. The results are presented relative to the expression of 18s rRNA with the standard deviation ( n = 3, technical replicates). * P < 0.05, ** P < 0.01 (Student’s t test).

Article Snippet: Naive CD8 T (CD44 low CD62L high ) cells and naive CD4 T (CD44 low CD62L high CD25 negative ) cells were prepared using a Naive CD8 + T-cell Isolation kit (cat# 130-096-543; Miltenyi Biotec, San Diego, CA, USA) and a Naive CD4 + T-cell Isolation kit (cat# 130-093-227; Miltenyi Biotec), respectively.

Techniques: Binding Assay, Western Blot, Control, Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Communications Biology

Article Title: T cell-specific deletion of Pgam1 reveals a critical role for glycolysis in T cell responses

doi: 10.1038/s42003-020-01122-w

Figure Lengend Snippet: a The intracellular amounts of glutamine and glutamate in WT and Pgam1 KO CD8 T cells stimulated with anti-TCR-β mAb plus anti-CD28 mAb for 6 or 24 h ( n = 3–4, biological replicate). b The intracellular amounts of glutamine and glutamate in WT CD8 T cells stimulated with anti-TCR-β mAb plus anti-CD28 mAb in the presence or absence of 10 mM 2-DG for 24 h ( n = 3, biological replicate). c WT naïve CD8 T cells were stimulated in the presence or absence of glutamine for 36 h, and then the ECAR was determined. The results are indicated with the standard deviation (right panel; n = 3, technical replicate). d The intracellular levels of lactate in activated CD8 T cells in the presence or absence of glutamine for 24 h are indicated with the standard deviation ( n = 3, biological replicate). e Representative results of the intracellular FACS analysis of phospho-S6 (Ser235/236) in CD8 T cells stimulated in the presence or absence of glutamine for the indicated number of hours. f The results of the immunoblot analysis of c-Myc, Hif1α, Srebp1, and β-actin in CD8 T cells cultured in the presence or absence of glutamine and rapamycin for 24 h. The protein amount of β-actin was used as a loading control. The results are presented as cropped images. The numbers below the bands indicate the densitometry ratio with CD8 T cells cultured under Gln (+), Rapa (−) conditions. The results of the immunoblot analyses are representative of at least three independent experiments with similar results. The full-length blots are presented in Supplementary Fig. . f The levels of Pgam1 , Gapdh , Scd1 , and Elovl6 mRNA in CD8 T cells stimulated in the presence or absence of glutamine for the indicated number of hours were determined by the quantitative RT-PCR analysis. The effect of rapamycin on glutamine-dependent alteration was also determined. The results are presented relative to the expression of 18s rRNA with the standard deviations ( n = 3, technical replicates). The results of the FACS analyses are representative of at least three independent experiments with similar results. * p < 0.05, ** p < 0.01 (Student’s t test).

Article Snippet: Naive CD8 T (CD44 low CD62L high ) cells and naive CD4 T (CD44 low CD62L high CD25 negative ) cells were prepared using a Naive CD8 + T-cell Isolation kit (cat# 130-096-543; Miltenyi Biotec, San Diego, CA, USA) and a Naive CD4 + T-cell Isolation kit (cat# 130-093-227; Miltenyi Biotec), respectively.

Techniques: Standard Deviation, Western Blot, Cell Culture, Control, Quantitative RT-PCR, Expressing

Journal: Communications Biology

Article Title: T cell-specific deletion of Pgam1 reveals a critical role for glycolysis in T cell responses

doi: 10.1038/s42003-020-01122-w

Figure Lengend Snippet: a The intracellular amounts of ATP in CD8 T cells stimulated with anti-TCR-β mAb plus anti-CD28 mAb in the presence or absence of glutamine for the indicated number of hours. The results are presented with the standard deviations ( n = 3, biological replicate). b The results of the immunoblot analysis of protein-tyrosine phosphorylation and β-actin in naïve CD8 T cells cultured with or without glutamine for the indicated number of hours. The protein amount of β-actin was used as a loading control. The numbers below the bands indicate the densitometry ratio with CD8 T cells cultured with glutamine. c The effect of rapamycin on glutamine-induced protein-tyrosine phosphorylation. Naïve CD8 T cells were stimulated under glutamine-sufficient conditions in the presence or absence of rapamycin for the indicated number of hours. The numbers below the bands indicate the densitometry ratio with CD8 T cells cultured without rapamycin. d The results of the immunoblot analysis of phospho-Zap70 (Tyr319), phosphor-Plcγ1 (Tyr783) and β-actin (control) in cells in (b). The numbers below the bands indicate the densitometry ratio with CD8 T cells cultured with glutamine. e The results of the immunoblot analysis of phospho-Zap70 (Tyr319), phosphor-Plcγ1 (Tyr783) and β-actin (control) in cells in ( c ). The numbers below the bands indicate the densitometry ratio with CD8 T cells cultured without rapamycin. f Representative results of the FACS analysis of CD25 and CD69 in CD8 T cells stimulated under the indicated conditions for 48 h. g Representative results of the intracellular FACS analysis of phosphor-Stat5 in CD8 T cells cultured with or without IL-2 stimulation. Naïve CD8 T cells were stimulated with anti-TCR-β mAb plus anti-CD28 mAb for 48 h, and then the cells were cultured with or without IL-2 for 24 h. The results of the FACS and immunoblot analyses are representative of at least three independent experiments with similar results. The results of the immunoblot analyses are presented as cropped images. The full-length blots are presented in Supplementary Fig. . * p < 0.05, ** p < 0.01 (Student’s t test).

Article Snippet: Naive CD8 T (CD44 low CD62L high ) cells and naive CD4 T (CD44 low CD62L high CD25 negative ) cells were prepared using a Naive CD8 + T-cell Isolation kit (cat# 130-096-543; Miltenyi Biotec, San Diego, CA, USA) and a Naive CD4 + T-cell Isolation kit (cat# 130-093-227; Miltenyi Biotec), respectively.

Techniques: Western Blot, Phospho-proteomics, Cell Culture, Control

Journal: Cancer cell

Article Title: Cathepsin S Regulates Antigen Processing and T Cell Activity in Non-Hodgkin Lymphoma.

doi: 10.1016/j.ccell.2020.03.016

Figure Lengend Snippet: Figure 3. Oncogenic Role of Mutated and Overexpressed CTSS Indolent Lymphoma For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.ccell.2020.03.016. (A) Graphical representation of experimental design using vavP-Bcl2 chimeric models expressing CTSSY132D or CTSSHIGH. (B) Tumor latencies for mice injected with empty vector (n = 23) or CTSSY132D (n = 16) or CTSSHIGH (n = 15) (log rank p value < 0.001). (C–E) (C) Immunohistochemistry of control, CTSSHIGH and CTSSY132D vavP-Bcl2 tumors. Dashed lines delineate the GCs. Scale bars, 100 mm. (D and E) Quantification of the percentage of GC B cells on the total number of B220+ B cells (D), and CD4+ Tfh cells on the total number of CD4+ T cells (E) in

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER LPS Sigma Aldrich cat #L4391 OVA InvivoGen cat #vac-stova SIINFEKL peptide InvivoGen cat #vac-sin CellTrace Violet Thermo Fisher Scientific cat #C34557 Z-FL-COCHO Anawa cat #A13502-5 LY300328 Lucerna Chem AG cat #MCE-HY-15533 PhosSTOP Roche cat #04906837001 cOmplete Mini protease inhibitor Sigma Aldrich cat #11836153001 Nonfat dried milk powder Applichem cat #A0830 Tween 20 Fisher scientific cat #BP337 Critical Commercial Assays MACS C Tubes miltenyi biotech cat #130-096-334 Mouse tumor dissociation kit miltenyi biotech cat #130-096-730 GentleMACS Dissociator miltenyi biotech N/A Mouse liver dissociation kit miltenyi biotech cat #130-105-807 RNeasy Mini Kit Qiagen cat #74104 SuperScript III First-Strand Synthesis System Invitrogen cat #18080051 mouse CD8 Tcell purification kit miltenyi biotech cat #130-096-427 mouse CD4 Tcell purification kit miltenyi biotech cat #130-104-454 mouse pan B cell purification kit miltenyi biotech cat #130-104-443 ChromoMap DAB kit Roche Diagnostics cat #760-159 DISCOVERY Rhodamine 6G Kit Ventana cat #760-244 DISCOVERY Red 610 Kit Ventana cat #760-245 DISCOVERY Cy5 Kit Ventana cat #760-238 DISCOVERY FAM Kit Ventana cat #760-243 Deposited Data Expression data of DLBCL patients GEO GSE12195 Single cell expression data GEO GSE115795 Expression data FL patients NCBI PRJNA278311 Expression data DLBCL patients N/A https://llmpp.nih.gov/FL/.

Techniques: Expressing, Injection, Plasmid Preparation, Immunohistochemistry, Control